Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (μ1), μ2, μNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus ‘piscine orthoreovirus 2’ (PRV-2). Reverse transcription–quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.

Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5–3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.

Ayu Plecoglossus altivelis were experimentally infected with Flavobacterium psychrophilum, which is the causative agent of bacterial cold water disease (CWD). The fish infected by immersion usually died within an hour after they became moribund. The blood volume and haematocrit values of moribund fish were low, while those values of many infected fish that were not moribund were in the range of controls. Most of the affected fish in the immersion-infected groups had ulcerative lesions on their lower jaw. No histological evidence of haemolysis was observed. These results suggest that rapid bleeding occurred through ulcerative lesions, probably causing hypoxia which killed the fish. Ulcerative lesions developed on the dorsal skin when this area had been slightly abraded artificially prior to immersion challenge. Histologically, F. psychrophilum was initially found on the skin that had microscopic injuries, but not on normal skin. The bacterium then entered the dermis and migrated through connective tissues. The lesions subsequently expanded into the underlying musculature through the myosepta, developed necrotic myositis and formed externally open ulcers. Only in later stages of infection did mild lesions develop in the internal organs and the gill, probably caused by the bacterium migrating through blood vessels. This suggests that infection with CWD through the gill or digestive tract is unlikely. Virtually no open lesions were found in ayu challenged by intramuscular injections except at the injection sites. The results suggest that skin injuries are major portals of entry for F. psychrophilum in ayu, and the bacterium has affinity for collagenous connective tissues. KEY WORDS: Cold water disease · Flavobacterium psychrophilum · Pathogenesis · Ayu · Plecoglossus altivelis Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [93][94][95][96][97][98][99][100][101][102][103][104] 2005 salmonids in which the disease occurs most frequently when the water temperature is between 4 and 10°C (Shotts & Starliper 1999). The most notable clinical sign of CWD in ayu is anaemia as revealed by pale gills (Iida & Mizokami 1996). Ulcerative lesions are often found on the caudal peduncle of affected wild ayu, including stocked fish (Iida & Mizokami 1996). However, when CWD occurs in cultured ayu, ulcerative lesions on the surface of the body trunk are often not observed, although haemorrhagic lesions or ulcers develop on the lower jaw of many of the affected fish. The ulcerative lesions found on the caudal peduncle of affected wild ayu might develop following infection by the bacteria into skin wounds caused by fishing (snagging) or by territorial aggression by other ayu. Erosion of fins (fin rot) or tail loss, which are typical clinical signs for CWD in salmonids (Nematollahi et al. 2003), are not observed in ayu. Furthermore, the F. psychrophilum strain(s) that causes CWD in ayu is distinct serologically and genetically from those in other fish species including salmonids...

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASfV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV). African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes a hemorrhagic fever with high mortality, with rates approaching 100% in domestic pigs 1. The virus infects domestic pigs and their relatives and ticks 2. ASF outbreaks had been recorded in Africa and Europe, but in recent years the disease has spread to China, Vietnam, Cambodia, Mongolia, Hong Kong, and Korea, becoming a threat to the swine industry worldwide 3. ASFV is a member of nucleocytoplasmic large DNA viruses (NCLDVs) with an average diameter of 200 nm. Although some related viruses, such as faustovirus 4 , kaumoebavirus 5 , and pacmanvirus 6 , have been reported, ASFV is the only member of the Asfarviridae family 7. In the present paper, we describe a virus likely to be the closest ASFV relative found to date; this novel virus was isolated as the presumptive causative agent of abalone amyotrophia. Mass mortalities of abalone have been reported since the early 1980s, during seed production in Japan. The disease was designated abalone amyotrophia because diseased abalone develop muscle atrophy in the mantle and foot 8. Diseased abalone show reduced ability to adhere to the substrate, and some diseased abalone exhibit incisions on the front margin of the shell and brown pigmentation inside of the shell 9. Histopathological evaluation has revealed the presence of abnormal cell masses that are produced extensively, primarily in the ganglion and peripheral nerve of the foot muscle 9. Cumulative mortality can reach 50% and higher 10. Abalone herpesvirus (AbHV) 11,12 and abalone shriveling syndrome-associated virus (AbSV) 13 also cause mortality ac...

Bottom-dwelling teleosts, particularly flatfishes or cod living in temperate to cold seawater, sometimes develop tumor-like lesions on the body surface or in the branchial cavity. These lesions usually contain masses of so called 'X-cells' of unknown origin. We amplified a gene for small subunit ribosomal RNA (18S rRNA) from X-cell lesions of the flathead flounder Hippoglossoides dubius. Phylogenetic analysis clearly classified the obtained sequence as a protozoan, although the organism had no clear affinity with any known protistan groups. In situ hybridization showed that probes specific for the protozoan 18S rRNA hybridized only with X-cells, and not with the host-fish cells, indicating that X-cells harbor the protozoan rRNA. On the other hand, a probe specific for vertebrate 18S rRNA hybridized with the host-fish cells, but not with X-cells. This is conclusive evidence that X-cells are parasitic protozoans.KEY WORDS: X-cell · 18S rRNA · In situ hybridization · Protozoa · Hippoglossoides dubius · Fishes · Pseudotumor Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: [165][166][167][168][169][170] 2004 In 2001 and 2002, many flathead flounder Hippoglossoides dubius caught by bottom-trawling in coastal waters of Niigata and Yamagata prefectures of Japan (eastern part of the Japan Sea), were found to have tumor-like lesions of the skin. The prevalence of the diseased fish ranged from 0 to 40% of the total catch, and histological examination revealed X-cells in the tumorous lesions. X-cell disease in the flathead flounder had already been reported (Ito et al. 1976, Katsura et al. 1984, although the disease had not previously been recorded in this area. In the present study, we examined the X-cell disease in the flathead flounder to clarify the origin of the X-cells. MATERIALS AND METHODSHistological examination. The flathead flounder used for general histological observations were caught by bottom-trawling and landed at a nearby port. Tissues of the lesions were taken from 10 affected fish, and fixed in Davidson's solution (330 ml 95% ethanol, 220 ml commercial formaldehyde solution containing 37 to 39% formaldehyde, 115 ml glacial acetic acid, and 335 ml distilled water). The tissues were then embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin (HE).DNA extraction and in situ hybridization. Immediately after capture with a bottom trawl, tissues of tumor-like lesions in flathead flounder were sampled on board the fishing vessel. Tissues were taken from the lesions of 3 affected flounder. For DNA extraction, excised tissues of the lesions were fixed and stored in 100% ethanol. For in situ hybridization, tissues of the lesions were fixed in Davidson's solution overnight, and incubated in 300 mM EDTA (pH 7.5) for 1 wk to decalcify bones or scales in the tissues. From 1 affected fish, pieces of liver, spleen, and kidney were also sampled and fixed as described above. Subsequently, the tissues were embedded in paraffin.PCR and phylo...