First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Vitamin B12 biosynthesis of Cetobacterium ceti isolated from the intestinal content of captive common bottlenose dolphins (Tursiops truncatus).

In comparison with terrestrial mammals, dolphins require a large amount of haemoglobin in blood and myoglobin in muscle to prolong their diving time underwater and increase the depth they can dive. The genus Cetobacterium is a common gastrointestinal bacterium in dolphins and includes two species: C. somerae and C. ceti. Whilst the former produces vitamin B12, which is essential for the biosynthesis of haem, a component of haemoglobin and myoglobin, but not produced by mammals, the production ability of the latter remains unknown. The present study aimed to isolate C. ceti from dolphins and reveal its ability to biosynthesize vitamin B12. Three strains of C. ceti, identified by phylogenetic analyses with 16S rRNA gene and genome-based taxonomy assignment and biochemical features, were isolated from faecal samples collected from two captive common bottlenose dolphins (Tursiops truncatus). A microbioassay using Lactobacillus leichmannii ATCC 7830 showed that the average concentration of vitamin B12 produced by the three strains was 11 (standard deviation: 2) pg ml-1. The biosynthesis pathway of vitamin B12, in particular, adenosylcobalamin, was detected in the draft genome of the three strains using blastKOALA. This is the first study to isolate C. ceti from common bottlenose dolphins and reveal its ability of vitamin B12 biosynthesis, and our findings emphasize the importance of C. ceti in supplying haemoglobin and myoglobin to dolphins.

Highly abundant core taxa in the blow within and across captive bottlenose dolphins provide evidence for a temporally stable airway microbiota.

BACKGROUND: The analysis of blow microbiota has been proposed as a biomarker for respiratory health analysis in cetaceans. Yet, we lack crucial knowledge on the long-term stability of the blow microbiota and its potential changes during disease. Research in humans and mice have provided evidence that respiratory disease is accompanied by a shift in microbial communities of the airways. We investigate here the stability of the community composition of the blow microbiota for 13 captive bottlenose dolphins over eight months including both sick and healthy individuals. We used barcoded tag sequencing of the bacterial 16S rRNA gene. Four of the dolphins experienced distinct medical conditions and received systemic antimicrobial treatment during the study. RESULTS: We showed that each dolphin harboured a unique community of zero-radius operational taxonomic units (zOTUs) that was present throughout the entire sampling period ('intra-core'). Although for most dolphins there was significant variation over time, overall the intra-core accounted for an average of 73% of relative abundance of the blow microbiota. In addition, the dolphins shared between 8 and 66 zOTUs on any of the sampling occasions ('inter-core'), accounting for a relative abundance between 17 and 41% of any dolphin's airway microbiota. The majority of the intra-core and all of the inter-core zOTUs in this study are commonly found in captive and free-ranging dolphins and have previously been reported from several different body sites. While we did not find a clear effect of microbial treatment on blow microbiota, age and sex of the dolphins did have such an effect. CONCLUSIONS: The airways of dolphins were colonized by an individual intra-core 'signature' that varied in abundance relative to more temporary bacteria. We speculate that the intra-core bacteria interact with the immune response of the respiratory tract and support its function. This study provides the first evidence of individual-specific airway microbiota in cetaceans that is stable over eight months.

Helicobacter delphinicola sp. nov., isolated from common bottlenose dolphins Tursiops truncatus with gastric diseases.

Gastritis and gastric ulcers are well-recognized symptoms in cetaceans, and the genus Helicobacter is considered as the main cause. In this study, we examined the gastric fluid of captive common bottlenose dolphins Tursiops truncatus with gastric diseases in order to isolate the organisms responsible for diagnosis and treatment. Four Gram-negative, rod-shaped isolates (TSBT, TSH1, TSZ, and TSH3) with tightly coiled spirals with 2-4 turns and 2-6 bipolar, sheathed flagella, were obtained from gastric fluids of common bottlenose dolphins with gastric diseases. Phylogenetic analysis, based on 16S rRNA, atpA, and 60 kDa heat-shock protein (hsp60) genes, demonstrated that these isolates form a novel lineage within the genus Helicobacter. Analyses of 16S rRNA, atpA, and hsp60 gene sequences showed that isolate TSBT was most closely related to H. cetorum MIT99-5656T (98.5% similarity), H. pylori ATCC 43504T (76.7% similarity), and H. pylori ATCC 43504T (78.0% similarity), respectively. Type strains of Helicobacter showing resistance to 2% NaCl have not been reported previously; however, these novel isolates were resistant to 2% NaCl. Culture supernatant of some isolates induced intracellular vacuolization in mammalian cultured cells. These data, together with the different morphological and biochemical characteristics of the isolates, reveal that these isolates represent a novel species for which we propose the name Helicobacter delphinicola sp. nov. with type strain TSBT (= JCM 32789T = TSD-183T). Future studies will confirm whether H. delphinicola plays a role in lesion etiopathogenesis in cetaceans.

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.

Molecular characterization of Brucella ceti from a bottlenose dolphin (Tursiops truncatus) with osteomyelitis in the western Pacific.

Although the presence of Brucella spp. in the western Pacific has been suggested by epidemiological studies on cetaceans, it has not been confirmed by bacterial isolation. Here, for the first time, we report that a marine Brucella strain was isolated in the western Pacific from a bottlenose dolphin with osteomyelitis. The isolate from the lesion was confirmed to be B. ceti of sequence type 27 by multilocus sequence typing and Bruce-ladder PCR. Infrequent-restriction-site PCR and omp2 gene sequencing revealed that molecular characteristics of this isolate were similar to those of Brucella DNA previously detected from minke whales in the western North Pacific. These results suggest that genetically related Brucella strains circulate in cetacean species in this region.

Isolation of Leptospira interrogans from a Bottlenose Dolphin (Tursiops truncatus) in the Mediterranean Sea.

The pathogenic Leptospira species are very widespread in nature, persisting in the renal tubules of many domestic and wild animal reservoirs. We report the isolation of Leptospira interrogans serovar Pomona in a bottlenose dolphin (Tursiops truncatus) stranded along the coast of Sardinia, Italy, in 2016.

First isolation of voriconazole-resistant Candida albicans, C. tropicalis, and Aspergillus niger from the blowholes of bottlenose dolphins (Tursiops truncatus).

Pulmonary mycosis is a fungal disease that commonly affects bottlenose dolphins (Tursiops truncatus) and is generally treated by the oral administration of azoles, such as itraconazole (ITZ) and voriconazole (VRZ). However, antifungal susceptibility testing of clinical isolates has not been well performed as a routine clinical examination in aquaria. In this study, we collected fungal species from the blowholes of 14 bottlenose dolphins, of which 12 were treated with ITZ or VRZ. All dolphins were housed in the Port of Nagoya Public Aquarium. The fungal species Candida albicans, C. tropicalis, C. glabrata, Aspergillus fumigatus, and A. niger were isolated. E-tests were performed to determine the minimum inhibitory concentrations (MICs) of ITZ and VRZ on these isolates. VRZ-resistant C. tropicalis (MIC: >32 µg/ml) and A. niger (MIC: >32 µg/ml) were isolated from three dolphins treated with ITZ or VRZ. Additionally, azole-resistant isolates of C. albicans and C. glabrata were collected from two dolphins that had never received azole therapy. To the best of our knowledge, our study is the first to report the isolation of VRZ-resistant C. albicans, C. tropicalis, and A. niger from the blowholes of bottlenose dolphins. Thus, antifungal susceptibility testing is a crucial strategy for selecting antifungal agents to treat respiratory fungal infections in bottlenose dolphins in aquaria.

Comparative Innate and Adaptive Immune Responses in Atlantic Bottlenose Dolphins (Tursiops truncatus) With Viral, Bacterial, and Fungal Infections.

Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.

Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus).

Aspergillosis is a fungal infection caused by Aspergillus molds that can affect both humans and animals. Despite advances in diagnostics and therapy, medical management of this disease remains difficult. Expansion of the basic knowledge regarding its pathophysiology in animals is critical to aid in the identification of new biomarkers of infection for diagnosis and therapeutic targets. For such a purpose, proteomics can be used by addressing protein changes during various disease processes. In the present study, a mass spectrometry analysis based on isobaric tagging for relative and absolute quantitation (iTRAQ®) was applied for direct identification and relative quantitation of proteins in blood collected from 32 Aspergillus-diseased common bottlenose dolphins (Tursiops truncatus, 32 samples) in comparison with blood from 55 other dolphins (55 samples from 41 clinically-normal controls and from 14 cetaceans with miscellaneous non-Aspergillus inflammation diseases) and ten convalescent dolphins (28 samples). Sixty-six and 40 proteins were found to be ≥2.0-fold over- and underrepresented versus miscellaneous non-Aspergillus inflammatory dolphins, respectively, and most were confirmed vs. clinically-normal controls and convalescents. Many proteins which play a role in the adaptive immune response were identified, including MHC proteins and others involved in catalytic activity like the NADPH-ubiquinone oxido-reductases. Overall, iTRAQ® appears to be a convenient proteomic tool greatly suited for exploratory ex vivo studies focusing on pathophysiology. This technique should be considered as a preliminary step before validation of new diagnostic markers.

Surgical Management of a Chronic Neck Abscess in a U.S. Navy Bottlenose Dolphin.

Surgical intervention on cetaceans is rarely performed due to challenges including general anesthesia and post-operative wound healing. This report describes the evaluation and treatment of an adult female bottlenose dolphin (Tursiops truncatus) with the US Navy Marine Mammal Program, with a chronic ventral cervical abscess caused by Candida glabrata. Despite aspiration and lavage along with multiple antifungal drugs, the patient developed inspiratory stridor with decreased performance level and surgical treatment was pursued. Under general anesthesia with the dolphin in dorsal recumbency position a 12-cm longitudinal ventral midline neck incision was used for exploration. Intraoperative ultrasound aided the identification of surgical landmarks and the abscess cavity. After adequate drainage and curettage, a closed-suction drain was placed in the surgical site. Retention sutures were used to close the incision and the external drain bulb was secured to a pectoral fin strap. One-year post-op, the dolphin was clinically normal and follow-up imaging showed no significant recurrence of the abscess. This case demonstrates a novel surgical approach of managing abscesses in dolphins, including placement and management of a negative suction drain in a submerged patient. The successful collaboration between veterinary anesthesiology, veterinary medicine, radiology, and general surgery allowed the patient to continue her normal activities as a full-duty service member.

Comparison of the gut microbiota of captive common bottlenose dolphins Tursiops truncatus in three aquaria.

AIMS: This study was conducted to assess the presence and extent of differences in the gut microbiota of common bottlenose dolphins depending on rearing facilities. METHODS AND RESULTS: Faecal samples were collected from 16 common bottlenose dolphins at three aquaria in Japan. After extracting DNA from the faeces, the V3-V4 region of bacterial 16S rRNA was amplified and sequenced using Illumina MiSeq platform. The constituent phyla of the gut microbiota were similar among aquaria; however, the most dominant phylum differed depending on the facility, and the compositions of microbiota were remarkably varied at the family level among aquaria. The alpha diversity indices tended to differ among aquaria. Some bacterial families observed in terrestrial mammalian carnivores or carnivorous fish were detected, as well as several bacterial species suspected of being pathogenic in dolphins. CONCLUSION: Our findings indicate that captive environmental conditions including prey and housing types may contribute to differences in the gut microbiota of the dolphins. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study revealing the differences in gut microbiota of captive dolphins among facilities. Our findings will provide valuable information for improving the health management of dolphins.

Death Associated to Methicillin Resistant Staphylococcus aureus ST8 Infection in Two Dolphins Maintained Under Human Care, Italy.

The present study describes the isolation of Methicillin-resistant Staphylococcus aureus (MRSA) from respiratory tract of 2 dolphins of different origin, a stranded juvenile Risso's dolphin (Grampus griseus) and a captive born common bottlenose dolphin (Tursiops truncatus) calf, which died in the same institution at 1-month distance from the other. A complete microbiological and genetic investigation confirmed the presence of MRSA clone-complex 8, sequence type (ST) 8, spa-type t008 in both individuals. This strain differs from the one previously reported in walruses and dolphins and has never been described in dolphins before, but it is randomly isolated from Italian human patients. Vertical transmission of the infection may also occurs in other species and considering the description and location of the pathological lesions, this seems to be the most likely route of transmission implied in the young bottlenose dolphin. Staphylococcus aureus is known as an opportunistic agent, usually secondary to other pathogens, but its multiple antibiotic resistance and its zoonotic implications suggest a thorough and strict application of animal management hygiene protocols.

Evaluation of a genus-specific ELISA and a commercial Aspergillus Western blot IgG® immunoblot kit for the diagnosis of aspergillosis in common bottlenose dolphins (Tursiops truncatus).

Aspergillosis is a fungal infection with high mortality and morbidity rates. As in humans, its definitive diagnosis is difficult in animals, and thus new laboratory tools are required to overcome the diagnostic limitations due to low specificity and lack of standardization. In this study of common bottlenose dolphins (Tursiops truncatus), we evaluated the diagnostic performance of a new commercial immunoblot kit that had been initially developed for the serologic diagnosis of chronic aspergillosis in humans. Using this in a quantitative approach, we first established its positive cutoff within an observation cohort of 32 serum samples from dolphins with "proven" or "probable" diagnosis of aspergillosis and 55 negative controls. A novel enzyme-linked immunosorbent assay (ELISA) test was also developed for detecting anti-Aspergillus antibodies, and results were compared between the two assays. Overall, the diagnostic performance of immunoblot and ELISA were strongly correlated (P < .0001). The former showed lower sensitivity (65.6% versus 90.6%), but higher specificity (92.7% vs. 69.1%), with no cross-reaction with other fungal infections caused by miscellaneous non-Aspergillus genera. When assessing their use in a validation cohort, the immunoblot kit and the ELISA enabled positive diagnosis before mycological cultures in 42.9% and 33.3% subjects addressed for suspicion of aspergillosis, respectively. There was also significant impact of antifungal treatment on the results of the two tests (P < .05). In all, these new serological methods show promise in aiding in the diagnosis of aspergillosis in dolphins, and illustrate the opportunity to adapt commercial reagents directed for human diagnostics to detect similar changes in other animals.

Bacterial Species Identified on the Skin of Bottlenose Dolphins Off Southern California via Next Generation Sequencing Techniques.

The dermis of cetaceans is in constant contact with microbial species. Although the skin of the bottlenose dolphin provides adequate defense against most disease-causing microbes, it also provides an environment for microbial community development. Microbial community uniqueness and richness associated with bottlenose dolphin skin is a function of varying habitats and changing environmental conditions. The current study uses ribosomal DNA as a marker to identify bacteria found on the skin of coastal and offshore bottlenose dolphins off of Southern California. The unique microbial communities recovered from these dolphins suggest a greater microbial diversity on the skin of offshore ecotype bottlenose dolphins, while microbial populations associated with the coastal ecotype include species that are more closely related to each other and that suggest exposure to communities that are likely to be associated with terrestrial runoff.

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)

Captive bottlenose dolphins and killer whales harbor a species-specific skin microbiota that varies among individuals.

Marine animals surfaces host diverse microbial communities, which play major roles for host's health. Most inventories of marine animal surface microbiota have focused on corals and fishes, while cetaceans remain overlooked. The few studies focused on wild cetaceans, making difficult to distinguish intrinsic inter- and/or intraspecific variability in skin microbiota from environmental effects. We used high-throughput sequencing to assess the skin microbiota from 4 body zones of 8 bottlenose dolphins (Tursiops truncatus) and killer whales (Orcinus orca), housed in captivity (Marineland park, France). Overall, cetacean skin microbiota is more diverse than planktonic communities and is dominated by different phylogenetic lineages and functions. In addition, the two cetacean species host different skin microbiotas. Within each species, variability was higher between individuals than between body parts, suggesting a high individuality of cetacean skin microbiota. Overall, the skin microbiota of the assessed cetaceans related more to the humpback whale and fishes' than to microbiotas of terrestrial mammals.

Novel Microbial Diversity and Functional Potential in the Marine Mammal Oral Microbiome.

The vast majority of bacterial diversity lies within phylum-level lineages called "candidate phyla," which lack isolated representatives and are poorly understood. These bacteria are surprisingly abundant in the oral cavity of marine mammals. We employed a genome-resolved metagenomic approach to recover and characterize genomes and functional potential from microbes in the oral gingival sulcus of two bottlenose dolphins (Tursiops truncatus). We detected organisms from 24 known bacterial phyla and one archaeal phylum. We also recovered genomes from two deep-branching, previously uncharacterized phylum-level lineages (here named "Candidatus Delphibacteria" and "Candidatus Fertabacteria"). The Delphibacteria lineage is found in both managed and wild dolphins; its metabolic profile suggests a capacity for denitrification and a possible role in dolphin health. We uncovered a rich diversity of predicted Cas9 proteins, including the two longest predicted Cas9 proteins to date. Notably, we identified the first type II CRISPR-Cas systems encoded by members of the Candidate Phyla Radiation. Using their spacer sequences, we subsequently identified and assembled a complete Saccharibacteria phage genome. These findings underscore the immense microbial diversity and functional potential that await discovery in previously unexplored environments.

Characterization and phylogenetic analysis of a Cunninghamella bertholletiae isolate from a bottlenose dolphin (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.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe.

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health.