Entrance and Survival of <i>Brucella pinnipedialis</i> Hooded Seal Strain in Human Macrophages and Epithelial Cells

<div><p>Marine mammal <i>Brucella</i> spp. have been isolated from pinnipeds (<i>B. pinnipedialis</i>) and cetaceans (<i>B. ceti</i>) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal <i>Brucella</i> spp. We examined the infective capacity of two genetically different <i>B. pinnipedialis</i> strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that <i>B. pinnipedialis</i> is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular <i>B. pinnipedialis</i> hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of <i>B. pinnipedialis</i> hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of <i>B. pinnipedialis</i> hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by <i>B. pinnipedialis</i> do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, <i>B. pinnipedialis</i> hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of <i>B. pinnipedialis</i> hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. <i>Brucella pinnipedialis</i> does not multiply or survive for prolonged periods intracellulary. </p>