Genome sequence of Chionoecetes opilio bacilliform virus, a nimavirus infecting the snow crab Chionoecetes opilio.

Nimaviridae (class Naldaviricetes) is a family of double-stranded DNA viruses infecting crustaceans, with the only officially recognized representative being white spot syndrome virus (WSSV). Chionoecetes opilio bacilliform virus (CoBV) was isolated as the causative agent of milky hemolymph disease in the snow crab Chionoecetes opilio, an economically important crustacean in the northwestern Pacific. Here, we present the complete genome sequence of CoBV and show that it is unambiguously a nimavirus. The CoBV genome is a 240-kb circular DNA molecule with 40% GC content that encodes 105 proteins, including 76 WSSV orthologs. Phylogenetic analysis based on eight naldaviral core genes established that CoBV is a member of the family Nimaviridae. The availability of the CoBV genome sequence provides a deeper understanding of CoBV pathogenicity and nimavirus evolution.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2 kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50 °C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50 °C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters.

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S-23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63 °C, and concentrations of the inner and loop primers were 1.4 and 1.0 µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35 min is 50 pg, and at 60 min it is 5 fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

Isolation and characterization of a Vibrio sp. strain MA3 associated with mass mortalities of the pearl oyster Pinctada fucata.

In the summers of 2019 and 2020, a previously undescribed disease occurred in both juvenile and adult shellfish, causing mass mortalities in cultured pearl production, characterized by the major symptom of extreme atrophy of the soft tissues, including the mantle. However, the causative organism was uncertain. We isolated Vibrio sp. strain MA3 from the mantles of diseased pearl oysters Pinctada fucata. Analyses of 16S rRNA gene and DNA gyrase sequence homologies and its biochemical and morphological characteristics suggested that strain MA3 is a new strain of Vibrio alginolyticus. In addition, a hemolysin gene (Vhe1) of strain MA3 was detected as one of the virulence factors, and the complete sequence was determined. BLAST searches showed that Vhe1 shares 99.8% nucleotide sequence identity with Vibrio alginolyticus strain A056 lecithin-dependent hemolysin (ldh) gene, complete cds. Experimental infection of healthy oysters via injection with strain MA3 indicated it could cause high mortalities of the typically affected oysters from which the strain was isolated. These results suggest that the newly isolated Vibrio sp. strain MA3 is a putative causal agent of the recent disease outbreaks in Akoya pearl oysters.

Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors.

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, 4 µg/µL of myoglobin, 10 µg/µL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

Histopathological and electron microscopy studies on sleepy disease of koi Cyprinus carpio koi in Japan.

Koi sleepy disease (KSD) usually occurs when koi Cyprinus carpio koi are taken from nursing earthen ponds and placed in cement-lined ponds containing fresh water in spring and autumn in Japan. We transfered koi from an earthen pond to tanks containing fresh water and 0.5 % salt water in an attempt to replicate KSD and prevent the onset of KSD, respectively, in the laboratory. KSD broke out after 4 to 5 d, followed by mass mortality (76%: 95/125 fish) within 17 d, in the fresh water. Diseased fish died within a few days. Examination revealed enlarged cells in the respiratory epithelia of gill lamellae; hyperplasia of interlammellar epithelia resulted in clubbing of gill filaments, which caused hypoxia when severe. Electron microscopy showed that enlarged cells contained immature particles (416-450 nm diameter) or mature virions (333-400 x 400-413 nm) of a pox-like virus in the cytoplasm. Mature virions were transported to the periphery of the cells. Hepatocytes, renal tubular epithelial cells, muscle cells of the lateral musculature and cardiac muscle cells were cloudy with mitochondrial degeneration. PCR assay using primer sets for a pox-like virus causing 'carp edema' determined that KSD-virus is the same as the carp edema virus. None of the koi held in 0.5% salt water showed sleepy disease during a 25 d experimental period; PCR assay revealed no KSD-virus in gills of koi in the same treatment.