pathogens Article Dermacentor reticulatus and Babesia canis in Bavaria (Germany)—A Georeferenced Field Study with Digital Habitat Characterization Cornelia Silaghi 1,2,*, Lisa Weis 1 and Kurt Pfister 1 1 Comparative Tropical Medicine and Parasitology, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, 80802 Munich, Germany; [email protected] (L.W.); kpfi[email protected] (K.P.) 2 Institute of Infectology, Friedrich-Loeffler-Institut, Greifswald Isle of Riems, 17493 Greifswald, Germany * Correspondence: cornelia.silaghi@fli.de; Tel.: +49-3835171172 Received: 27 April 2020; Accepted: 30 June 2020; Published: 7 July 2020 Abstract: The hard tick Dermacentor reticulatus transmits Babesia canis, the causative agent of canine babesiosis. Both the occurrence and local distribution of D. reticulatus as well as infection rates of questing ticks with B. canis are thus far poorly known in Bavaria, Germany. The objectives of this study were to conduct (1) a georeferenced field study on the occurrence of D. reticulatus with digital habitat characterization and (2) a PCR analysis of D. reticulatus collected in Bavaria for infection with B. canis. Dermacentor reticulatus were collected by flagging at 60 sites specifically selected according to habitat conditions and screened individually for Babesia DNA. A digital habitat characterization for D. reticulatus was performed according to results of the field analysis including the parameters land use, proximity to water, “potential natural vegetation”, red deer corridors and climate data. Altogether, 339 D. reticulatus ticks (214 females and 125 males) were collected between 2010 and 2013 at 12 out of 60 sampling sites. All 12 sites were characterized by high humidity with marshy areas. Babesia canis DNA was detected in 1 out of 301 (0.3%) questing D. reticulatus in Bavaria. The digital habitat characterization revealed 15 forest areas in Bavaria with similar ecological characteristics as the sites positive for D. reticulatus. Keywords: Dermacentor reticulatus; Canine babesiosis; georeferenced; ecotones; habitat characterization; red deer 1. Introduction The hard tick Dermacentor reticulatus can transmit zoonotic pathogens such as Rickettsia raoultii and Francisella tularensis as well as the protozoon Babesia canis, the causative agent of canine babesiosis, a severe infectious disease in dogs [1,2]. Canine babesiosis is increasingly also reported as an autochthonous disease in Germany [1]. Possible reasons for such autochthonous infections include an expanding distribution of D. reticulatus due to changes of climate, land use and host density and distribution [1,3,4]. A similar pattern has been shown for the distribution of Ixodes scapularis and the incidence of Lyme disease caused by Borrelia burgdorferi sensu lato. An increase in temperature due to climate change led to an increase in the incidence of Lyme disease [5]. Hence, accurate and updated knowledge on the distribution of this tick species as well as its potential infections rates with B. canis is of utmost importance, particularly with regard to potentially necessary prevention measures. First investigations on the distribution of D. reticulatus in Germany initially reported its absence in Germany [6], but since then, several D. reticulatus foci have been reported [7–13]. Furthermore, there are also some reports of D. reticulatus on dogs, roe deer and red deer [3,14–16]. Pathogens 2020, 9, 541; doi:10.3390/pathogens9070541 www.mdpi.com/journal/pathogens Pathogens 2020, 9, 541 2 of 10 Dermacentor reticulatus prefer rather humid habitats (e.g., alluvial forests and swamps) where they can survive flooding for certain periods of time [17]. Originally, D. reticulatus was reported only from areas with a lot of humidity, but there have also been reports from habitats with a drier character [7,9,18]. Since such conditions are largely present throughout Germany, further spreading of D. reticulatus is to be expected [19]. In Bavaria, a federal state in the southeast of Germany, D. reticulatus was first reported in the area of Regensburg and near Munich [20,21]. These findings raised the question of whether D. reticulatus has meanwhile also become endemic in Bavaria. To estimate the local infection risk for autochthonous canine babesiosis, prevalence rates must be determined in questing D. reticulatus. Until now, B. canis has only been detected in questing D. reticulatus in Germany in Saarland in western Germany [8]. Other detections were from engorged ticks collected on dogs [16], which, however, may reflect the infection status of the dog and not necessarily the infection rates of ticks. To update the knowledge on the occurrence of D. reticulatus in Bavaria, Germany, the objectives of this study were to establish (1) a field study on the occurrence of D. reticulatus including a digital habitat characterization using both analog and digital climate data and (2) a PCR analysis of all collected D. reticulatus for a possible infection with B. canis. 2. Material and Methods 2.1. Field Study for Dermacentor reticulatus 2 The federal state of Bavaria (70,550 km ) in the southeast of Germany is located between 47◦160 and 50◦340 northern latitude and between 8◦580 and 13◦500 eastern longitude. The field study for collection of D. reticulatus was prepared by compiling a habitat list based on three different approaches with the aim of including areas with an increased probability of finding questing D. reticulatus: (i) habitats as described in historic and current scientific literature; (ii) geographic locations of previous descriptions and observations by veterinarians, pet owners and hunters; and (iii) Bavarian rivers with both natural and renatured alluvial forests (data from the Bavarian State Ministry of the Environment and Consumer Protection). Due to the large number of river areas, widely renatured areas containing large alluvial forests were chosen. According to these three search criteria, an initial habitat list was put together. Due to the vast number of sites, they were evaluated individually according to existing vegetation and terrain using the satellite maps from Google Maps of each region of interest (https://www.google.com/maps/). Following this, altogether 60 sampling sites were chosen, which were summed up in 17 sampling areas G1–G17 (Figure1). All sites were sampled once yearly between March and June and again between August and November during the years 2010–2013, reflecting the two peak activity periods (spring and autumn) of D. reticulatus. In negative sites, D. reticulatus was assumed pseudoabsent. According to Wisz and Guisan (2009) [22], pseudoabsence describes the fact that absence of any species cannot definitely be proven. As far as tick sampling is concerned, the targeted tick species might be present in a sampling site, but might be temporarily unavailable to sampling efforts, e.g., due to climate conditions or any other reason. Positive sites were re-evaluated in one of the following activity periods. Sites which were positive in two consecutive activity periods were considered endemic foci. Ticks were collected by the flagging method, and all ticks found on the flag were collected with tweezers and added to 15 mL Falcon tubes containing 70% ethanol and stored at room temperature. For each collection site, the coordinates and altitude were documented with a mobile navigation device (Garmin® GPSmap 60CSx, Garmin, Garching, Germany). The temperature as well as the relative humidity were taken at a 50 cm height with a handheld thermohygrometer at the beginning of sampling (P33 Handmessgerät, Carl Roth GmbH, Karlsruhe, Germany). Additionally, the soil temperature at a 5 cm depth was taken with an insertion thermometer, also at the beginning of Pathogens 2020, 9, 541 3 of 10 sampling. All collected ticks were identified in the laboratory under a stereomicroscope according to morphologicalPathogens 2020 criteria, 9, x FOR [ 23PEER,24 REVIEW]. 3 of 10 Figure 1.FigureLocation 1. Location of the of 17the sampling 17 sampling areas areas (including (including th thee 60 60 sampling sampling sites sites shown shown in the insupplementary the supplementary table) in the federal state of Bavaria (Germany), sampled from 2010–2013. table) in the federal state of Bavaria (Germany), sampled from 2010–2013. 2.2. PCR2.2. Analysis PCR Analysis for Babesia for Babesia canis canis All D. reticulatus ticks were washed twice in distilled water and air-dried, and DNA was extracted AllindividuallyD. reticulatus usingticks the QIAamp were washed DNA Mini twice Kit in (Qiagen, distilled Hilden, water Germany) and air-dried, according and to DNAthe was extractedmanufacturer’s individually instructions using the for QIAamp tissue. Ticks DNA were Mini disrupted Kit (Qiagen, in a TissueLyser Hilden, (Qiagen) Germany) with 80 according µL PBS to the manufacturer’sand a 5 mm instructions stainless steel forbead tissue. in 2 mL Ticks Eppendorf were tubes disrupted for 5 min in at a 20 TissueLyser bpm. Incubation (Qiagen) was carried with 80 µL PBS andout a overnight 5 mm stainless at 56 °C. steelEvery bead 24 to in48 2samples, mL Eppendorf a negative tubesextraction for control 5 min containing at 20 bpm. distilled Incubation and was sterile water was included. The quality and quantity of the extracted DNA were checked with a carried out overnight at 56 C. Every 24 to 48 samples, a negative extraction control containing distilled photospectrometer (NanoDrop◦ ®ND-1000; PeqLab, Erlangen, Germany). and sterile waterAll DNA was extracts included. were Thescreened