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Fatal marine Brucella infections with histologic lesions specific to the central nervous system (CNS), known as neurobrucellosis, have been described in 5 species of odontocete cetaceans in the UK: striped dolphins Stenella coeruleoalba, Atlantic white-sided dolphins Lagenorhynchus acutus, short-beaked common dolphins Delphinus delphis, long-finned pilot whale Globicephala melas and Sowerby’s beaked whale Mesoplodon bidens. To date, these CNS lesions have only been associated with Brucella ceti ST26 and not with B. pinnipedialis, which is rarely isolated from cetaceans and, although commonly found in various seal species, has never been associated with any pathology. This paper describes the first report of neurobrucellosis in a common minke whale Balaenoptera acutorostrata which was associated with the isolation of Brucella pinnipedialis ST24 and co-infection with Balaenoptera acutorostrata gamma-herpesvirus 2. This is the first report of neurobrucellosis in any species of mysticete and the first report of Brucella pinnipedialis in association with any pathology in any species of marine mammal, which may be due to co-infection with a herpesvirus, as these are known to be associated with immunosuppression.

Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammal species. We report the first complete genome sequence of a Brucella strain isolated from a hooded seal (Cystophora cristata), Brucella pinnipedialis strain 23a-1 of sequence type 54, found in the North Atlantic Ocean surrounding Norway.

The following article, Larsen, A.K., Nymo, I.H., Sørensen, K.K., Seppola, M., Rødven, R., Jiménez de Bagüés, M.P., ... Godfroid, J. (2018). Concomitant Temperature Stress and Immune Activation may Increase Mortality Despite Efficient Clearance of an Intracellular Bacterial Infection in Atlantic Cod. Frontiers in Microbiology, 9(2963), can be accessed at https://doi.org/10.3389/fmicb.2018.02963. Licensed CC BY-NC-ND 4.0. The environmental temperature has profound effects on biological systems of marine aquatic organisms and plays a critical role in species distribution and abundance. Particularly during the warmer seasons, variations in habitat temperature may introduce episodes of stressful temperatures which the organisms must adapt to and compensate for to maintain physiological homeostasis. The marine environment is changing and predicted raises in water temperatures will affect numerous marine species. Translocation of pathogens follow migration of species and alternations in physical environmental parameters may have influence upon the virulence of pathogens, as well as the hosts immune responses. While pathogenicity of many true pathogens is expected to increase following climate induced temperature stress, the impact from environmental stressors on the occurrence and severity of opportunistic infections is unknown. Here we describe how thermal stress in the cold-water species Atlantic cod influenced the fish immune responses against an opportunistic intracellular bacterium. Following experimental infection with Brucella pinnipedialis at normal water temperature (6°C) and sub-optimal temperature (15°C), cod cleared the intracellular bacteria more rapidly at the highest temperature. The overall immune response was faster and of higher amplitude at 15°C, however, a significant number of cod died at this temperature despite efficient clearance of infection. An increased growth rate not affected by infection was observed at 15°C, confirming multiple energy demanding processes taking place. Serum chemistry suggested that general homeostasis was influenced by both infection and increased water temperature, highlighting the cumulative stress responses (allostatic load) generated by simultaneous stressors. Our results suggest a trade-off between resistance and tolerance to survive infection at sub-optimal temperatures and raise questions concerning the impact of increased water temperatures on the energetic costs of immune system activation in aquatic ectotherms.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

Source at https://doi.org/10.3389/fvets.2018.00008 . Brucella pinnipedialis was first isolated from true seals in 1994 and from eared seals in 2008. Although few pathological findings have been associated with infection in true seals, reproductive pathology including abortions, and the isolation of the zoonotic strain type 27 have been documented in eared seals. In this study, a Brucella enzyme-linked immunosorbent assay (ELISA) and the Rose Bengal test (RBT) were initially compared for 206 serum samples and a discrepancy between the tests was found. Following removal of lipids from the serum samples, ELISA results were unaltered while the agreement between the tests was improved, indicating that serum lipids affected the initial RBT outcome. For the remaining screening, we used ELISA to investigate the presence of Brucella antibodies in sera of 231 eared and 1,412 true seals from Alaskan waters sampled between 1975 and 2011. In eared seals, Brucella antibodies were found in two Steller sea lions (Eumetopias jubatus) (2%) and none of the 107 Northern fur seals (Callorhinus ursinus). The low seroprevalence in eared seals indicate a low level of exposure or lack of susceptibility to infection. Alternatively, mortality due to the Brucella infection may remove seropositive animals from the population. Brucella antibodies were detected in all true seal species investigated; harbor seals (Phoca vitulina) (25%), spotted seals (Phoca largha) (19%), ribbon seals (Histriophoca fasciata) (16%), and ringed seals (Pusa hispida hispida) (14%). There was a low seroprevalence among pups, a higher seroprevalence among juveniles, and a subsequent decreasing probability of seropositivity with age in harbor seals. Similar patterns were present for the other true seal species; however, solid conclusions could not be made due to sample size. This pattern is in accordance with previous reports on B. pinnipedialis infections in true seals and may suggest environmental exposure to B. pinnipedialis at the juvenile stage, with a following clearance of infection. Furthermore, analyses by region showed minor differences in the probability of being seropositive for harbor seals from different regions regardless of the local seal population trend, signifying that the Brucella infection may not cause significant mortality in these populations. In conclusion, the Brucella infection pattern is very different for eared and true seals.

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals ( Phoca vitulina richardii) and North Atlantic hooded seals ( Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS) 711 gene and sequence type (ST)27 primer–probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS 711 and ST27 primer–probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS 711 primer–probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS 711 primer–probe was used to test Atlantic cod ( Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS 711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

Brucella infection in seals was reported for the first time in 1994 around the coast of Scotland. Since then, marine mammal Brucella infections were found to be widely distributed in the northern hemisphere. Two Brucella species affect marine mammals: Brucella pinnipedialis in pinnipeds and Brucella ceti in cetaceans. We examined the livers of Baltic grey seals (Halichoerus grypus) from the Finnish coast (n=122) hunted, found dead, or killed as by-catch in fishing gear in 2013–15 as part of population health monitoring. We detected B. pinnipedialis in the livers of three grey seals. The bacterium was isolated from livers displaying parasitic cholangitis. We also detected Brucella DNA in liver flukes (Pseudamphistomum truncatum) obtained from a Brucella-infected grey seal, suggesting that flukes might be possible vectors of this pathogen in the marine environment.

There are three major lineages of marine mammal strains of Brucella spp.: Brucella ceti ST23, found predominantly in porpoises; B. ceti ST26, in pelagic delphinids and ziphiids; and Brucella pinnipedialis ST24/25, predominantly in seals. The isolation of Brucella spp. in mysticetes has been described only in common minke whales (Balaenoptera acutorostrata) in Norway and Scotland. We report a third case of Brucella infection and isolation in a minke whale associated with a large abscess. In contrast to the two previous reports that involved isolates of B. pinnipedialis ST24 or the porpoise-associated B. ceti complex ST23, this case was associated with the dolphin-associated B. ceti ST26. Thus, minke whales can be infected naturally with members of all the distinct major lineages of Brucella associated with marine mammals. This report is unique in that the B. ceti ST26 did not originate from a pelagic delphinid or a beaked whale.

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.

In 1994 a novel Brucella sp., later named B. pinnipedialis, was identified in stranded harbor seals (Phoca vitulina). This Brucella sp. is a potential zoonotic pathogen and is capable of causing disease in domestic animals. Serologic, microbiologic, and pathologic data collected from live captured and stranded harbor seals were used to better describe the epizootiology of B. pinnipedialis in harbor seals from Washington State, USA, in 1994 through 2006. We found no sex predilection in harbor seal exposure or infection with B. pinnipedialis but noted a significant difference in prevalence among age classes, with weaned pups, yearlings, and subadults having highest exposure and infection. The most common postmortem finding in 26 Brucella-positive animals (culture and/or PCR) was verminous pneumonia due to Parafilaroides spp. or Otostrongulus circumlitus. Our data are consistent with exposure to B. pinnipedialis post-weaning, and it is likely that fish or invertebrates and possibly lungworms are involved in the transmission to harbor seals. Brucella pinnipedialis was cultured or detected by PCR from seal salivary gland, lung, urinary bladder, and feces, suggesting that wildlife professionals working with live, infected seals could be exposed to the bacterium via exposure to oral secretions, urine, or feces. Endangered sympatric wildlife species could be exposed to B. pinnipedialis via predation on infected seals or through a common marine fish or invertebrate prey item involved in its transmission. More work is required to elucidate further potential fish or invertebrates that could be involved in the transmission of B. pinnipedialis to harbor seals and better understand the potential risk they could pose to humans or sympatric endangered species who also consume these prey items.

Bruceloza je prepoznata i dokazana u kitova, dupina, tuljana, morskih lavova, morževa i vidra. Klinički se po svemu sudeći očituje placentitisom i pobačajem, neonatalnom smrtnošću, meningoencefalitisom, apscesima po koži. Poznato je da su brucele izdvojene iz morskih sisavaca bez ikakvih simptoma i tkiva bez patoloških promjena. Potencijalni domaćini Brucella u vodenim sustavima mogu uključiti čak oko 130 vrsta morskih sisavaca koji žive i hrane se u morima, rijekama i jezerima. U početku su svi izolati izdvojeni iz morskih sisavaca nazvani B. maris. Kasnije su studije pokazale da se radi o dvije vrste brucela. Izolati iz kitova nazvani su Brucella ceti, a prirodni domaćini su im kitovi (Cetaceans). Izolati iz perajara nazvani su B. pinnipedialis, a najčešći domaćini su tuljani (Phocidae). Nakon gotovo dva desetljeća istraživanja i pronalaska bruceloze u morskih sisavaca sada je dobro poznato da je bruceloza u morskih sisavaca globalno rasprostranjena. Dokazano je da ljudi iz 114 različitih zemalja često imaju dodir s morskim sisavcima. Profesionalna izloženost ljudi odgovorna je i za prvi opisani slučaj bruceloze prouzročen brucelama podrijetlom iz mora. Ljudi koji love morske sisavce, kitolovci i lovci na tuljane su rizična skupina, a osobito kada obrađuju trupla ili konzumiraju sirovo meso. U druge ugrožene skupine ubrajaju se zoolozi, veterinari, laboratorijski radnici, ribari, ljudi koji rade s morskim sisavcima u različitim centrima, kao i svi oni koji pristupaju nasukanim životinjama ili lešini na plaži.

License:Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/) Background: Marine Brucella spp. have been isolated from numerous pinniped and cetacean species, but pathological findings in association with infection with Brucella pinnipedialis in pinnipeds have been sparse. The capacity of brucellae to survive and replicate within host macrophages underlies their important ability to produce chronic infections, but previous work has shown that B. pinnipedialis spp. are rapidly eliminated from hooded seal (Cystophora cristata) alveolar macrophages. Results: To investigate if multiplication could take place in other hooded seal cell types, primary epithelial cells were isolated, verified to express the epithelial marker cytokeratin and challenged with three different strains of B. pinnipedialis; B. pinnipedialis sp. nov., B. pinnipedialis hooded seal strain B17, and B. pinnipedialis hooded seal strain 22F1. All strains were steadily eliminated and the amounts of intracellular bacteria were reduced to less than one-third by 48 h post infection. Intracellular presence was verified using immunocytochemistry. Conclusions: So far, intracellular multiplication in seal cells has not been documented for B. pinnipedialis. The lack of intracellular survival in macrophages, as well as in epithelial cells, together with the fact that pathological changes due to B. pinnipedialis infection is not yet identified in seals, suggests that the bacteria may only cause a mild, acute and transient infection. These findings also contribute to substantiate the hypothesis that seals may not be the primary host of B. pinnipedialis and that the transmission to seals are caused by other species in the marine environment.

Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea. This work was supported by the grants RTA 2013-00065-C02-01 from The National Institute for Agricultural and Food Research and Technology (INIA) and agrifood research in Spain (MPJdB) and SAF2014-54763-C2 from the European Social Fund (JP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Published

Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammals and were recently recognized as two different species, i.e. Brucella pinnipedialis for pinniped isolates and Brucella ceti for cetacean isolates. The aim of this study was to identify specific DNA fragments of marine mammal Brucella strains using a previously described infrequent restriction site-PCR (IRS-PCR) method but with three new couples of restriction enzymes applied on a larger panel of marine mammal Brucella isolates ( n = 74) and one human isolate from New Zealand likely from marine mammal origin. This study revealed five DNA fragments specific of Brucella strains isolated from marine mammals. Among them two new DNA fragments were specific of B. pinnipedialis but were not detected in hooded seal isolates. DNA fragment I identified in the previous IRS-PCR study and fragment VI of this study were located on a cloned and sequenced 6 kb SacI fragment. Its nucleotide sequence revealed that it is likely part of a putative genomic island. Sequence analysis showed that it carries four ORFs coding for putative metabolic functions. Although hooded seal isolates are classified within B. pinnipedialis it was shown in this study that they do not carry this genomic island and this raises the question about their evolutionary history within B. pinnipedialis .

A Brucelose é uma zoonose de origem bacteriana frequentemente reportada tanto em animais terrestres como marinhos. Foram reportados casos de Brucelose marinha em países do Sul da Europa e América do Sul. É uma bactéria causadora de arrojamentos de animais marinhos, por originar problemas de natação e flutuabilidade. Como potenciais foram identificadas 130 espécies de animais marinhos, entre os quais 86 espécies de cetáceos e 36 espécies de pinípedes. Brucella ceti e Brucella pinnipedialis são as duas espécies mais comuns e importantes de brucelose em animais marinhos. Este agente patogénico dissemina-se principalmente para os órgãos e tecidos, como o baço, gânglios linfáticos, útero, testículos, glândula mamária e tecido nervoso através de inclusões intrafagocitárias. Nos dias de hoje, existe um grande contacto entre o Homem e animais marinhos, como tal, a redução ou eliminação desta bactéria é um passo essencial para minimizar e prevenir o problema, evitando assim a perda de espécies tão importantes e prevenindo uma questão de saúde pública. Este trabalho de revisão tem como objetivo rever o conhecimento atual sobre brucelose em animais marinhos, as respetivas espécies infetantes, as suas manifestações clinicas, epidemiologia, diagnóstico e a terapêutica utilizada. Adicionalmente pretende-se abordar o que poderá ser feito para aumentar a eficácia de diagnóstico e tratamento desta patologia nestes animais, melhorando as suas condições de vida em cativeiro e evitando a sua morte em liberdade. Brucellosis, is a zoonosis of bacterial origin, frequently reported both in terrestrial and marine animals. It has the ability to infect humans. Marine brucellosis has already been reported in countries from South Europe and South America. This pathogenic agent is the cause of live and dead animal strandings due to swimming disorders and buoyancy problems. It is already recognized 130 species of marine mammal hosts of Brucella spp., of which, 86 species are cetaceans and 36 species are Pinnipeds. Brucella ceti and Brucella pinnipedialis are the two most important species in marine mammals brucellosis. This pathogenic agent spreads primarily through organs and tissue like spleen, lymph nodes, uterus, testicles, mammary gland and nervous system due to intra-phagocytic inclusions. In fact, nowadays, there is a great relationship and contact between humans and marine mammals, therefore, it is very important to avoid the spreading of the disease, and control and eliminate this agent, which is crucial to minimize losses, such as, dead animals and infected Humans, avoiding a public health problem. The aim of this study was to perform a review over brucellosis in marine mammals, its infection and clinical signs, its manifestation in different species of marine mammals, define its transmission from host to host, available treatment and what can be done in the near future to create new ways to diagnose and treat this disease in this animals, contributing to better health and living conditions in captivity and avoiding death in the wild.

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti , the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella , 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum , were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti , Brucella pinnipedialis , and newly described species ( B. microti , Brucella inopinata BO1, B. inopinata -like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

Fifty years ago, bacteria in the genus Brucella were known to cause infertility and reproductive losses. At that time, the genus was considered to contain only 3 species: Brucella abortus, Brucella melitensis, and Brucella suis. Since the early 1960s, at least 7 new species have been identified as belonging to the Brucella genus ( Brucella canis, Brucella ceti, Brucella inopinata, Brucella microti, Brucella neotomae, Brucella ovis, and Brucella pinnipedialis) with several additional new species under consideration for inclusion. Although molecular studies have found such high homology that some authors have proposed that all Brucella are actually 1 species, the epidemiologic and diagnostic benefits for separating the genus based on phenotypic characteristics are more compelling. Although pathogenic Brucella spp have preferred reservoir hosts, their ability to infect numerous mammalian hosts has been increasingly documented. The maintenance of infection in new reservoir hosts, such as wildlife, has become an issue for both public health and animal health regulatory personnel. Since the 1960s, new information on how Brucella enters host cells and modifies their intracellular environment has been gained. Although the pathogenesis and histologic lesions of B. abortus, B. melitensis, and B. suis in their preferred hosts have not changed, additional knowledge on the pathology of these brucellae in new hosts, or of new species of Brucella in their preferred hosts, has been obtained. To this day, brucellosis remains a significant human zoonosis that is emerging or reemerging in many parts of the world.

Brucellosis is a worldwide infectious zoonotic disease caused by Gram-negative bacteria of the genus Brucella, and Brucella infections in marine mammals were first reported in 1994. A serosurvey investigating the presence of anti-Brucella antibodies in 3 Antarctic pinniped species was undertaken with a protein A/G indirect enzyme-linked immunosorbent assay (iELISA) and the Rose Bengal test (RBT). Serum samples from 33 Weddell seals Leptonychotes weddelli were analysed, and antibodies were detected in 8 individuals (24.2%) with the iELISA and in 21 (65.6%) with the RBT. We tested 48 southern elephant seal Mirounga leonina sera and detected antibodies in 2 animals (4.7%) with both the iELISA and the RBT. None of the 21 Antarctic fur seals Arctocephalus gazella was found positive. This is the first report of anti-Brucella antibodies in southern elephant seals. The potential impact of Brucella infection in pinnipeds in Antarctica is not known, but Brucella spp. are known to cause abortion in terrestrial species and cetaceans. Our findings suggest that Brucella infection in pinnipeds is present in the Antarctic, but to date B. pinnipedialis has not been isolated from any Antarctic pinniped species, leaving the confirmation of infection pending. Publisher PDF

Brucella organisms which differed from the recognised species of the genus, were isolated from nine seals, eight cetaceans and one otter. A method is described for the isolation of Brucella species from sea mammals and the first isolations of Brucella species are recorded from an Atlantic white-sided dolphin ( Lagenorhynchus acutus ), two striped dolphins ( Stenella coeuleoalba ), a hooded seal ( Cystophora cristata ), a grey seal ( Halichoerus grypus ) and a European otter ( Lutra lutra ). There were differences in the culture media required for the primary isolation of the organisms and in their dependency on carbon dioxide. Subcutaneous lesions, when present, always yielded a confluent growth. The organisms were isolated from seven of 14 spleen samples and also from the mammary glands, uterus, testes and blood and the mandibular, gastric, iliac, sub-lumbar and colorectal lymph nodes.

PLoS one 11(7), e0159272 (2016). doi:10.1371/journal.pone.0159272, Published by PLoS, Lawrence, Kan.

Background Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus. Results We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a) genome segments unshared between B. microti and B. pinnipedialis, b) gene deletion/fusion events and c) positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups. Conclusions In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups.

Infection with Brucella pinnipedialis and Brucella ceti in marine mammals of the northern hemisphere has been extensively reported. In Chile, the National Antarctic Institute (INACH) and the University of Chile have developed a collaborative study establishing the presence of anti- Brucella

To the Editor: Brucellosis is a zoonotic disease that remains endemic to many parts of the world. There are 6 classic Brucella species described with different preferred hosts. Human disease is most commonly associated with consumption of unpasteurized dairy products or with occupational exposure for veterinarians, agricultural workers, laboratory workers, meat industry workers, and hunters. In recent years, it has become clear that novel members of the genus, yet to be formally named, are associated with a variety of marine mammal species, particularly dolphins, porpoises, and seals (1). To date there are 3 reports in the literature of naturally acquired infection of humans with Brucella species originating from marine mammals (2,3) One other case, representing infection of a laboratory worker, has also been reported (4). Two of the naturally acquired cases were reported in Peru (2). One person had consumed raw shellfish and swam in the Pacific Ocean but did not report any direct contact with marine mammals; the second person reported infrequent visits to the coast and no contact with marine mammals but had consumed raw shellfish. An additional naturally acquired case was recently reported from New Zealand, where extensive molecular testing characterized the strain involved as a marine mammal type (3). This patient again reported no exposure to marine mammals but did report that he fished regularly, had contact with uncooked fish bait, and consumed raw snapper. The cases in Peru were notable for severe, atypical symptoms; both patients had symptoms of neurobrucellosis. The New Zealand case was associated with spinal osteomyelitis (3). In contrast, the laboratory-acquired infection was mild and uncomplicated (4). We have characterized these isolates by a variety of molecular approaches in conjunction with ongoing studies, which examine genetic diversity within Brucella species isolated from marine mammals. Multilocus sequence analysis (5) showed that all 3 isolates from naturally acquired human infection with Brucella species from marine mammals shared an identical genotype (ST27). In previous characterization of 56 Brucella isolates from marine mammals, ST27 was found only once. Strain F5/99, originally isolated from an aborted bottlenose dolphin fetus off the western coast of the United States (6), shares this genotype. Use of an alternative typing approach, based on restriction fragment length polymorphism analysis of outer membrane protein–encoding genes (7), gave identical findings. Again, all 3 isolates derived from naturally acquired human infection represent an identical genotype. This genotype is shared only by strain F5/99 among a collection of 120 Brucella isolates from marine mammals characterized by this method. Finally, isolates were characterized by a variable number of tandem repeats–based typing approach (8). When comparing profiles at 6 loci with a relatively slow evolutionary speed, previously shown to be useful for dividing Brucella isolates into species groups (8), we determined that F5/99 and the 3 naturally acquired human isolates share a unique profile not seen in any of >1,400 isolates of marine or terrestrial Brucella species examined to date. In contrast, the strain associated with laboratory-acquired infection (4) was not a member of ST27 but belonged to ST23, a genotype that is predominantly associated with porpoises (9). It is clear from these findings that the 3 cases of naturally acquired infection with Brucella species originating from marine mammals reported to date were caused by closely related organisms. The particular genotype concerned, ST27, is rare in our collection, having been noted only once in marine mammals. However, this may reflect the fact that most isolates examined to date originated from northern Europe; only 5 isolates in our collection originated from Pacific waters. It is possible that isolates of this genotype are predominantly or exclusively associated with regions other than those extensively sampled to date. Indeed, examination of the literature provides further evidence for the presence of this genotype in marine mammals in the Pacific. BLAST (www.ncbi.nlm.nih.gov/blast) comparison of outer membrane protein sequences from a minke whale isolate originating in the North Pacific (10) showed a close match with the equivalent sequence from F5/99. Although numbers are currently small, the isolation of an identical genotype from all 3 cases of naturally acquired human infection derived from marine mammal Brucella species raises the possibility of increased zoonotic potential associated with this genotype. Furthermore, where diagnosis is based on serologic testing alone, it is possible that human infection with marine mammal Brucella species may go unnoticed. Members of ST27 may be more pathogenic to man, per se. Alternatively, they may be associated with natural hosts or circulate through intermediaries that make contact with humans more likely. Notably, none of the 3 patients reported direct contact with marine mammals, though all had consumed raw seafood. These findings clearly suggest that more extensive studies of the presence and distribution of marine mammal Brucella genotypes, particularly ST27, in waters other than those of northern Europe would be valuable for clarifying the natural habitat of ST27. Furthermore, relevant authorities should be aware of the potential for zoonotic disease caused by this Brucella genotype particularly, but not exclusively, where occupation or lifestyle may make exposure more likely. Brucellosis research at the Veterinary Laboratories Agency is supported by the UK Department of Environment, Food and Rural Affairs (Defra).

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2180/8/17, [Background] The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose., [Results] In this study we obtained evidence of transposition of IS711 from the B. ovis and B. pinnipedialis chromosomes by using the "transposon trap" plasmid pGBG1. This plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain) and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR). TcR mutants due to IS711 transposition were only detected in B. ovis and B. pinnipedialis strains., [Conclusion] Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1. This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis., This work has been supported by grants from the "Instituto de Salud Carlos III" of the Spanish Ministry of Health to the "Network for Brucellosis research/G03-204" and PI05/2499.

Investigations for Brucella-infections were conducted in 29 hooded seals (Cystophora cristata) caught between Svalbard and Greenland (North Atlantic Ocean; Greenland Sea) autumn 2002, and from 20 ringed seals (Phoca hispida) caught in Billefjord, Svalbard, spring 2003. All animals were apparently healthy and were caught in their natural habitat. Bacteriology on tissue samples from ringed seals was negative, whereas Brucella sp. were recovered in tissues from 11 of the 29 hooded seals (38%), with the highest tissue prevalence in spleen (9/29) and lung lymph nodes (9/24). Anti-Brucella antibodies were detected in sera from 9 hooded seals (31%) (EDTA-modified Slow Agglutination test of Wright, Rose Bengal test, Complement Fixation Test, and Protein-A ELISA). The bacterial isolates all belonged to the genus Brucella according to classical biotyping and PCR analysis based on Insertion Sequence IS711, and were shown to be typical marine mammal strains, based on the occurrence of an IS711 element downstream of the bp26 gene. Their dependency on CO2 for growth, and the presence of one copy each of the omp2a and omp2b gene finally classified them as Brucella pinnipediae. Furthermore, all the hooded seal isolates showed an A+ M+ agglutination profile, which is different from the profile of reference seal strain 2/94 (harbour seal, Phoca vitulina). Thus, these results indicate that B. pinnipediae may contain different biovars. The present results suggest that infection with B. pinnipediae is enzootic in this population. Since the hooded seal is commercially hunted and consumed in Norway, the pathological impact of such infections and their zoonotic potential should be further addressed.

Brucellae recovered from sea mammals were first reported in 1994. In the years since both culture and serological analysis have demonstrated that the infection occurs in a wide range of species of marine mammals inhabiting a vast amount of the world's oceans. Molecular studies have demonstrated that the isolates differ from those found amongst terrestrial animals and also distinguish between strains which have seals and cetaceans as their preferred hosts. At the phenotypic level seal and cetacean strains can also be differed with respect to their CO(2) requirement, primary growth on Farrells medium and metabolic activity on galactose. Two new species B. cetaceae and B. pinnipediae have been proposed as a result. This paper provides a review of Brucella in sea mammals and updates findings from the study of sea mammals from around the coast of Scotland.

A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale. These strains were identified as brucellae by conventional typing tests. However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called 'Brucella maris'. In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species. This observation was also recently made for a minke whale Brucella isolate. The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals. By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates. Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals. Therefore, grouping the marine mammal Brucella isolates into one species 'Brucella maris' seems inappropriate unless the currently recognized Brucella species are grouped. With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B. pinnipediae and B. cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus. © 2001 Editions scientifiques et medicales Elsevier SAS

DNA polymorphism of the bp26 gene, coding for a diagnostic protein antigen for brucellosis, was assessed by PCR and restriction fragment length polymorphism analysis using primers to amplify the bp26 gene with its flanking regions. Surprisingly, whereas PCR performed on DNA of the reference strains of the six recognized Brucella species produced a product of the expected size (1,029 bp), PCR performed on DNA of three representative strains from marine mammals (from a seal, a dolphin, and a porpoise) produced a larger product, of about 1,900 bp. Nucleotide sequencing of the 1,900-bp PCR products revealed the presence of an insertion sequence, IS 711 , downstream of the bp26 gene and adjacent to a Bru-RS1 element previously described as being a hot spot for IS 711 insertion. PCR performed on a large number of field strains from different geographic origins and from marine mammal isolates indicated that the occurrence of an IS 711 element downstream of the bp26 gene was a feature specific to the marine mammal Brucella strains. Thus, this PCR assay is able to differentiate Brucella terrestrial isolates from marine mammal isolates and could be applied for diagnostic purposes.

Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus , B. melitensis , B. canis , and B. suis . IS 711 -based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new Brucella species may be appropriate.

Small Gram negative coccobacilli isolated from seals, porpoises, dolphins and from an otter road casualty were identified as Brucellae by their colonial and cell morphology, staining characteristics, biochemical activity, agglutination by monospecific antisera and susceptibility to lysis by Brucella specific bacteriophage. Their characterisation, including metabolic profiles, is described. These strains could not be assigned to recognised nomen species of the genus Brucella and it is suggested that they comprise a new nomen species to be called B. maris (sp. nov., type strain 2/94). It is further suggested the nomen species be subdivided into three biovars corresponding to their CO2 requirement, metabolic activity on galactose, dominant antigen and animal host.

Summary: Isolation of Brucella spp. in marine mammals has been reported during the past several years. A Brucella strain from the spleen and liver of a minke whale (Balaenoptera acutorostrata) was isolated. Conventional typing methods indicated that this isolate was related to the genus Brucella but did not match the profiles of any known Brucella species or biovar. Successful PCR amplification of the Brucella rrs-rrl spacer sequence and of the insertion sequence IS6501 also indicated that the minke whale strain was related to the genus Brucella. In addition, the rrs gene of this strain shared a very high degree of nucleotide identity (>98%) with published Brucella spp. rrs sequences. However, RFLP studies using an IS6501-specific probe showed a unique profile for this strain in comparison with the profiles of the six known Brucella species. Moreover, analysis of the omp2 locus by PCR-RFLP, by Southern hybridization using omp2a- and omp2b-specific probes, and by DNA sequencing showed that the minke whale isolate possesses two copies of the omp2b gene instead of one omp2a and one omp2b gene copy or two copies of the omp2a gene described in the six known Brucella species. Thus, molecular typing methods showed that this isolate is clearly distinct from all other known Brucella species and strains. The specific molecular features of this minke whale Brucella isolate raise questions about the lineage between the Brucella strains isolated from marine mammals and the Brucella species isolated from terrestrial mammals.

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 culture isolated from an aborted fetus of a bottlenose dolphin ( Tursiops truncatus) was characterized. The isolate was a gram-negative coccobacillus, and the colonial morphology was typical of a smooth Brucella. The isolate was positive for catalase, oxidase, nitrate reduction, and urease. Hydrogen sulfide was not produced. It grew in air at 37 C but required 72 hours for good growth. There was growth on media containing basic fuchsin, thionin, thionin blue, penicillin, and erythritol. The M antigen was dominant, and the isolate was lysed by 4 of 10 brucellaphages tested. The oxidative metabolic profile of the isolate was similar to that for B. abortus but differed in utilization of L-asparagine, L-glutamic acid, and DL-citrulline. Whole-cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles were markedly different from the protein profiles of reference strains of Brucella species. Biochemical and oxidative metabolism profiles indicated that the isolate belongs in the genus Brucella but did not match the profiles of any established species or biovars. This isolate may be an atypical strain of a recognized Brucella species or a new biovar or species of Brucella.

Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population.

BMC microbiology 9(145), (2009). doi:10.1186/1471-2180-9-145, Published by BioMed Central, London