Influence of tick age and land-use on Borrelia burgdorferi s.l. in Ixodes ricinus ticks from the Swabian Alb, Germany

Background: In Europe, Ixodes ricinus ticks transmit various zoonotic pathogens, including Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis (LB). However, the relationship between Borrelia prevalence, bacterial load in unfed nymphs of different physiological ages, and the influence of season and land-use remains poorly understood. The B. burgdorferi s.l. complex exhibits significant genetic diversity, with genospecies varying in distribution and pathogenicity. This study aimed to examine physiological tick age in relation to land-use, Borrelia infection rates, and genetic diversity. Furthermore, small and large mammal diversity as well as environmental factors such as shrub cover and tree species richness were incorporated in the analyses. Methods: Ticks were collected using the flagging method on 25 experimental plots in the Biodiversity Exploratory Swabian Alb in Baden-Wuerttemberg, Germany, during spring, summer, and autumn of 2023, as well as spring 2024. This was followed by morphometric age measurement of the nymphs as well as by molecular biological analyses for Borrelia spp. and subsequent multilocus sequence typing (MLST) to detect Borrelia genospecies. Generalized linear models (GLM), and generalized linear mixed models (GLMM) were implemented to assess the effects of season and land-use on Borrelia prevalence and tick age and their reciprocal interactions as well as on effects of small and large mammal diversity on Borrelia diversity. Proportional odds logistic regression evaluated the impact of environmental factors on morphometric tick age. Model averaging was specifically applied to Borrelia genospecies to address uncertainty and refine coefficient estimates. Results: A total of 1,816 Ixodes spp. ticks were collected [63 females (3.5%), 48 males (2.6%), 1,439 nymphs (79.2%), 266 larvae (14.7%)]. The nymphs examined varied in the age groups, with age group II (young) for 1.0%, age group IV (old) accounting for 7.6% and age group III (middle-aged) for 91.4%. The overall Borrelia prevalence was 6.5%, but it varied among the developmental stages. The GLMM revealed that Borrelia prevalence in age-measured nymphs differed significantly between seasons, with the highest prevalence in autumn (11.9%; confidence intervals, CI 7.83–17.52) compared with spring (P = 0.0177) and summer (P = 0.0478). MLST analyses revealed five different genospecies: B. garinii, B. afzelii, B. valaisiana, B. burgdorferi sensu stricto and B. lusitaniae. For 44 samples, sequence type (ST) assignment was possible, revealing 34 different STs, all of which except for 12 have not been detected previously. Further analyses using a conditional averaged generalized linear regression model revealed a significant increase in the diversity of Borrelia genospecies with higher Shannon diversity indices of large mammals (P = 0.00824). Conclusions: Our study revealed high Borrelia diversity in Ixodes ricinus ticks in the Swabian Alb, Germany, with a peak prevalence in autumn. Large mammal diversity influenced genospecies diversity, while tree composition seemed to affect tick age, highlighting key ecological drivers of Borrelia transmission.