We here report the first detection of A. phagocytophilum and Mycoplasma sp. in seal lice E. horridus.
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 [39, 40]. 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. As tick infestations have never been reported to occur in marine seals, potential role of hematophagous echinophthtiriid lice in A. phagocytophilum transmission can here be hypothesised.
Clinical signs of granulocytic anaplasmosis can include high fever, inappetence, anorexia, dullness, reduced weight gain, coughing and abortion. In addition, leucopenia, thrombocytopenia and/or anaemia have been frequently reported to occur in certain A. phagocytophilum strain-infections [41]. 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 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) [1]. In this context, red fox activity was observed in UK within a mainland grey seal breeding colony [42] and satellite tracking showed that tagged grey seals in the Netherlands leave to breed in the UK [43].
Mycoplasma sp. was also molecularly identified in collected seal lice specimens and in contrast to A. phagocytophilum there are previous reports on occurrence of mycoplasmal infection in pinnipeds [44, 45, 46, 47, 48, 49]. 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 [46]. 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% [50], evidencing rather cosmopolitan distribution of mycoplasmas in pinnipeds. 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 [51]. 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 [49, 52], 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. He previously hunted and harvested ringed seals without protective gloves in an area where Mycoplasma infected seals were noticed before [34]. For universal detection of mycoplasmas the highly specific and sensitive PCR assay of van Kuppenveld was used based on the conserved region of the 16S gene [26]. 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 Northern Wadden Sea. Whether seal lice might be potentially involved in the transmission of Mycoplasma needs further investigations.
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 described as natural obligate intermediate host of A. spirocauda [19, 20, 21], and different stages of A. spirocauda larvae were found in dissected E. horridus seal lice [19, 21]. So far, the heartworm A. spirocauda has been reported from different phocid species such as harbour seals (P. vitulina), hooded seals (Cystophora cristata), bearded seals (Erignathus barbatus), ribbon seals (P. fasciata), harp seals (P. groenlandica), ringed seals (P. hispida), spotted seals (P. largha), monk seals (Monachus monachus), and recently from grey seals (H. grypus) [20, 37, 53]. Furthermore, there is a significant positive correlation between heartworm infection and infestation of harbour seals with seal lice [21, 22]. The designed nested PCR on basis of the mitochondrial cox1 gene was superior in detection of A. spirocauda-DNA in lice compared to a single PCR and had a confident sensitivity of five microfilariae corresponding to 0.5 ng DNA [54]. Recently Keroack et al. [37] 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), supporting 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 [7]. Our SEM analysis confirmed unique morpho-physiological 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 [5, 6, 20]. Access to oxygen is to be considered limiting factor for survival and reproduction strategies of this marine ectoparasite. Presence of numerous and stout setae and tiny scales covering almost the whole body could be useful for accumulation of air bubbles, as reported [5]. Females of E. horridus are suggested to lay their eggs only when seals are on land [6, 11]. The predilection sites are different compared to genera Antarctophthirius and Lepidophthirius as E. horridus parasitize mainly the head of animals and thereby being more frequently exposed to oxygen while seals are in the sea. In this context, presence of several micropyles on egg lids could also improve air diffusion from accumulated air bubbles in fur coat of seals during short dives from haul-out sites, when the tide comes in or when animals are completely outside sea water. Regarding its obligate haematophagous feeding habits, extrudable and strong developed proboscis of E. horridus in all parasitic stages is clearly indicating an efficient obligate stationary permanent ectoparasite behavior. The stationarity of Echinophthirius is also underlined by diving experiments performed by Messner et al. [55] who showed that submerged E. horridus adopt a sudden immobility and do not trap or retain a layer of air (i.e. a plastron). The authors postulate that E. horridus will not leave the air layer of the dense, wooly ground hair once the seals are offshore. In this context, the restructured strong forelegs with claws could facilitate the fixation of adult E. horridus in the down coat.
During the present study, sampling of E. horridus specimens was carried out using fixation techniques of stranded seals during a rehabilitation period. In general, many studies on echinophthiriid lice necessitated either death [56, 57], anesthesia of free-living seals or were detected as pathological findings [58]. Most recently, studies on A. microchir from juvenile South American sea lions (O. flavescens) focused on fixation techniques with low handling times implying conservation aspects [12, 59]. Ebmer et al. [60] showed the successful application of a non-invasive sampling method by using a lice comb screwed on a telescopic rod for taking lice samples within a unique ‘urban‘ colony of South American sea lions (O. flavescens) in Valdivia, Chile. This technique could definitively be applied during sampling of other synanthropic seals. Analysis of seal pathogens in lice sampled during a rehabilitation period or from synanthropic seals is a less invasive method for epidemiological studies compared to blood collections or to be reliant on stranded death animals.
Regarding epidemiology and pathogenicity of echinophthiriosis, it is more frequently reported in young and weak animals [19, 20, 61], showing no seasonal variations for adult seals, but pups and immature seals have higher prevalences in spring. In contrast, Dailey and Fallace [22] 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 [11].