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Neisseria animaloris is considered to be a commensal of the canine and feline oral cavities. It is able to cause systemic infections in animals as well as humans, usually after a biting trauma has occurred. We recovered N . animaloris from chronically inflamed bite wounds on pectoral fins and tailstocks, from lungs and other internal organs of eight harbour porpoises. Gross and histopathological evidence suggest that fatal disseminated N . animaloris infections had occurred due to traumatic injury from grey seals. We therefore conclude that these porpoises survived a grey seal predatory attack, with the bite lesions representing the subsequent portal of entry for bacteria to infect the animals causing abscesses in multiple tissues, and eventually death. We demonstrate that forensic microbiology provides a useful tool for linking a perpetrator to its victim. Moreover, N . animaloris should be added to the list of potential zoonotic bacteria following interactions with seals, as the finding of systemic transfer to the lungs and other tissues of the harbour porpoises may suggest a potential to do likewise in humans.

Marine mammals are sentinel species representing the “health” of our oceans on which we are dependent. There are many threats to marine mammals including infectious diseases that increase with climate change and pollution of the marine environment. Streptococcus phocae has frequently been isolated from diseased or dead marine mammals. However, its pathogenicity and contribution to disease in marine mammals is still unknown. As bacteria including (potential) pathogens has to deal with different host environments during colonization or infection, we investigated the survival of S . phocae in fresh porcine and phocid blood, in seawater and in the presence of macrophages and (epithelial) cells from harbor seals and pigs. Furthermore, we tested adherence on and invasion of different (marine) mammalian cells by S . phocae . Our results showed that S . phocae can survive in seawater for at least 11 and 28 days at 16°C and 4°C, respectively. It is able to grow in blood of harbor and grey seals, but not in porcine blood. Furthermore, S . phocae is adherent and invasive to cells from seals and pigs, while the portion of invasive cells was higher in seal derived cells. Macrophages of harbor seals were more efficient in killing S . phocae than porcine macrophages. Our results indicate that S . phocae has strategies enabling it to adapt to the marine environment and seal hosts.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

Canine distemper virus (CDV), Leptospira interrogans, and Toxoplasma gondii are potentially lethal pathogens associated with decline in marine mammal populations. The Caspian Sea is home for the endangered Caspian seal (Pusa caspica). In the late 1990s and early 2000s, CDV caused a series of mortality events involving at least several thousand Caspian seals. To assess current infection status in Caspian seals, we surveyed for antibodies to three pathogens with potential to cause mortality in marine mammals. During 2015-2017, we tested serum samples from 36, apparently healthy, Caspian seals, accidentally caught in fishing nets in the Caspian Sea off Northern Iran, for antibodies to CDV, L. interrogans, and T. gondii, by virus neutralization, microscopic agglutination, and modified agglutination, respectively. Twelve (33%), 6 (17%), and 30 (83%) samples were positive for CDV, L. interrogans and T. gondii antibodies, respectively. The highest titers of CDV, L. interrogans, and T. gondii antibodies were 16, 400, and 50, respectively. Frequencies of antibody to these pathogens were higher in seals >1 year old compared to seals <1 year old. Two serovars of L. interrogans (Pomona and Canicola) were detected. Our results suggest a need for additional studies to clarify the impact of these pathogens on Caspian seal population decline and the improvement of management programs, including systematic screening to detect and protect the remaining population from disease outbreaks.

Pathology has not been observed in true seals infected with Brucella pinnipedialis. A lack of intracellular survival and multiplication of B. pinnipedialis in hooded seal (Cystophora cristata) macrophages in vitro indicates a lack of chronic infection in hooded seals. Both epidemiology and bacteriological patterns in the hooded seal point to a transient infection of environmental origin, possibly through the food chain. To analyse the potential role of fish in the transmission of B. pinnipedialis, Atlantic cod (Gadus morhua) were injected intraperitoneally with 7.5 x 107 bacteria of a hooded seal field isolate. Samples of blood, liver, spleen, muscle, heart, head kidney, female gonads and feces were collected on days 1, 7, 14 and 28 post infection to assess the bacterial load, and to determine the expression of immune genes and the specific antibody response. Challenged fish showed an extended period of bacteremia through day 14 and viable bacteria were observed in all organs sampled, except muscle, until day 28. Neither gross lesions nor mortality were recorded. Anti-Brucella antibodies were detected from day 14 onwards and the expression of hepcidin, cathelicidin, interleukin (IL)-1β, IL-10, and interferon (IFN)-γ genes were significantly increased in spleen at day 1 and 28. Primary mononuclear cells isolated from head kidneys of Atlantic cod were exposed to B. pinnipedialis reference (NCTC 12890) and hooded seal (17a-1) strain. Both bacterial strains invaded mononuclear cells and survived intracellularly without any major reduction in bacterial counts for at least 48 hours. Our study shows that the B. pinnipedialis strain isolated from hooded seal survives in Atlantic cod, and suggests that Atlantic cod could play a role in the transmission of B. pinnipedialis to hooded seals in the wild.

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) 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 Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis 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 B. pinnipedialis is internalized, but is then gradually eliminated during the next 72-96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis 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 B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary.

A high prevalence of Brucellapinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal (Cystophoracristata); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B. pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B. pinnipedialis reference strain (NCTC 12890) from harbor seal (Phocavitulina), B. ceti reference strain (NCTC 12891) from harbor porpoise (Phocoenaphocoena) and a B. ceti Atlantic white-sided dolphin (Lagenorhynchusacutus) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B. pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal.

Dominant colonic bacteria in wild hooded (n = 9), harbour (n = 1) and grey (n = 1) seals were identified using 16S rRNA gene clone libraries (313 clones), revealing 52.7% Bacteroidetes, 41.5% Firmicutes, 4.5% Proteobacteria and 1.0% Fusobacteria. Thirty (77%) of the 39 phylotypes identified were novel, showing <97% sequence similarity to their nearest cultivated relatives. Mean colonic bacterial cell density, determined by real-time PCR, was high (12.8 log₁₀ cells/g wet wt) for the hooded seals, while the number of methanogenic Archea was low (4.0 log₁₀ cells/g wet wt). The level of ampicillin (ampr) and tetracycline-resistant (tetr) isolates was investigated by cultivation. Aerobic ampr isolates were only detected in colon contents from four hooded seals, whereas aerobic tetr isolates were found in seven of the nine hooded seals. These data provide novel insight to the gut microbiota of Arctic and sub-Arctic seals living in the wild.

Representative portions of pelvic, iliac, bronchial and gastric lymph nodes, spleen, lung, kidney, liver, testis, pelvic muscle and skin wound were fixed in 10 per cent neutral buffered formalin and routinely processed for histological examination. Iliac, bronchial and gastric lymph nodes, spleen, lung, kidney, liver and testis showed multifocal to coalescing small to moderate aggregates of epithelioid macrophages, lymphocytes and plasma cells, which in the case of the lung, effaced 60-70 per cent of the alveolar parenchyma (Fig 2). M bovis has been isolated from other domestic, feral and wild species in the UK (Delahay and others 2007, Defra 2011a), but species other than cattle and badgers are generally considered only to be spillover hosts (Defra 2011b). [...]although there is a wide range of species from which the seal pup may have been infected with M bovis, cattle and badgers are the most likely sources. International Journal of Systematic and Evolutionary Microbiology 53, 1305-1314 Defra ( 2011a ) Incidents of M. bovis infection in non-bovine domestic ani-mals & wild deer in GB confirmed by laboratory culture. http://archive.defra.gov.uk/foodfarm/­farmanimal/diseases/atoz/tb/documents/tb-otherspecies.pdf Accessed October 31, 2012 Defra ( 2011b ) Bovine TB Eradication Programme for England. https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/69443/pb13601-bovinetb-eradication-programme-110719.pdf Accessed May 27, 2013 Delahay R. J. Smith G. C. Barlow A. M. Walker N. Harris A. Clifton-Hadley R. S. Cheeseman C. L. ( 2007 ) Bovine tuberculosis in-fection in wild mammals in the South-West region of England: A survey of prevalence and a semi-quantitative assessment of the relative risks to cattle.