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Background  Elucidating which wildlife species significantly contribute to the maintenance of  Ixodes ricinus  populations and the enzootic cycles of the pathogens they transmit is imperative in understanding the driving forces behind the emergence of tick-borne diseases. Here, we aimed to quantify the relative contribution of four mustelid species in the life-cycles of  I. ricinus  and  Borrelia burgdorferi  ( sensu lato ) in forested areas and to investigate their role in the transmission of other tick-borne pathogens .  Road-killed badgers, pine martens, stone martens and polecats were collected in Belgium and the Netherlands. Their organs and feeding ticks were tested for the presence of tick-borne pathogens.    Results   Ixodes hexagonus  and  I. ricinus  were found on half of the screened animals ( n  = 637). Pine martens had the highest  I. ricinus  burden, whereas polecats had the highest  I. hexagonus  burden. We detected DNA from  B. burgdorferi  ( s.l. ) and  Anaplasma phagocytophilum  in organs of all four mustelid species ( n  = 789), and  Neoehrlichia mikurensis  DNA was detected in all species, except badgers. DNA from  B. miyamotoi  was not detected in any of the investigated mustelids. From the 15 larvae of  I. ricinus  feeding on pine martens ( n  = 44), only one was positive for  B. miyamotoi  DNA, and all tested negative for  B. burgdorferi  ( s.l. ),  N. mikurensis  and  A. phagocytophilum . The two feeding larvae from the investigated polecats ( n  = 364) and stone martens ( n  = 39) were negative for all four pathogens. The infection rate of  N. mikurensis  was higher in feeding nymphs collected from mustelids compared to questing nymphs, but not for  B. burgdorferi  ( s.l. ),  B. miyamotoi  or  A. phagocytophilum.Conclusions  Although all stages of  I. ricinus  can be found on badgers, polecats, pine and stone martens, their relative contribution to the life-cycle of  I. ricinus  in forested areas is less than 1%. Consequently, the relative contribution of mustelids to the enzootic cycles of  I. ricinus -borne pathogens is negligible, despite the presence of these pathogens in organs and feeding ticks. Interestingly, all four mustelid species carried all stages of  I. hexagonus , potentially maintaining enzootic cycles of this tick species apart from the cycle involving hedgehogs as main host species.    Electronic supplementary material  The online version of this article (10.1186/s13071-018-3126-8) contains supplementary material, which is available to authorized users.

Role of mustelids in the life-cycle of ixodid ticks and transmission cycles of four tick-borne pathogens