This could reflect the bactericidal efficiency of the cell type, as HeLa cells are not professional phagocytes and PMA-induced THP-1 cells show increased macrophage maturation 5 days post treatment [60]. challenged with bacteria in a gentamicin protection assay. Our results show thatB. pinnipedialisis internalized, but is usually then gradually eliminated during the next 72 96 hours. Confocal microscopy revealed that intracellularB. pinnipedialishooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after contamination. Intracellular presence ofB. pinnipedialishooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance ofB. pinnipedialishooded seal strain in human macrophages was significantly reduced by 65.8 % ( 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded byB. pinnipedialisdo not 20-HETE release nitric oxide (NO) and intracellular bacterial presence 20-HETE does not induce cell death. In summary,B. pinnipedialishooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry ofB. pinnipedialishooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages.Brucella pinnipedialisdoes not multiply or survive for prolonged periods intracellulary. == Introduction == Brucellosis is an infectious disease that affects a wide range of mammalian species and is regarded as the worlds most common bacterial zoonotic disease [1]. For decades, the genusBrucellaincluded six species with different preferred terrestrial mammalian hosts, four of which are pathogenic for humans [2]. Recently four additional species have been included [35]. The occurrence of human disease is dependent on animal reservoirs, including wildlife [6]. Brucellaspp. were isolated from marine mammals for the first time in 1994 [7,8] and validly published as members of genusBrucellawith the namesB. pinnipedialis(pinnipeds; seals, sea lions and walruses) andB. ceti(cetaceans; whales, dolphins, and porpoises) in 2007 [3]. Marine mammal brucellae have since been serologically indicated in and isolated from pinnipeds and cetaceans from multiple locations; however gross pathology in association withBrucellainfection in marine mammals is usually exclusively found in cetaceans [9,10]. The results from experimental infections in various animal species are diverging and the zoonotic potential of the marine mammal brucellae is largely unknown [7,1113]. However, reports of human disease exist [1416]. Interestingly, none of the naturally infected human cases reported contact with marine mammals, but consumption NF2 of raw seafood was noted [15,16]. All threeBrucella-isolates from the naturally acquired infections shared an identical genotype (sequence type 27), to date only being found in marine mammals in Pacific waters [17]. The infectious cycle of marine mammal brucellae is usually to a large extent uncertain and unknown hosts or carriers may exist.Brucella melitensishas been isolated from Nile catfish (Clarias gariepinus) [18], whileB.cetiandB. pinnipedialishave been isolated from lungworms in cetaceans [19] and pinnipeds [20] respectively, pointing at other possible reservoirs in the marine ecosystem besides pinnipeds and cetaceans. In light of the extensive use of marine resources, including products from marine mammals, increased knowledge aboutBrucellaspp. in the marine environment and their possible implications in human disease, is an important issue in the One Health perspective. Brucellaspp. are facultative intracellular bacteria that can survive and replicate within membrane-bound compartments in phagocytes and epithelial cells [2123]. Studies of the mechanisms of bacterial invasion and intracellular multiplication involving the marine mammal brucellae are sparse and previously only investigated by Maquart and co-workers (2009). They observed different contamination dynamics between the marine mammal brucellae during macrophage infectionin vitro[24].Brucella pinnipedialisreference strain (NCTC 12890), isolated from a common seal (Phoca vitulina), was able to infect and multiply in human and murine macrophage cell lines to the same extent as the virulent terrestrialBrucellaspp. In contrast,B. pinnipedialisisolated from hooded seal (Cystophora cristata) showed absence of invasive potential. This particular strain displayed a high prevalence in the declined Northeast Atlantic hooded seal stock [25] and has been isolated from various organs of young seals; howeverBrucella-associated pathology has not been identified [26]. The Northeast Atlantic stock of hooded seal 20-HETE is now 10 15 % of the 1946 population and has been stable at this low level since the 1980s [27]. HowB. pinnipedialishooded seal strain (HS) may affect the population dynamics is unknown, as we lack knowledge about the strains ability to establish contamination and cause pathology. Entering macrophages after a transient.