A case report of live-stranded rough-toothed dolphin (Steno bredanensis) with plastic debris ingestion on the Pacific coast of Japan.

A juvenile rough-toothed dolphin (Steno bredanensis) was live-stranded and rescued in Kanagawa Prefecture, Japan. From the results of diagnostic examinations, blood tests indicated that the dolphin was malnourished, dehydrated, and anemic. The dolphin died on sixth day of rescue despite treatment. At autopsy, 570g of foreign material, including 34 pieces of cellophanes and plastic debris (PD), were found in the forestomach. Additional gross findings, including some endoparasitism and presence of accessory spleens were also identified. This is the first case in Japan which accidental ingestion of foreign bodies, including PD, was suspected to be the cause of death in a cetacean.

Brucella infection in rough-toothed dolphin (Steno bredanensis) with severe orchitis stranded on the Pacific coast of Japan.

In the western North Pacific, prominent granulomatous testes have been detected in many Brucella-infected common minke whales (Balaenoptera acutorostrata), but there have been no reports in toothed cetaceans. We found severe orchitis with granulomatous lesions in a rough-toothed dolphin (Steno bredanensis) stranded on the Pacific coast of Japan in 2011. Histopathological examination revealed leukocyte infiltration of the lesions. DNA from the lesion was analyzed by PCR and it showed molecular biological similarities with those of Brucella-infected common minke whales and Brucella ceti of sequence-type 27 (ST27). These results suggest that the type of Brucella ceti that infected the dolphin was ST27, which may have caused severe orchitis. This study adds to our understanding of Brucella infections in marine mammals.

Foot extension and retraction in the clam Calyptogena okutanii without any Keber's valve: an inflatable fastener bag model.

In order to investigate the foot manipulation of a clam without a Keber's valve, Calyptogena okutanii was examined by light microscopy, magnetic resonance imaging and computed tomography. The foot chamber was divided into two compartments by a dense muscle fastener zone (FZ) comprising a pedal artery and sinuses in the mid-sagittal plane in between muscles running in the anterior-posterior oblique direction. The distal part of the foot chamber (inflatable fastener bag, IFB) had a loose superficial muscle layer. The proximal part of the foot chamber (visceral reservoir, VR) was covered by a dense superficial muscle layer. The outlet of the VR was connected with the hinge ligament duct, consisting of the hinge ligament, a pair of shells and the pericardium. Based on these anatomical structures, foot extension starts from contraction of muscles in the FZ, so that flow in the FZ is stopped. Then, the superficial muscles of the foot contract, and the pressure of the IFB increases so that the foot can extend. Foot retraction starts from the relaxation of muscles in the FZ so that the hemolymph returns to the VR. The hinge ligament duct allows a constant return flow from the foot chamber to the gills and the heart. The heart rate and the flow in the FZ, which decreased and increased during the foot extension and retraction, respectively, supported this model. In conclusion, the FZ of Calyptogena okutanii could be an alternative to Keber's valve in Anodonta, playing a similar role.

Anti-Brucella antibodies in seals at coastal locations of Hokkaido, Japan, with focus on life stages.

The enzyme-linked immunosorbent assay (ELISA) was applied to detect antibodies against Brucella abortus in serum samples from four seal species at nine coastal locations of Hokkaido, Japan. These antibodies were detected in 27% (32/118) of Western Pacific harbor seals (Phoca vitulina stejnegeri) at Cape Erimo. The antibodies were observed in spotted seals (P. largha) in one out of six at Nemuro, in two out of three at Rebun Island, in one out of two at Bakkai, and in examined one at Soya. They were also found in respective examined one ribbon seal (Histriophoca fasciata) and one ringed seal (Pusa hispida) at Akkeshi. Harbor seals that tested positive were mostly yearlings (35%, 20/57) and juveniles (45%, 10/22), while only one pup (1/13) and one subadult (1/5) tested positive with low titers of the antibody; no antibodies were observed in adults (n=21). These results suggest that Brucella mainly infected harbor seals from the environment while weaning, and the bacteria were cleared during the early life stage of the seals. In spotted seals, however, antibodies were also detected in adults, suggesting that spotted seals could become infected with Brucella even as adults. It is also possible that a different, more persistent strain of Brucella may have infected the spotted seals.

Draft Genome Sequence of Brucella ceti Isolated in the Western Pacific Ocean.

In 2018, Brucella ceti was isolated from a bottlenose dolphin from the western Pacific Ocean. Here, we report a draft genome sequence of the isolate BD1442 of sequence type 27, which is the only sequence type known to have been isolated from human clinical cases.

Measles Virus Hemagglutinin Protein Establishes a Specific Interaction With the Extreme N-Terminal Region of Human Signaling Lymphocytic Activation Molecule to Enhance Infection.

Measles virus (MV) is a human pathogen that is classified in the genus Morbillivirus in the family Paramyxoviridae together with several non-human animal morbilliviruses. They cause severe systemic infections by using signaling lymphocytic activation molecule (SLAM) and poliovirus receptor-like 4 expressed on immune and epithelial cells, respectively, as receptors. The viral hemagglutinin (H) protein is responsible for the receptor-binding. Previously determined structures of MV-H and SLAM complexes revealed a major binding interface between the SLAM V domain and MV-H with four binding components (sites 1-4) in the interface. We studied the MV-H and human SLAM (hSLAM) complex structure in further detail by in silico analyses and determined missing regions or residues in the previously determined complex structures. These analyses showed that, in addition to sites 1-4, MV-H establishes a unique interaction with the extreme N-terminal region (ExNTR) of hSLAM. The first principles calculation-based fragment molecular orbital computation method revealed that methionine at position 29 (hSLAM-Met29) is the key residue for the interaction. hSLAM-Met29 was predicted to establish a CH-π interaction with phenylalanine at position 549 of MV-H (MVH-Phe549). A cell-cell fusion assay showed that the hSLAM-Met29 and MVH-Phe549 interaction is important for hSLAM-dependent MV membrane fusion. Furthermore, Jurkat cell lines expressing hSLAM with or without Met29 and recombinant MV possessing the H protein with or without Phe549 showed that the hSLAM-Met29 and MVH-Phe549 interaction enhanced hSLAM-dependent MV infection by ~10-fold. We speculate that in the evolutionary history of morbilliviruses, this interaction may have contributed to MV adaptation to humans because this interaction is unique for MV and only MV uses hSLAM efficiently among morbilliviruses.

Identification of correlated inter-residue interactions in protein complex based on the fragment molecular orbital method.

A theoretical scheme to systematically describe correlated (network-like) interactions between molecular fragments is proposed within the framework of the fragment molecular orbital (FMO) method. The method is mathematically based on the singular value decomposition (SVD) of the inter-fragment interaction energy (IFIE) matrix obtained by the FMO calculation, and can be applied to a comprehensive description of protein-protein interactions in the context of molecular recognition. In the present study we apply the proposed method to a complex of measles virus hemagglutinin and human SLAM receptor, thus finding a usefulness for efficiently eliciting the correlated interactions among the amino-acid residues involved in the two proteins. Additionally, collective interaction networks by amino-acid residues important for mutation experiments can be clearly visualized.

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.

Phocine distemper virus uses phocine and other animal SLAMs as a receptor but not human SLAM.

Morbilliviruses use the signaling lymphocyte activation molecule (SLAM) as a receptor to infect their hosts. Seals are almost the only animal species that show apparent infection with phocine distemper virus (PDV). Seal SLAM functioned as a PDV receptor. However, dolphin- and dog-SLAM molecules, but not human SLAM, were also fully functional PDV receptors. These data suggest that the host range of PDV is not simply determined by its SLAM usage. However, human nonsusceptibility to PDV infection may be at least partly attributable to the inability of PDV to use human SLAM as a receptor.

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.

Identification of cells expressing two peptidoglycan recognition proteins in the gill of the vent mussel, Bathymodiolus septemdierum.

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

Marine Morbilliviruses: Diversity and Interaction with Signaling Lymphocyte Activation Molecules.

Epidemiological reports of phocine distemper virus (PDV) and cetacean morbillivirus (CeMV) have accumulated since their discovery nearly 30 years ago. In this review, we focus on the interaction between these marine morbilliviruses and their major cellular receptor, the signaling lymphocyte activation molecule (SLAM). The three-dimensional crystal structure and homology models of SLAMs have demonstrated that 35 residues are important for binding to the morbillivirus hemagglutinin (H) protein and contribute to viral tropism. These 35 residues are essentially conserved among pinnipeds and highly conserved among the Caniformia, suggesting that PDV can infect these animals, but are less conserved among cetaceans. Because CeMV can infect various cetacean species, including toothed and baleen whales, the CeMV-H protein is postulated to have broader specificity to accommodate more divergent SLAM interfaces and may enable the virus to infect seals. In silico analysis of viral H protein and SLAM indicates that each residue of the H protein interacts with multiple residues of SLAM and vice versa. The integration of epidemiological, virological, structural, and computational studies should provide deeper insight into host specificity and switching of marine morbilliviruses.

Detection of serum antibodies to Brucella in Russian aquatic mammals.

A serologic survey of Brucella infection was performed in Caspian seals (Pusa caspica, n=71), Baikal seals (P. sibirica, n=7), ringed seals (P. hispida hispida, n=6), and beluga whales (Delphinapterus leucas, n=4) inhabiting Russian waters, by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis as antigens. The sera of 4 Caspian seals (4%) tested positive for B. abortus. The same sera samples demonstrated weaker yet detectable affinity for B. canis antigens. Several discrete bands against B. abortus and B. canis antigens were detected on Western blot analysis of the ELISA-positive seal sera; the bands against B. canis were weaker than those against B. abortus. The sera of 3 beluga whales (75%) were positive for B. abortus antigens but showed no binding to B. canis antigens in the ELISA. The positive whale sera showed a strong band appearance only against B. abortus antigens in the 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 Caspian seals and beluga whales inhabiting Russian waters, and that the Brucella strains infecting the seals and the whales were antigenetically distinct.

Computational Analysis of the Interaction Energies between Amino Acid Residues of the Measles Virus Hemagglutinin and Its Receptors.

Measles virus (MV) causes an acute and highly devastating contagious disease in humans. Employing the crystal structures of three human receptors, signaling lymphocyte-activation molecule (SLAM), CD46, and Nectin-4, in complex with the measles virus hemagglutinin (MVH), we elucidated computationally the details of binding energies between the amino acid residues of MVH and those of the receptors with an ab initio fragment molecular orbital (FMO) method. The calculated inter-fragment interaction energies (IFIEs) revealed a number of significantly interacting amino acid residues of MVH that played essential roles in binding to the receptors. As predicted from previously reported experiments, some important amino-acid residues of MVH were shown to be common but others were specific to interactions with the three receptors. Particularly, some of the (non-polar) hydrophobic residues of MVH were found to be attractively interacting with multiple receptors, thus indicating the importance of the hydrophobic pocket for intermolecular interactions (especially in the case of Nectin-4). In contrast, the electrostatic interactions tended to be used for specific molecular recognition. Furthermore, we carried out FMO calculations for in silico experiments of amino acid mutations, finding reasonable agreements with virological experiments concerning the substitution effect of residues. Thus, the present study demonstrates that the electron-correlated FMO method is a powerful tool to search exhaustively for amino acid residues that contribute to interactions with receptor molecules. It is also applicable for designing inhibitors of MVH and engineered MVs for cancer therapy.

Serologic evidence of Brucella infection in pinnipeds along the coast of Hokkaido, the northernmost main island of Japan.

Brucella infection in Hokkaido was serologically surveyed in four species of pinnipeds inhabiting Cape Erimo during 2008-2013 and the Shiretoko Peninsula in 1999 by ELISA using Brucella abortus and B. canis as antigens. Anti-Brucella positive sera showed higher absorbance to B. abortus than B. canis in almost all samples. Anti-B. abortus antibodies were detected in serum samples from 24% (n = 55) of Western Pacific harbor seals (Phoca vitulina stejnegeri) in Cape Erimo and from 66% (n = 41) of spotted seals (P. largha), 15% (n = 20) of ribbon seals (Histriophoca fasciata) and 18% (n = 17) of Western Steller's sea lions (Eumetopias jubatus jubatus) in the Shiretoko Peninsula. Anti-Brucella antibodies were detected at higher absorbance in 1- to 4-year-old harbor seals than in the pups and mature animals, suggesting either that Brucella infection mainly occurs after weaning or that it is maternally transmitted to pups with premature or suppressed immunity. Anti-Brucella antibodies were detected in both immature and mature spotted seals and ribbon seals, with higher absorbance in the former. The antibodies were detected only in mature Western Steller's sea lions. Western blot analysis of the serum samples showed some differences in band appearances, namely discrete versus smeary, and in the number of bands, indicating that multiple different Brucella may be prevalent in pinnipeds in Hokkaido. Alternatively, the Brucella of pinnipeds may have some intra-species diversity.

Long-term Cultivation of the Deep-Sea Clam Calyptogena okutanii: Changes in the Abundance of Chemoautotrophic Symbiont, Elemental Sulfur, and Mucus.

Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans.

In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994-2010 offshore surveys (21%, 285/1353) and in the 2006-2010 Japanese coastal surveys (20%, 86/436), in Bryde's whales (B. edeni brydei) in the 2000-2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002-2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000-2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000-2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde's whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level.

Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 µm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 µm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 µm) and contained small basophilic granules (diameter, 0.3-0.4 µm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 µm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed lower phagocytic activity and lower rates of phagosome-lysosome fusion than the EGs. These findings indicate that the two types of granulocyte play distinct roles in the defense system.

Magnetic resonance imaging analysis of water flow in the mantle cavity of live Mytilus galloprovincialis.

Water flow inside the shell of Mytilus galloprovincialis was measured by phase-contrast magnetic resonance imaging (MRI). In seawater without algal cells at 23 °C, water approached the mussel from the posterior-ventral side, and entered through the inhalant aperture at a velocity of 40-20 mm s(-1). The flow rate in the lower mantle cavity decreased to 10-20 mm s(-1), the water flowed in the anterior-dorsal direction and approached the demibranches at a velocity of 5-10 mm s(-1). After passing through the lamellae to the upper mantle cavity, the water stretched the interlamellar cavity, turned to the posterior-dorsal direction and accumulated in the epibranchial cavity. The water flows came together at the ventral side of the posterior adductor muscle. The velocity increased more to than 50 mm s(-1) in the exhalant siphon, and exhaled out in the posterior-dorsal direction. The anterior-posterior direction of the flow was imaged every 1.92 s by the inflow effect of T1-weighted MRI. The flow seemed to be constant, and no cyclic motion of the mantles or the gills was detected. Spontaneous closure of the shells caused a quick drop of the flow in the mantle cavity. In the opening process of the shells, water flow in the interlamellar cavities increased before the opening, followed by an increase of flows in the exhalant siphon and inhalant aperture with minimum delay, reaching a plateau within 1 min of the shells opening. This provides direct evidence that the lateral cilia drive water in the mussel M. galloprovincialis.

Recent host range expansion of canine distemper virus and variation in its receptor, the signaling lymphocyte activation molecule, in carnivores.

The signaling lymphocyte activation molecule (SLAM) is a receptor for morbilliviruses. To understand the recent host range expansion of canine distemper virus (CDV) in carnivores, we determined the nucleotide sequences of SLAMs of various carnivores and generated three-dimensional homology SLAM models. Thirty-four amino acid residues were found for the candidates binding to CDV on the interface of the carnivore SLAMs. SLAM of the domestic dog (Canis lupus familiaris) were similar to those of other members of the suborder Caniformia, indicating that the animals in this group have similar sensitivity to dog CDV. However, they were different at nine positions from those of felids. Among the nine residues, four of domestic cat (Felis catus) SLAM (72, 76, 82, and 129) and three of lion (Panthera leo persica) SLAM (72, 82, and 129) were associated with charge alterations, suggesting that the felid interfaces have lower affinities to dog CDV. Only the residue at 76 was different between domestic cat and lion SLAM interfaces. The domestic cat SLAM had threonine at 76, whereas the lion SLAM had arginine, a positively charged residue like that of the dog SLAM. The cat SLAM with threonine is likely to have lower affinity to CDV-H and to confer higher resistance against dog CDV. Thus, the four residues (72, 76, 82, and 129) on carnivore SLAMs are important for the determination of affinity and sensitivity with CDV. Additionally, the CDV-H protein of felid strains had a substitution of histidine for tyrosine at 549 of dog CDV-H and may have higher affinity to lion SLAM. Three-dimensional model construction is a new risk assessment method of morbillivirus infectivity. Because the method is applicable to animals that have no information about virus infection, it is especially useful for morbillivirus risk assessment and wildlife conservation.