Teruo Miyazaki

Histopathology, Biochemistry, and Pathogenicity of Vibrio harveyi Infecting Black Tiger Prawn Penaeus monodon

Abstract Vibrio harveyi infection was found to occur in pond-reared black tiger prawn in Thailand. The diseased prawns with a hepatopancreatic infection of V. harveyi showed bacterial invasions and multiplication in the tubular lumens. This condition was followed by necrosis of hepatopancreatic cells and the thickened basal lamina, subsequent granulomatous encapsulation of the invaded tubules, and production of granulation tissue around granulomatous lesions. Heavy bacterial multiplication in the hepatopancreatic tubules caused systemic bacterial dissemination, which resulted in marked necrosis in the heart and lymphoid organ. On the other hand, two prawns with percuticular infection by V. harveyi had bacterial invasions in the subcuticular, spongy connective tissue in the telson, and systemic dissemination was followed by the occurrence of bacteriaphagocytizing hemocytes in the various tissues. Both types of isolates (chitinase positive and negative) were moderately pathogenic to prawn; intramuscular inj...

Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries

Summary. Tropical iridovirus infection causes severe epizootic resulting in mass mortalities and large economic losses in freshwater ornamental fishes cultured in Southeast Asian countries, in wild fish seedlings captured in South China Sea, and in marine fishes farmed in Japan, Singapore, and Thailand. All of tropical iridovirus-infected fishes histopathologically showed the systemic formation of inclusion body-bearing cells and necrosis of virus-infected splenocytes and hematopoietic cells. We designed primer sets for the ATPase gene and the major capsid protein (MCP) gene and sequenced the PCR products derived from 5 iridovirus isolates from sea bass in South China Sea, red sea bream in Japan, brown-spotted grouper with a grouper sleepy disease in Thailand, dwarf gourami from Malaysia and African lampeye from Sumatra Island, Indonesia. The ATPase gene and the MCP gene of these 5 viral isolates were highly homologous (> 95.8%, > 94.9% identity, respectively) and the deduced amino acid sequences of the ATPase and the MCP were also highly identical (> 98.1%, > 97.2% identity, respectively). Based on the high homology, these 5 isolates of tropical iridovirus from various fishes in geographically different regions were determined to have a single origin and to be native to Southeast Asian regions. However, these sequences were far different from those of members of the genera Ranavirus, Lymphocystivirus and Iridovirus in the Family Iridoviridae. We propose a new genus “Tropivirus” for tropical iridovirus in the Family Iridoviridae.

Responses of Japanese eels to oral challenge with Edwardsiella tarda after vaccination with formalin-killed cells or lipopolysaccharide of the bacterium.

Abstract Vaccination with formalin-killed cells (FKC) or the lipopolysaccharide (LPS) of Edwardsiella tarda was evaluated for its effectiveness against edwardsiellosis in orally challenged Japanese eels Anguilla japonica. The vaccination procedure was an intramuscular injection of 10 mg FKCs/ 100 g body weight or 1 mg LPS/ 100 g body weight, and a booster of the same type and dose 7 d later. The oral challenge with viable E. tarda was performed with 105-106 colony-forming units per fish 21 d after vaccination. At this time, agglutination titers of serum antibody ranged from 1:1,280 to 1:2,560 in the FKC-vaccinated fish and from 1:3,840 to l:5,120 in the LPS-vaccinated fish. In the FKC experiment, mortality of vaccinated fish ranged from 60 to 87.5%, whereas the unvaccinated control group had 80–100% mortality; these results indicated slight efficacy of the vaccine. The FKC-vaccinated fish showed bacterial phagocytosis by macrophages, a process that contributes to a slight decrease in mortality. In the LPS...

Histopathological studies on Vibrio harveyi– infected tiger puffer, Takifugu rubripes (Temminck et Schlegel), cultured in Japan

Vibrio harveyi infection occurred with a moderate mortality in tiger puffer, Takifugu rubripes (Temminck et Schlegel), in autumn 2007, at a mariculture farm in western Japan. The diseased fish showed nodular lesions in the branchial chamber and the inner surface of the operculum. Histopathologically, the lesions comprised granulation tissue containing many suppurative foci allowing propagation of the bacteria and granuloma encapsulating abscesses with a decrease in bacteria. The bacteria were disseminated in visceral organs including the spleen, kidney, liver, and myocardium, resulting in the formation of granulomatous lesions. Two groups of tiger puffer juveniles were artificially infected by an intramuscular injection with an isolate (1.0_10(8) CFU/fish). During the experimental period, 20% mortality occurred within 4-6 days post-infection (d.p.i). The fish sampled on 4 d.p.i showed abscesses in the lateral musculature at the injection site. The fish sampled 5 d.p.i. displayed the production of granulation tissue containing many suppurative foci, which replaced the necrotic dermis and lateral musculature. Surviving fish (15 d.p.i.) had granulomatous lesions in the lateral musculature at the injection site. Pyogranulomatosis is pathognomonic in V. harveyi infection of tiger puffer.

Histopathology Associated with Two Viral Diseases of Larval and Juvenile Fishes: Epidermal Necrosis of the Japanese Flounder Paralichthys olivaceus and Epithelial Necrosis of Black Sea Bream Acanthopagrus schlegeli

Abstract Epidermal necrosis was detected in hatchery-reared larvae and juveniles of the Japanese flounder in 1986–1988 in Japan. Because healthy fish could be experimentally infected by immersion in an ultrafiltrate from diseased fish, it was concluded that the disease was caused by a viral infection. Diseased fish had white cloudy fins and body surfaces, as well as occasional ascites. Skin lesions contained necrotic epidermal cells that had intranuclear inclusion granules and displayed vacuolar degeneration. These damaged cells retained their interdigitations and adhered to one another to form papillate cell aggregates in the epidermis. Necrotic cells examined by electron microscopy contained intranuclear and intracytoplasmic viral particles that displayed features of a herpesvirus. A different disease, epithelial necrosis, occurred among hatchery-reared larval black sea bream in 1987. Fish became experimentally infected with the disease organism by immersion in an ultrafiltrate of naturally infected lar...

Vitamin A Deficiency in Cherry Salmon

Abstract Cherry salmon Oncorhynchus masou (also called amago salmon) were fed a diet with no vitamin A, and control fish were fed the same purified diet but with 5,000 IU retinyl acetate/ 100 g of diet. After 15 weeks of feeding, the fish deprived of vitamin A displayed nutritional deficiency signs, including reduced growth, pale body color, red fins, and truncated snout. The group of vitamin A-deficient fish had 5.6% mortality by the end of 22 weeks, whereas the control group had none. Hematological examination revealed severe anemia. Histological examination revealed separation of hepatic laminae and formation of thin collagen fibers between the separated laminae, atrophic and necrotic lesions in the hepatic parenchyma, numerical atrophy in the hematopoietic tissue and compensatory production of fibroadipose tissue, poorly formed bony tissue in the skull and vertebral protuberance, degeneration of skeletal and cardiac muscle fibers, numerical atrophy of pancreatic acinar cells, and thinning of the epide...

The effect of water temperature on rhabdoviral dermatitis in the Japanese eel, Anguilla japonica Temminck and Schlegel

Abstract Rhabdoviral dermatitis has been occurring in the Japanese eel, Anguilla japonica Temminck and Schlegel during the post-harvest stocking stage when the eels are kept in cages that are sprayed with well water (about 15°C) while this disease never occurred in the warm water, farming ponds. This study evaluated the thermal effect on the outbreak of this disease using a pathogenic rhabdovirus that was isolated from the cutaneous lesions. In in vitro tests, this virus successfully replicated in the EPC cell line at temperatures of 15°C, 20°C and 25°C showing titer of 10 8.05 TCID 50 /ml after the 5th day post-inoculation at 25°C while 10 8.55 TCID 50 /ml on the 9th day at 15°C. In vivo, eels were inoculated by an intracutaneous injection at viral levels of 10 4.0 , 10 6.4 and 10 8.4 TCID 50 /ml/fish and placed at three different water temperatures at 15°C, 20°C, and 25°C. At 15°C, inoculated fish became moribund and displayed cutaneous lesions accompanied by necrosis of the dermal fibrocytes, hemorrhage and inflammatory cellular infiltration, and diffuse necrosis of the hematopoietic tissue, splenic pulps and hepatic parenchyma. At 20°C, only one inoculated fish at 10 8.4 TCID 50 /ml was moribund and displayed dermal lesions and splenic necrosis. At 25°C, there were no moribund fish.

DNA cloning and characterization of an allograft inflammatory factor-1 homologue in red sea bream (Chrysophrys major)

We isolated allograft inflammatory factor-1 (AIF-1) homologue cDNA from lipopolysaccharide (LPS)-stimulated red sea bream leukocytes. We then determined that this cDNA encodes 444 bp in length and has an open reading frame that encodes 142 amino acids. The deduced amino acid sequence was homologous to the AIF-1s in carp and mammals (>65.5% sequence identity). The nucleotide sequence of the protein-coding region in the AIF-1 gene was determined to have six exons and five introns. The exon–intron structure resembles those of the mouse and the human. Genomic Southern blot analysis revealed the presence of a single AIF-1 gene per haploid in red sea bream. A time course evaluation indicated that the expression level of AIF-1 gene in LPS-stimulated leukocytes was static in the first hour, but gradually increased from 3 to 24 h. The similarities between AIF-1s in mammals and red sea bream suggest that the AIF-1 in red sea bream may have a similar function in activated leukocytes as AIF-1s do in mammals.

Histopathological and ultrastructural features of enlarged cells of humpback grouper Cromileptes altivelis challenged with Megalocytivirus (family Iridoviridae) after vaccination

The genus Megalocytivirus in the family Iridoviridae encompasses isolates of red sea bream iridovirus (RSIV) and grouper sleepy disease iridovirus (GSDIV). In the present study, humpback grouper Cromileptes altivelis juveniles were challenged with GSDIV after vaccination with a commercial RSIV vaccine. The unvaccinated group (in duplicate) showed higher mortalities (59.3 to 66.7%) than the vaccination group (0% mortalitiy, in duplicate). Surviving fish in the vaccinated group displayed masses of enlarged cells in the spleen. Electron microscopy revealed that they contained hemosiderin granules within the cytoplasm. In contrast, moribund fish from the unvaccinated group exhibited large numbers of inclusion body-bearing cells (IBCs) in the spleen, while surviving fish displayed masses of enlarged cells in which a small number of GSDIV virions were assembled.