Lyme disease, or Lyme borreliosis, is the most prevalent tick-borne infection of humans. In the early stages, Lyme disease is clinically manifested by an erythema migrans, an expanding skin lesion occurring after several days or weeks at the site of the tick bite. Late and more serious Lyme disease is a multi-system disorder with skin, neurological, cardiac and musculoskeletal manifestations [1, 2]. Over the last decade, the incidence of Lyme disease has increased in at least nine European countries . In The Netherlands, a long-term retrospective study among all general practitioners (GP) has shown a strong and continuing increase in GP consultations for erythema migrans during the past fifteen years from 4 per 10,000 inhabitants in 1994 up to 13 per 10,000 inhabitants in 2009 [4, 5]. A similar magnitude of increase was observed in the number of people that reported a tick bite: The incidence of GP consultations for tick bites was 19 per 10,000 inhabitants in 1994, and increased to 56 tick bites per 10,000 inhabitants in 2009 [4, 5]. The most straightforward explanation for the increase in Lyme disease is therefore the increase in the exposure to infected ticks. Despite considerable efforts during the past decades in education of the public, aiming to reduce human exposure to ticks and promote timely removal of ticks from the human skin, the rise in the incidence of Lyme disease continued.
The risk of acquiring Lyme disease depends on many different biological, environmental, and societal factors [6–14]. In short, however, it depends on two main factors: the abundance of questing Ixodes ricinus ticks infected with the causal agent B. burgdorferi sensu lato (s.l.), and the level of human exposure to ticks. In this study, we assessed whether the number of questing ticks infected with B. burgdorferi s.l. in The Netherlands has increased during the past decades. Besides direct methods such as tick sampling from the field, we explore several indirect measures to investigate longitudinal trends in the number of questing Borrelia-infected nymphs.
The number of questing ticks in an area is determined by the tick density and their level of activity, which, in turn, is determined by a complex interplay between vegetation, climatic conditions and the presence of blood hosts. For example, vegetation provides questing sites for ticks, but it also affects micro-climatic conditions, such as humidity and temperature, which determines tick survival and activity [15–18]. Vegetation also influences utilization by host animals and affects questing times by providing different degrees of shelter for ticks. An increase in tick bites may be due to an increase in tick density or activity but could also be due to an increase of tick suitable areas or exposure.
Temporal weather and climate conditions appear to be predictors for the tick activity, and to a lesser extent tick density [19–22]. Nymphal and adult ticks tend to quest for a blood meal once the weekly mean daily maximum temperature exceeds 7°C [23–25]. The development of ticks depends upon the consumption of vertebrate blood. Therefore, the abundance of feeding hosts can affect the abundance of ticks. All mobile life stages of Ixodes ricinus can feed on a broad range of warm- and cold-blooded vertebrate hosts [12, 21, 26, 27]. Ixodes ricinus larvae infest small mammals, but also feed on larger animals such as roe deer. Nymphs and adults usually feed on medium-sized and large mammals . These differences are probably due to the differential vertical distribution of instars . To investigate trends in the relative abundance of blood hosts during the past decades, we analyzed readily available data on the abundance of roe deer and fallow deer, birds, rodents, and proxies for the abundance of rodents such as birds of prey [30, 31].
The best available estimate for the tick density is the activity of questing ticks measured by standard blanket dragging, despite the limitation that moulting, resting and feeding ticks are not caught [32, 33]. Furthermore, this technique is not equally efficient in different vegetation types, and therefore will not provide absolute numbers of questing ticks from a given area. Measuring the number of ticks searching for a blood meal, and testing these ticks for infection with B. burgdorferi s.l. provides an estimate of local and temporal variation in public health risk. To search for longitudinal trends in the number of questing ticks and tick infection with B. burgdorferi s.l., tick sampling data from two field studies was analyzed. Our analyses involved a long-term field study at a single location [18, 34, 35], and an ongoing longitudinal field study at thirteen locations geographically spread throughout The Netherlands .
Finally, we applied a population genetic approach as a complementary indirect exploration for changes in the abundance of B. burgdorferi s.l. infected ticks that were collected in these field studies. For this purpose, we used genetic information of Borrelia-DNA found in ticks to construct phylogenetic trees, mapping the genetic divergence within and between B. burgdorferi genospecies. Using this reconstruction of the recent demographic history of B. burgdorferi, we looked for indications of changes in the genetic diversity over time, which may suggest changes in the sylvatic transmission of B. burgdorferi s.l. in The Netherlands.