The present study reports on the presence of pathogens in the seal louse E. horridus and discusses the potential vector role of this ectoparasite in the marine habitat. The presence of A. spirocauda larvae in E. horridus was already described in the past [7, 8, 9] and, for first time, molecularly confirmed in the present study. Thereby, our results strongly support the hypothesis that E. horridus functions as intermediate host of A. spirocauda. Furthermore, A. spirocauda, the only filarial nematode parasite of phocid seals [8, 34], was molecularly characterized for the first time. Before this survey started no molecular data were available from the filariae A. spirocauda.
Moreover, our results represent the first detection of A. phagocytophilum and Mycoplasma sp. in seal lice. However, based on the limited knowledge of vector-borne pathogens occurring in marine habitats, our findings of A. phagocytophilum and Mycoplasma sp. should not directly be equated with the presence of vector-borne pathogens in seal lice. Analog to mosquitoe vectors , evidence of vector competence of E. horridus and the ability of transmitting these pathogens could only be proved by experimental infections or the morphological verification of pathogens in salivary glands of seal lice under controlled settings. Using molecular methods, it cannot be excluded that pathogens detected were located in the gastro-intestinal tract of E. horridus individuals after blood-consumption of infected seals, but are not able to be transmitted to other host individuals. Nevertheless, molecular surveys of the present study constitute important baseline studies in the field of marine mammal parasitology to initially reveal a spectrum of pathogens, which could possibly be transmitted by seal lice. Thereby, our results can also help to encourage other researchers to extend the knowledge of vector-borne pathogens in the field of marine mammal parasitology.
To our current knowledge, there are neither reports on A. phagocytophilum or Mycoplasma spp. in any other ectoparasite affecting marine mammals nor evidence of anaplasmosis occurring in stranded phocid seals [36, 37]. A. phagocytophilum (Rickettsiales, Anaplasmataceae) constitutes an emerging globally distributed pathogen transmitted mainly by Ixodes ticks and leads to granulocytic anaplasmosis, which is one of the most relevant tick-borne diseases of veterinary and public health significance worldwide . Practically, nothing is known on anaplasmosis in seals as nobody has investigated clinical relevance of A. phagocytophilum in determining marine seal population health status, like the ones occurring in the Dutch Wadden Sea. No tick infestations are known to occur in pinnipeds, nonetheless it might be speculated that haemotophagous ectoparasites other than ticks might become involved in transmission of A. phagocytophilum among marine mammals.
Variable clinical signs of granulocytic anaplasmosis can include high fever, lethargy, inappetence, anorexia, dullness, reduced weight gain, coughing and abortion in different animal species in Europe, including domestic ruminants, horses, dogs and cats [39, 40]. In humans, symptoms were reported as non-specific and include influenza-similar symptoms with fever and myalgia. In addition, leucopenia, thrombocytopenia and/or anaemia have been frequently reported to occur in certain A. phagocytophilum strain-infections . Many of these clinical signs coincided well with the ones observed in the highly E. horridus infested grey seal pup at Sealcentre Pieterburen and might have been linked to an acute granulocytic anaplasmosis infection. However, the zoonotic potential of the A. phagocytophilum genetic variant detected here cannot be assessed by the msp2 sequence and needs further characterisation. In this context, it would also be interesting to investigate if red foxes — hosts of A. phagocytophilum — living in the Dutch coastal dune area occasionally feed on dead seal pups and get infested by E. horridus as had been reported for Arctic foxes from Alaska and the fur seal louse (Antarctophthirus callorhini) . Thereby, red fox activity was observed in UK within a mainland grey seal breeding colony  and satellite tracking showed that tagged grey seals in the Netherlands leave to breed in the UK .
Mycoplasma sp. was also molecularly identified in collected seal lice specimens in the current study, and in contrast to A. phagocytophilum there are previous reports on occurrence of mycoplasmal infections in pinnipeds [43, 44, 45, 46, 47, 48]. Thus, Mycoplasma spp. are common inhabitants of respiratory, gastrointestinal and genital tract of marine mammals, and a study on Australian fur seals (Arctocephalus pusillus doriferus) demonstrated the presence of different Mycoplasma species such as M. zalophi, M. phocidae, M. phocicerebrale, and Mycoplasma sp. in tested animals . Furthermore, PCR testing of nasal swabs detected presence of Mycoplasma spp.-DNA in South American fur seal (Arctocephalus australis) populations in Peru with an estimated prevalence of 37.9% , evidencing rather cosmopolitan distribution of mycoplasmas in pinnipeds. For universal detection of mycoplasmas the highly specific and sensitive PCR assay of van Kuppenveld  was used in the current study based on the conserved region of the 16S gene. Direct sequencing and sequencing of several clones indicated a single species only. However, better species differentiation would be possible selecting further housekeeping genes (e.g. rpoB, rpoC) and culturing for phenotypical characterisation and serological testing. Therefore, our findings on Mycoplasma-positive E. horridus lice might suggest presence of these bacterial infections in pinnipeds of the Dutch Wadden Sea. Whether seal lice might be potentially involved in the transmission of Mycoplasma needs further investigations.
However, some mycoplasmas (e.g. M. phocicerebrale) are associated with seal mortality and zoonotic ‘seal-finger’ infection, a disease known among people who handle seals for more than hundred years . Seal-finger lesions could progress to septic arthritis of joints if tetracycline-based treatment is not received. Accordingly, in more recent published studies on a series of bites and contact abrasion in open-water swimmers caused by California sea lions and harbour seals revealed presence of Mycoplasma spp. in human wounds [48, 51], demonstrating its zoonotic potential. GenBank database search using the partial Mycoplasma sp. 16S sequence, detected in seal lice collected from three harbour seals in the current study, resulted in 100% identity to the sequence of an unnamed Mycoplasma species (GenBank accession no. KP292569) obtained from a patient with ‘seal finger‘ and infected hip joint. The patient previously hunted and harvested ringed seals (Phoca hispida) without protective gloves in an area where Mycoplasma infected seals were noticed before .
Molecular analyses on collected seal lice from harbour and grey seals revealed presence of DNA of the seal heartworm A. spirocauda. In this context, E. horridus has previously been proposed to be the natural obligate intermediate host of A. spirocauda [7, 8, 9], and different stages of A. spirocauda larvae were found in dissected E. horridus seal lice [7, 9]. So far, the heartworm A. spirocauda has been reported from different phocid species such as harbour seals, hooded seals (Cystophora cristata), bearded seals, ribbon seals (Phoca fasciata), harp seals (Phoca groenlandica), ringed seals, spotted seals (Phoca largha), monk seals (Monachus monachus), and recently from grey seals [8, 33, 52]. Furthermore, there is a significant positive correlation between heartworm infection and infestation of harbour seals with seal lice [9, 10]. Tested on 10 pools the designed nested PCR on basis of the mitochondrial cox1 gene was more sensitive in detection of A. spirocauda-DNA in lice pools compared to a single PCR and had a confident sensitivity of 10 microfilariae. This number will correspond to approximately 1.0 ng DNA . Recently Keroack et al.  developed a more sensitive A. spirocauda real-time quantitative PCR based on a highly repetitive genomic DNA repeat identified using whole genome sequencing which will improve future monitoring of seal heartworm infections. These authors also identified the first time an A. spirocauda adult worm in a presumed grey seal carcass from the coast of Cape Cod (Massachusetts, USA). However, the authors mentioned that the carcass was in very poor condition due to extensive decomposition and could not be fully identified and reported just the evidence for possible A. spirocauda infection in the grey seal. Nevertheless, the results of Keroack et al.  support our detection of A. spirocauda-DNA in the seal lice collected from the grey seal which implied that grey seals could also get infected.
Previous SEM studies of E. horridus described the antennal structures [15, 16]. Our SEM analysis confirmed unique morphological adaptation features of E. horridus as for presence of strong legs with potent and well-developed claws acting almost as padlocks, allowing this marine seal louse to hold tight to the host fur coat while diving activities as previously postulated [10, 13, 14].
Regarding epidemiology and pathogenicity of echinophthiriosis, it is more frequently reported in young and weak animals [7, 8, 54], showing no seasonal variations for adult seals, but pups and immature seals have higher prevalences in spring. In contrast, Dailey and Fallace  reported highest prevalence in autumn and winter months, but no significant differences between examined age classes of seals and their seal lice burden were detected. Interestingly, in closely related species A. microchir from the South American sea lion over 60% of 1-day-old pups were infested with lice, and recruitment increased in pups up to three days old and leveled off onwards. In 1-day-old pups, significantly more adults than nymphs were found, but this pattern was reversed in older pups, documenting importance of vertical transmission most probably through their mother .