Meta-analysis of the Cetacea gut microbiome: Diversity, co-evolution, and interaction with the anthropogenic pathobiome.

Despite their critical roles in marine ecosystems, only few studies have addressed the gut microbiome (GM) of cetaceans in a comprehensive way. Being long-living apex predators with a carnivorous diet but evolved from herbivorous ancestors, cetaceans are an ideal model for studying GM-host evolutionary drivers of symbiosis and represent a valuable proxy of overall marine ecosystem health. Here, we investigated the GM of eight different cetacean species, including both Odontocetes (toothed whales) and Mysticetes (baleen whales), by means of 16S rRNA-targeted amplicon sequencing. We collected faecal samples from free-ranging cetaceans circulating within the Pelagos Sanctuary (North-western Mediterranean Sea) and we also included publicly available cetacean gut microbiome sequences. Overall, we show a clear GM trajectory related to host phylogeny and taxonomy (i.e., phylosymbiosis), with remarkable GM variations which may reflect adaptations to different diets between baleen and toothed whales. While most samples were found to be infected by protozoan parasites of potential anthropic origin, we report that this phenomenon did not lead to severe GM dysbiosis. This study underlines the importance of both host phylogeny and diet in shaping the GM of cetaceans, highlighting the role of neutral processes as well as environmental factors in the establishment of this GM-host symbiosis. Furthermore, the presence of potentially human-derived protozoan parasites in faeces of free-ranging cetaceans emphasizes the importance of these animals as bioindicators of anthropic impact on marine ecosystems.

Brucella ceti and Brucella pinnipedialis genome characterization unveils genetic features that highlight their zoonotic potential.

The Gram-negative bacteria Brucella ceti and Brucella pinnipedialis circulate in marine environments primarily infecting marine mammals, where they cause an often-fatal disease named brucellosis. The increase of brucellosis among several species of cetaceans and pinnipeds, together with the report of sporadic human infections, raises concerns about the zoonotic potential of these pathogens on a large scale and may pose a threat to coastal communities worldwide. Therefore, the characterization of the B. ceti and B. pinnipedialis genetic features is a priority to better understand the pathological factors that may impact global health. Moreover, an in-depth functional analysis of the B. ceti and B. pinnipedialis genome in the context of virulence and pathogenesis was not undertaken so far. Within this picture, here we present the comparative whole-genome characterization of all B. ceti and B. pinnipedialis genomes available in public resources, uncovering a collection of genetic tools possessed by these aquatic bacterial species compared to their zoonotic terrestrial relatives. We show that B. ceti and B. pinnipedialis genomes display a wide host-range infection capability and a polyphyletic phylogeny within the genus, showing a genomic structure that fits the canonical definition of closeness. Functional genome annotation led to identifying genes related to several pathways involved in mechanisms of infection, others conferring pan-susceptibility to antimicrobials and a set of virulence genes that highlight the similarity of B. ceti and B. pinnipedialis genotypes to those of Brucella spp. displaying human-infecting phenotypes.

Bacteriological and histopathological findings in cetaceans that stranded in the Philippines from 2017 to 2018.

The relatively high frequency of marine mammal stranding events in the Philippines provide many research opportunities. A select set of stranders (n = 21) from 2017 to 2018 were sampled for bacteriology and histopathology. Pertinent tissues and bacteria were collected from individuals representing eight cetacean species (i.e. Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to histopathological examination and antibiotic resistance screening, respectively. The antibiotic resistance profiles of 24 bacteria (belonging to genera Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n = 24) and susceptible to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n = 24). The resistance profiles of these bacteria show the extent of antimicrobial resistance in the marine environment, and may inform medical management decisions during rehabilitation of stranded cetaceans. Due to inadequate gross descriptions and limited data gathered by the responders during the stranding events, the significance of histopathological lesions in association with disease diagnosis in each cetacean stranding or mortality remained inconclusive; however, these histopathological findings may be indicative or contributory to the resulting debility and stress during their strandings. The findings of the study demonstrate the challenges faced by cetacean species in the wild, such as but not limited to, biological pollution through land-sea movement of effluents, fisheries interactions, and anthropogenic activities.

Modes of genetic adaptations underlying functional innovations in the rumen.

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We analyzed 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen functional innovations. We identified genes with relatively high expression in the rumen, of which many appeared to be recruited from other tissues. These genes show functional enrichment in ketone body metabolism, regulation of microbial community, and epithelium absorption, which are the most prominent biological processes involved in rumen innovations. Several modes of genetic change underlying rumen functional innovations were uncovered, including coding mutations, genes newly evolved, and changes of regulatory elements. We validated that the key ketogenesis rate-limiting gene (HMGCS2) with five ruminant-specific mutations was under positive selection and exhibits higher synthesis activity than those of other mammals. Two newly evolved genes (LYZ1 and DEFB1) are resistant to Gram-positive bacteria and thereby may regulate microbial community equilibrium. Furthermore, we confirmed that the changes of regulatory elements accounted for the majority of rumen gene recruitment. These results greatly improve our understanding of rumen evolution and organ evo-devo in general.

The use of Unmanned Aerial Vehicles (UAVs) to sample the blow microbiome of small cetaceans.

Recent studies describe the use of UAVs in collecting blow samples from large whales to analyze the microbial and viral community in exhaled air. Unfortunately, attempts to collect blow from small cetaceans have not been successful due to their swimming and diving behavior. In order to overcome these limitations, in this study we investigated the application of a specific sampling tool attached to a UAV to analyze the blow from small cetaceans and their respiratory microbiome. Preliminary trials to set up the sampling tool were conducted on a group of 6 bottlenose dolphins (Tursiops truncatus) under human care, housed at Acquario di Genova, with approximately 1 meter distance between the blowing animal and the tool to obtain suitable samples. The same sampling kit, suspended via a 2 meter rope assembled on a waterproof UAV, flying 3 meters above the animals, was used to sample the blows of 5 wild bottlenose dolphins in the Gulf of Ambracia (Greece) and a sperm whale (Physeter macrocephalus) in the southern Tyrrhenian Sea (Italy), to investigate whether this experimental assembly also works for large whale sampling. In order to distinguish between blow-associated microbes and seawater microbes, we pooled 5 seawater samples from the same area where blow samples' collection were carried out. The the respiratory microbiota was assessed by using the V3-V4 region of the 16S rRNA gene via Illumina Amplicon Sequencing. The pooled water samples contained more bacterial taxa than the blow samples of both wild animals and the sequenced dolphin maintained under human care. The composition of the bacterial community differed between the water samples and between the blow samples of wild cetaceans and that under human care, but these differences may have been mediated by different microbial communities between seawater and aquarium water. The sperm whale's respiratory microbiome was more similar to the results obtained from wild bottlenose dolphins. Although the number of samples used in this study was limited and sampling and analyses were impaired by several limitations, the results are rather encouraging, as shown by the evident microbial differences between seawater and blow samples, confirmed also by the meta-analysis carried out comparing our results with those obtained in previous studies. Collecting exhaled air from small cetaceans using drones is a challenging process, both logistically and technically. The success in obtaining samples from small cetacean blow in this study in comparison to previous studies is likely due to the distance the sampling kit is suspended from the drone, which reduced the likelihood that the turbulence of the drone propeller interfered with successfully sampling blow, suggested as a factor leading to poor success in previous studies.

Does sociality drive diversity and composition of airway microbiota in cetaceans?

The number of social contacts of mammals is positively correlated with the diversity of their gut microbes. There is some evidence that sociality also affects microbes in the respiratory tract. We tested whether the airway microbiota of cetacean species differ depending on the whales' level of sociality. We sampled the blow of blue (Balaenoptera musculus), grey (Eschrichtius robustus), humpback (Megaptera novaeangliae) and long-finned pilot whales (PWs) (Globicephala melas) and analysed the blow microbiota by barcode tag sequencing targeting the V4 region of the bacterial 16S rRNA gene. Humpback whales (HWs) show higher levels of sociality than blue (BW) and grey (GW), while PWs are the most gregarious among the four species. The blow samples of the HWs showed the highest richness and diversity. HWs were also the only species with a species-specific clustering of their microbial community composition and a relatively large number of core taxa. Therefore, we conclude that it cannot be sociality alone shaping the diversity and composition of airway microbiota. We suggest the whale species' lung volume and size of the plume of exhaled air as an additional factor impacting the transmission potential of blow microbiota from one individual whale to another.

Serologic survey of Brucella infection in cetaceans inhabiting along the coast of Japan.

A serologic investigation of Brucella infection was performed in 7 species of cetaceans inhabiting along the coast of Japan. A total of 32 serum samples were examined by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis antigens. One serum sample from five melon-headed whales (Peponocephala electra) was positive for B. abortus. No serum sample showed positive for B. canis. The ELISA-positive melon-headed whale serum demonstrated a strong band appearance only against B. abortus antigens in Western blot analysis. Many detected bands were discrete, while some of them had a smeared appearance. The present results indicate that Brucella infection occurred in melon-headed whale population and the bacterial antigenicity is more similar to that of B. abortus than B. canis.

Leptospira spp. and Toxoplasma gondii in stranded representatives of wild cetaceans in the Philippines.

BACKGROUND: The stranding events of cetaceans in the Philippines provide opportunities for gathering biological information and specimens, especially from the pelagic forms. As part of an effort to monitor the health of wild cetaceans, this study detected Leptospira spp. and Toxoplasma gondii, causative agents of the emerging zoonotic diseases leptospirosis and toxoplasmosis respectively, in their stranded representatives. From October 2016-August 2018, 40 cetaceans (representing 14 species) that stranded nationwide were sampled for brain, cardiac muscle, skeletal muscle, kidney, and blood tissues, urine, and sera. These were subjected to molecular, serological, culture, and histopathological analyses to detect the target pathogens. RESULTS: T. gondii was detected in 20 (71%) of the 28 cetaceans with biological samples subjected to either molecular detection through RE gene amplification or IgG antibodies detection through agglutination-based serological assay. On the other hand, Leptospira was detected in 18 (64%) of 28 cetaceans with biological samples subjected to bacterial culture, molecular detection through 16S rDNA amplification, or IgM antibodies detection through ELISA-based serological assay. CONCLUSIONS: There is the plausibility of toxoplasmosis and leptospirosis in cetacean populations found in the Philippines, however, acute or chronic phases of infections in sampled stranded individuals cannot be confirmed in the absence of supporting pathological observations and corroborating detection tests. Further studies should look for more evidences of pathogenicity, and explore the specific mechanisms by which pelagic cetacean species become infected by Leptospira spp. and T. gondii. As there is growing evidence on the role of cetaceans as sentinels of land-sea movement of emerging pathogens and the diseases they cause, any opportunity, such as their stranding events, should be maximized to investigate the health of their populations. Moreover, the role of leptospirosis or toxoplasmosis in these stranding events must be considered.

Microbiota fingerprints within the oral cavity of cetaceans as indicators for population biomonitoring.

The composition of mammalian microbiota has been related with the host health status. In this study, we assessed the oral microbiome of 3 cetacean species most commonly found stranded in Iberian Atlantic waters (Delphinus delphis, Stenella coeruleoalba and Phocoena phocoena), using 16S rDNA-amplicon metabarcoding. All oral microbiomes were dominated by Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria bacteria, which were also predominant in the oral cavity of Tursiops truncatus. A Constrained Canonical Analysis (CCA) showed that the major factors shaping the composition of 38 oral microbiomes (p-value < 0.05) were: (i) animal species and (ii) age class, segregating adults and juveniles. The correlation analysis also grouped the microbiomes by animal stranding location and health status. Similar discriminatory patterns were detected using the data from a previous study on Tursiops truncatus, indicating that this correlation approach may facilitate data comparisons between different studies on several cetacean species. This study identified a total of 15 bacterial genera and 27 OTUs discriminating between the observed CCA groups, which can be further explored as microbiota fingerprints to develop (i) specific diagnostic assays for cetacean population conservation and (ii) bio-monitoring approaches to assess the health of marine ecosystems from the Iberian Atlantic basin, using cetaceans as bioindicators.

Pathology and molecular epidemiology of Mycobacterium pinnipedii tuberculosis in native New Zealand marine mammals.

Mycobacterium pinnipedii causes tuberculosis in a number of pinniped species, and transmission to cattle and humans has been reported. The aims of this study were to: characterize the pathology and prevalence of tuberculosis in New Zealand marine mammals; use molecular diagnostic methods to confirm and type the causal agent; and to explore relationships between type and host characteristics. Tuberculosis was diagnosed in 30 pinnipeds and one cetacean. Most affected pinnipeds had involvement of the pulmonary system, supporting inhalation as the most common route of infection, although ingestion was a possible route in the cetacean. PCR for the RD2 gene confirmed M. pinnipedii as the causal agent in 23/31 (74%) cases (22 using DNA from cultured organisms, and one using DNA from formalin-fixed paraffin-embedded (FFPE) tissue), including the first published report in a cetacean. RD2 PCR results were compared for 22 cases where both cultured organisms and FFPE tissues were available, with successful identification of M. pinnipedii in 7/22 (31.8%). In cases with moderate to large numbers of acid-fast bacilli, RD2 PCR on FFPE tissue provided a rapid, inexpensive method for confirming M. pinnipedii infection without the need for culture. VNTR typing distinguished New Zealand M. pinnipedii isolates from M. pinnipedii isolated from Australian pinnipeds and from common types of M. bovis in New Zealand. Most (16/18) M. pinnipedii isolates from New Zealand sea lions were one of two common VNTR types whereas the cetacean isolate was a type detected previously in New Zealand cattle.

Characterization and phylogenetic analysis of a Cunninghamella bertholletiae isolate from a bottlenose dolphin (Tursiops truncatus) / Tipificación y análisis filogenético de una cepa de Cunninghamella bertholletiae aislada de un delfín mular (Tursiops truncatus)

Background. Cunninghamella is a genus of the order Mucorales which includes saprophytic species, rarely causing mycoses. The most frequently reported in human mycoses is the thermophilic species Cunninghamella bertholletiae. However, this species does not appear to cause mucormycosis in animals, so there is scarce information about C. bertholletiae isolates from animals. Aims. In this paper we describe the phenotypic and genotypic characterization, and the phylogenetic analysis, of an isolate of C. bertholletiae involved in a central nervous system mucormycosis in a dolphin. Methods. The isolate studied in this publication was characterized using the current morphological and physiological identification system for Cunninghamella species. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene and the ITS-5.8S rRNA gene sequences were also performed. Results. Colonies were fast-growing, white at first, although they became tannish-gray, covering the whole plate after 7 days of incubation at 30 and 40°C. Limited growth was observed after 7 days at 45°C. The micromorphology showed characteristic erect sporangiophores. The identification of the isolate was confirmed by DNA sequencing of the D1/D2 regions of the 26S and the ITS-5.8S (ITS) rRNA gene sequencing. Conclusions. In the phylogenetic study, the isolate clustered in the same clade as C. bertholletiae neotype strain although some differences were observed in the ITS sequences. In the cetacean cases, the possible sources of infection are unclear. The reasons why this pathogen has been found only in cetaceans and not in other domestic or wild animals are at the moment unknown and need further study (AU)

Antecedentes. Cunninghamella es un género perteneciente al orden Mucorales, que incluye especies saprófitas que raramente causan micosis. De este género, Cunninghamella bertholletiae es la especie termófila más frecuentemente citada en micosis humanas. No obstante, no parece que sea una causa habitual de mucormicosis en animales, ya que es escasa la información sobre cepas de esta especie procedentes de estos. Objetivos. En esta publicación describimos la tipificación fenotípica, genotípica y el análisis filogenético de una cepa de C. bertholletiae causante de una mucormicosis del sistema nervioso central en un delfín. Métodos. La cepa fue tipificada mediante los criterios morfológicos y fisiológicos actualmente utilizados para la identificación de estas especies. También se llevó a cabo la secuenciación y el análisis de los fragmentos génicos D1/D2 26S e ITS-5.8S del ARN ribosómico. Resultados. Las colonias presentaron un crecimiento rápido; eran blanquecinas al principio y se volvieron de color marrón agrisado con el tiempo, y cubrieron totalmente las placas a los 7 días de incubación a las temperaturas de 30 y 40°C. A 45°C, después de 7 días de incubación, el crecimiento fue limitado. Al microscopio se pudieron observar los característicos esporangióforos de esta especie. La identificación de la cepa se confirmó mediante la secuenciación de los fragmentos génicos D1/D2 26S e ITS-5.8S del ARN ribosómico. Conclusiones. En el estudio filogenético, la cepa se agrupó en el mismo clado que la cepa neotipo de C. bertholletiae, aunque se detectaron algunas diferencias en las secuencias correspondientes a los ITS. En los casos causados por esta especie en cetáceos, se desconocen las posibles fuentes de infección. Tampoco se conoce por el momento por qué este patógeno ha sido aislado solo de cetáceos y no de otros animales domésticos o salvajes (AU)

Retrospective seroepidemiological investigations against Morbillivirus, Toxoplasma gondii and Brucella spp. in cetaceans stranded along the Italian coastline (1998-2014).

This study reports the results of seroepidemiological investigations carried out against Morbillivirus, Toxoplasma gondii and Brucella spp. on blood serum samples collected from 70 cetacean specimens found stranded along the Italian coastline between 1998 and 2014. A total number of 23 serum samples (32.8%) obtained from Stenella coeruleoalba, Tursiops truncatus, Balaenoptera physalus and Globicephala melas harboured anti-Morbillivirus neutralizing antibodies. Ten sera (16%) collected from S. coeruleoalba and T. truncatus were found positive against T. gondii, while no antibodies against Brucella spp. were found. These data reveal that stranded cetaceans provide a unique opportunity for monitoring the health status of free-ranging animals living in the Mediterranean Sea, in order to investigate the level of exposure of cetacean populations to selected infectious agents representing a serious threat for aquatic mammals.

Wildlife reservoirs of brucellosis: Brucella in aquatic environments.

Neurobrucellosis and osteomyelitis are common pathologies of humans and cetaceans infected with Brucella ceti or B. pinnipedialis. Currently, 53 species of marine mammal are known to show seropositivity for brucellae, and B. ceti or B. pinnipedialis have been isolated or identified in polymerase chain reaction assays in 18 of these species. Brucellae have also been isolated from fish and identified in lungworm parasites of pinnipeds and cetaceans. Despite these circumstances, there are no local or global requirements for monitoring brucellosis in marine mammals handled for multiple purposes such as capture, therapy, rehabilitation, investigation, slaughter or consumption. Since brucellosis is a zoonosis and may be a source of infection to other animals, international standards for Brucella in potentially infected marine mammals are necessary.

Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility.

In May of 2011, a live mass stranding of 26 short-finned pilot whales (Globicephala macrorhynchus) occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria cultured during the routine clinical care of the whales and necropsy of a deceased whale included methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). In order to investigate potential sources or reservoirs of MSSA and MRSA, samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal. Samples were collected on 3 days. The second collection day was 2 weeks after the first, and the third collection day was 2 months after the last animal was removed from the facility. MRSA and MSSA were isolated on each day from the facility when animals and volunteers were present. MSSA was found at an adjacent beach on all three collection days. Isolates were characterized by utilizing a combination of quantitative real-time PCR to determine the presence of mecA and genes associated with virulence, staphylococcal protein A typing, staphylococcal cassette chromosome mec typing, multilocus sequence typing, and pulsed field gel electrophoresis (PFGE). Using these methods, clonally related MRSA were isolated from multiple environmental locations as well as from humans and animals. Non-identical but genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. PFGE indicated that the majority of MRSA isolates were clonally related to the prototype human strain USA300. These studies support the notion that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.

A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals.

Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

Brucella ceti and brucellosis in cetaceans.

Since the first case of brucellosis detected in a dolphin aborted fetus, an increasing number of Brucella ceti isolates has been reported in members of the two suborders of cetaceans: Mysticeti and Odontoceti. Serological surveys have shown that cetacean brucellosis may be distributed worldwide in the oceans. Although all B. ceti isolates have been included within the same species, three different groups have been recognized according to their preferred host, bacteriological properties, and distinct genetic traits: B. ceti dolphin type, B. ceti porpoise type, and B. ceti human type. It seems that B. ceti porpoise type is more closely related to B. ceti human isolates and B. pinnipedialis group, while B. ceti dolphin type seems ancestral to them. Based on comparative phylogenetic analysis, it is feasible that the B. ceti ancestor radiated in a terrestrial artiodactyl host close to the Raoellidae family about 58 million years ago. The more likely mode of transmission of B. ceti seems to be through sexual intercourse, maternal feeding, aborted fetuses, placental tissues, vertical transmission from mother to the fetus or through fish or helminth reservoirs. The B. ceti dolphin and porpoise types seem to display variable virulence in land animal models and low infectivity for humans. However, brucellosis in some dolphins and porpoises has been demonstrated to be a severe chronic disease, displaying significant clinical and pathological signs related to abortions, male infertility, neurobrucellosis, cardiopathies, bone and skin lesions, strandings, and death.

Biologically threatened dolphins and whales.

Among the several threats to which free-ranging cetaceans are exposed, a number of biological noxae are believed to represent a serious hazard to their health and conservation on a global scale, with special emphasis on the Mediterranean Sea. These pathogens include viral agents such as Morbillivirus, which during the last 25 years have caused dramatic epidemics and die-offs among several aquatic mammal species and populations worldwide, as well as Herpesvirus, protozoan agents such as Toxoplasma gondii and bacterial pathogens such as Brucella spp.

The genome sequence of Brucella pinnipedialis B2/94 sheds light on the evolutionary history of the genus Brucella.

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.

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans.

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.