The Spatial Distribution of Dermacentor Ticks (Ixodidae) in Germany—Evidence of a Continuing Spread of Dermacentor reticulatus

In Europe, two tick species of the genus Dermacentor occur, Dermacentor marginatus and Dermacentor reticulatus. When the spatial distribution of both species in Germany was studied comprehensively for the first time in 1976, D. marginatus populations were recorded along the Rhine and Main river valleys in southwestern Germany, while D. reticulatus was very rare. In the last 50 years, however, a considerable range expansion of D. reticulatus has been noted in several European countries. To assess the current distribution of Dermacentor spp. in Germany, citizens were asked to send in ticks suspected to belong to the genus Dermacentor or that were of “unusual” appearance. From February 2019 until February 2020, 3,902 Dermacentor ticks were received in total. Of those, 15.48% (604/3,902) were identified as D. marginatus and 84.24% (3,287/3,902) as D. reticulatus, while 11 specimens could not be identified to species level. The majority of D. reticulatus specimens was collected from dogs (1,212/2,535; 47.12%), while D. marginatus was mostly collected from horses (184/526; 34.98%). Our results confirm that the adults of both Dermacentor species are active all year round. D. reticulatus specimens were sent in from all federal states except the Free and Hanseatic City of Hamburg, while D. marginatus specimens were only received from locations in southwestern Germany. Overall, data obtained from this citizen-science study show that D. reticulatus has significantly expanded its range, especially in northern Germany. Regarding D. marginatus, new locations northwest of the previous range were detected, although the distribution has remained rather stable as compared to D. reticulatus. The spread of D. reticulatus, the vector of Babesia canis, is of major importance for veterinarians and dog owners in terms of canine babesiosis outbreaks or endemization in hitherto B. canis-free areas. Thus, veterinarians and veterinary students need to be informed about the new situation to be able to give adequate advice to dog owners on the extended D. reticulatus range and appropriate control measures.


INTRODUCTION
In Europe, the hard tick genus Dermacentor is represented by two species, Dermacentor marginatus (Sulzer, 1776) and Dermacentor reticulatus (Fabricius, 1794). The range of the ornate sheep tick, D. marginatus, extends from Portugal in the west throughout southern Europe and northern Africa into Central Asia. The species' southern and northern distribution limits are currently considered to be in Morocco and at the northern extension of the Rhine basin in Germany (1). Within this range, the species typically inhabits steppes, meadows, open forests, and semi-desert areas (2). The ornate dog tick, D. reticulatus, has a more northern distribution, occurring from northern Portugal to southern Latvia (1). It is found in a wide range of habitats, including meadows, open forests, heath-, and marshland, clearings, and suburban wasteland (3).
Immature stages of both D. marginatus and D. reticulatus are almost exclusively endophilic parasites of rodents. As adults, both sexes commonly infest larger mammals such as sheep, dogs, horses, goats, cattle (2), and occasionally humans (4). They play a role as vectors of various pathogens of considerable veterinary and medical importance. For example, both D. marginatus and D. reticulatus are competent vectors of protozoa of the order Piroplasmida, which may cause potentially fatal disease in animals. The most important causative agent of canine babesiosis in Europe, Babesia canis, is transmitted by D. reticulatus (5), while both Dermacentor species may transmit causative agents of equine piroplasmosis (6). Additionally, the vector function of D. reticulatus for tickborne encephalitis virus (TBEV) has recently been proven (7). Although Ixodes ricinus (Linnaeus, 1758) is the main vector for TBEV in Europe, the virus has repeatedly been isolated from D. reticulatus in a TBEV-endemic area in Germany (8). Furthermore, Dermacentor spp. are the most relevant vectors for two causative agents of tick-borne lymphadenopathy in central Europe, Rickettsia slovaca transmitted by D. marginatus and R. raoultii transmitted by D. reticulatus (2,3). In addition, both species are relevant in central Europe as vectors of Francisella tularensis (9), and D. marginatus may contribute to the transmission of Coxiella burnetii, the causative agent of zoonotic Q fever (10).
The first comprehensive study on the spatial distribution of the genus Dermacentor in Germany was published by Liebisch and Rahman (11). The authors reported a mosaic-like pattern of D. marginatus occurrence along the Rhine and Main river valleys in southwestern Germany. In contrast, an established D. reticulatus population was found at only one location in Germany at that time, in a forest near Tübingen. In the 1960s, D. reticulatus was also reported from the area of Potsdam in the former German Democratic Republic (12). In the recent past, comprehensive data on the distribution of both species in Germany have been gathered from either citizen-science approaches (13) or literature reviews (1,14). These data, including reports on Dermacentor occurrence up to the year 2014, showed a considerable range expansion of D. reticulatus, which is in accordance with reports from other European countries, e.g., Slovakia and Poland (15,16).
In 2019, we received indications of a further significant spread of D. reticulatus in Germany and thus aimed to assess the current distribution of D. reticulatus and D. marginatus in Germany by involving the general public. Citizens were asked to send in ticks belonging to the genus Dermacentor or of unusual appearance to allow mapping the distribution of both Dermacentor species in detail and identifying new areas of occurrence.

Citizen-Science Call
In February 2019, a single male specimen of D. reticulatus collected from a dog in the city of Hanover, northern Germany, was received by the Institute for Parasitology, University of Veterinary Medicine Hannover. This was an unusual finding, as hitherto the region of Hanover has not been considered within the range of this tick species. Upon request, the owner stated that the tick was found crawling on the dog after a walk and that the dog had not traveled recently. Additionally, in March 2019, a member of the institute noticed one female and three male Dermacentor ticks on her dog after a walk in Clausthal-Zellerfeld, located about 75 km southeast of Hanover, also hitherto not recognized as within the German Dermacentor range. To investigate whether these were accidental findings or if a further range expansion of D. reticulatus has occurred in Germany, a call to send in Dermacentor ticks was published in the May issue of the gazette of the Federal Chamber of Veterinarians, which is sent to every veterinarian in Germany. Furthermore, a press release was issued at the beginning of May 2019, asking citizens to send in Dermacentor ticks, which was shared through several print and social media.
Additionally, as of the end of February 2019, the Department of Parasitology at the University of Hohenheim near Stuttgart, southern Germany, released a call to send in Hyalomma ticks as well as ticks of unusual appearance. Again, respective press releases were circulated in various regional and national media, and, additionally, a website was designed for further information, where citizens were also specifically asked to send in ticks of the genus Dermacentor.
All media releases included pictures to help citizens distinguish between different tick genera. Along with the ticks, citizens were asked to provide information on the date and location of collection [Global Positioning System (GPS) data or postal code], the involvement of potential hosts, and details about the circumstances under which the tick was discovered. To increase motivation to participate, citizens were informed about the tick species of their specimen(s).
The accuracy of the reported locations where ticks were found was categorized based on the details provided by the senders as follows: (i) the accuracy was estimated to be high if there was a high probability that a natural habitat of the collected tick was in close proximity to the location where it was found. For example, a high accuracy was assumed for ticks collected from cattle and horses, which did not leave their pasture in the days before an infestation was detected, as well as for ticks found on vegetation, but only when the sender provided a GPS reference or precise address. (ii) A medium accuracy was assumed for unengorged ticks found on dogs or humans during or immediately after a walk, as well as for ticks from cats or wild terrestrial animals, or if the location met the criteria for a high accuracy ranking but was reported only in the form of a postal code. (iii) The reported location was considered to be of low accuracy in cases of engorged ticks found on dogs or ticks found in an unsuitable habitat (e.g. inside a house), as the origin of these ticks was often unclear. (iv) If no information on the location or the circumstances of tick detection was provided or the ticks were detected on dogs or humans travelling large distances, the accuracy was categorized as unknown.
Only locations with a high or medium accuracy were used for distribution maps. These distribution maps were compared with the results reported by Rubel et al. (1) and Naucke (13). Maps were generated in R v. 3.5.1 (20) with spatial data retrieved via the rworldmap package (21), via the eurostat package (22), and from the Global Administrative Areas Database (23).

Tick Collection and Identification
From mid-February 2019 until the end of February 2020, 3,902 ticks of the genus Dermacentor were received. With a total of 3,287 (84.24%) specimens, D. reticulatus was sent in much more frequently than D. marginatus (604 specimens; 15.48%). The remaining 11 specimens (0.28%) could not be identified to species level, as essential morphological features had been destroyed. The sex ratio of D.    Figure 1B).

Geographic Distribution
In contrast to D. reticulatus, D. marginatus specimens were received from only six federal states located in southern and western Germany (Baden-Wuerttemberg, Bavaria, Hesse, North Rhine-Westphalia, Rhineland-Palatinate, and Saarland) (Table 1, Figure 1). Compared with the distribution maps provided by Rubel et al. (1) and Naucke (13), several additional sites of D. reticulatus occurrence are evident, especially in the north of Germany (Figure 2A). The spatial distribution of D. marginatus is largely comparable to the data provided by Rubel et al. (1) and Liebisch and Rahman (11). However, additional areas of occurrence were identified, for example, in the vicinity of Cologne ( Figure 2B).  Table 2.

DISCUSSION
The present study aimed to assess the current distribution of Dermacentor spp. in Germany. A continuing range expansion of D. reticulatus has been observed in several European countries (24)(25)(26). This range expansion has been attributed to climatic changes as well as changes in land use, travel activities of humans and pets, and an increase in available wildlife hosts, e.g., red foxes and wild boar (3). The spread of D. reticulatus is of considerable veterinary importance, since it is the vector of Babesia canis, a life-threatening protozoan blood parasite of dogs. Currently, B. canis transmission only occurs in restricted areas in Germany (27,28), while autochthonous infections with other piroplasms transmitted by Dermacentor spp., such as B. caballi and T. equi causing equine piroplasmosis, are rare in Germany (29). Nevertheless, introduction of these pathogens with infected animals or ticks from endemic areas may lead to the emergence of new transmission foci in areas where Dermacentor populations are present, especially since Babesia spp. are transmitted transovarially in ticks (30). Furthermore, D. reticulatus may pose a risk for humans due to its vector role for R. raoultii, F. tularensis, and TBEV, among other tick-borne pathogens (2, 3). Although D. reticulatus is considered to have been part of the German tick fauna for at least 100 years (31), it was limited to only few reported locations during most of the 20th century (12,32,33). After 1976, D. reticulatus apparently started spreading probably from at least two different populations, one in southwestern (11) and one in northeastern Germany (12). In the 1990s, several previously unknown D. reticulatus populations were described following autochthonous cases of canine babesiosis (28,34), although comprehensive studies on the species' distribution during the last quarter of the 20th century are lacking. Since the turn of the millennium, the distribution of  D. reticulatus in Germany has been the subject of several studies, especially with regard to its increased spread (1,13,32,35,36). As compared to field studies or literature surveys, studies involving citizens can cover a wider spatial extent and result in a larger number of records (37,38), although the quality of the obtained data can be variable. In the current study, only records presumably reflecting the true occurrence of the tick species, i.e., those assigned to a high or medium geographic accuracy, were taken into account for distribution maps. Our results confirm earlier studies (1,13,32), indicating that D. reticulatus is continuing its spread throughout Germany.
When compared with the most recent data provided by the European Centre for Disease Prevention and Control (39) (ECDC, cf. Figure 1A), our results show that multiple new foci of D. reticulatus occurrence have appeared in northern, western, and southern Germany. However, the comparability of the ECDC's map with the data generated in the present study is limited, as the resolution of the map provided by the ECDC is based on government districts and is therefore comparatively low (39).
The obtained data show notable clusters of D. reticulatus occurrence around the cities of Hanover and Brunswick (eastern part of the northern German federal state of Lower Saxony), Stuttgart, Mannheim, Freiburg, and Karlsruhe (western part of the southern German federal state of Baden-Wuerttemberg). In this context, however, it has to be kept in mind that citizens were asked to participate in the study via press releases, which were covered by various regional and national media. Regional media coverage was probably enhanced in the vicinity of the involved research institutions (located in Hanover and Stuttgart); thus, the clusters near Hanover and Stuttgart may reflect this bias in media attention. Nevertheless, D. reticulatus was detected for the first time in the greater Hanover area, where occurrence was also verified by flagging of questing ticks in 2019 (data not shown). In addition, the data indicate several other potentially new locations outside the hitherto known range, especially in northwestern Germany. D. reticulatus was even found in the northernmost part of Germany, on the island of Sylt in the North Sea (∼8.34 • E/54.91 • N). Two independent submissions of unfed male ticks from Sylt indicate that a D. reticulatus population may be present on the island. However, Sylt is also a popular tourist destination, and it cannot be entirely excluded that the unfed male ticks did not originate on the island, but reached it together with traveling dog hosts.
A further cluster of D. reticulatus occurrence was noted in the eastern part of Germany, around the federal state of Berlin. Interestingly, the citizen-science data do not confirm the presence of D. reticulatus in the area between the cities of Leipzig and Berlin, where the species was previously reported (1,32). However, this may be due to the low population density in this area, limiting the number of participants in the study. Alternatively, D. reticulatus may be so widespread in this region that the local population did not consider respective findings worth reporting.
The distribution of D. marginatus as indicated by this study is still very similar to the data presented by Liebisch and Rahman (11), also included in the distribution map by Rubel et al. (1). Nevertheless, additional locations were found in the federal state Rhineland-Palatine and in North Rhine-Westphalia, near the city of Cologne (6.96 • E/50.94 • N). To date, the current northern distribution limit of D. marginatus was believed to be near Giessen, federal state of Hesse, Germany, at coordinates 8.32 • E/50.65 • N (40). Thus, a slight northward spread, probably along the Rhine, did occur. Interestingly, the ecological niche model by Walter et al. (40) identified most of Rhineland-Palatinate as suitable habitat for D. marginatus, as well as a large area to the northeast of the distribution limit, including the entire federal state of Hesse and even the southern part of Lower Saxony. In contrast, North Rhine-Westphalia, which is located to the northwest of Giessen, was not identified as a suitable habitat by Walter et al. (40). Further studies should continue to examine whether stable populations of D. marginatus are permanently established in North Rhine-Westphalia.
The seasonal activity of both Dermacentor species in Central Europe was studied multiple times in the past (2,3). The data presented here accord with former reports (41), as D. reticulatus numbers in Germany peaked in September and October and, to a lesser extent, from March to May, whereas D. marginatus numbers peaked in February and March. Similar patterns were observed in field studies on questing Dermacentor ticks in other parts of Europe [e.g. (42,43)]. Likewise, the current study confirms winter activity of both tick species (3,43). However, it must be kept in mind that media coverage and human behavior, among other factors, can bias data gathered by a citizen-science approach, which limits the comparability to data from field collections. Sendings in March and May 2019 were probably influenced by the preceding press releases issued by the involved research institutions.
D. reticulatus was found predominantly on dogs, whereas D. marginatus was found mostly on equids. Among domestic animals, adult D. reticulatus seems to prefer dogs and may even outnumber I. ricinus on these hosts in areas where both species occur (44). In contrast, the main hosts of adult D. marginatus are ungulates, especially sheep (11). The fact that no D. marginatus was collected from sheep in the present study may be attributed to the study design, as horse or dog owners are far more likely to notice Dermacentor ticks on their animals as something unusual and worth reporting than shepherds, who probably do not consider D. marginatus ticks as unusual. In addition, sheep are probably less often checked for tick infestation than dogs or horses, and their thick wool makes ticks hard to spot if the infestation is not severe.
In light of the zoonotic pathogens that may be transmitted by Dermacentor spp., it is worth noting that human tick bites were only rarely reported. Although 4.34% of D. reticulatus and 12.55% of D. marginatus for which information on host association was provided were found crawling on humans, the proportions of ticks that had actually bitten humans were only 0.36 and 1.71%, respectively. In Spain, where both D. reticulatus and D. marginatus occur, these species accounted for 2.22 and 12.52% of 4,049 ticks found on humans, respectively (4). These numbers are comparable to our data; however, no information on the proportion of ticks that had actually bitten humans appears in the Spanish study. In the areas of Liguria and Tuscany, Italy, D. marginatus was identified as the second most important anthropophagic tick, after I. ricinus, accounting for 9.1% of 565 human tick bites (45). In contrast, among 2,547 ticks removed from humans in Germany between 2013 and 2017, only 0.16% were identified as Dermacentor spp. (46). Overall, the available data indicate that D. marginatus is more likely to attach to or even bite humans than D. reticulatus.

CONCLUSIONS
The present study shows that D. reticulatus is continuing to spread in Germany, especially in the northwestern part of the country. Overall, this tick was found in all federal states except the Free and Hanseatic City of Hamburg. In contrast, the distribution of D. marginatus is still restricted to southwestern Germany; however, newly identified locations in North Rhine-Westphalia show that this species has also undergone a geographical spread. A range expansion of both species is particularly worrying in light of their role as vectors. Both species may transmit human pathogens; however, they rarely seem to bite humans in Germany. Thus, the implications for public health may be considered of minor importance. By contrast, the spread of D. reticulatus is of major importance for veterinarians and dog owners in terms of canine babesiosis outbreaks or endemization in hitherto B. canis-free areas. Thus, veterinarians and veterinary students need to be informed about this situation, with updates during continuing education. Similarly, dog owners need to be advised on the expanding D. reticulatus range and the need for careful tick control measures by veterinarians and, where appropriate, by veterinary associations such as the German chapter of the European Scientific Counsel Companion Animal Parasites (ESCCAP).

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.