An organism, measuring 0.86±0.32×0.63 ±0.24 μm, likely to be member of family Rickettsiaceae, was detected for the first time from an outbreak of a disease with mass mortality among pond-reared tilapia in Taiwan. The pathogen was demonstrated to be Gram-negative and stained positively with Liu's solution and to grow on To2 and EPC cell cultures but not on nine artificial media selected and the pathogenicity was confirmed by Koch's postulates. Haematocrit in affected fish was decreased to 16.1±5.2% versus 28.3±5.1% for normal value. Typical lesions in moribund fish included marked white nodules and microscopical granulomatous formation all over the organs. The spleen enlarged to 5-50 times of the normal organ. Horizontal transmission without parasite vectors occurred. The experimental infection showed higher mortality at 15°C than at 30°C. In vitro experiments revealed that oxytetracycline which is legally permitted would probably be an effective antibiotic to control this disease. A field test showed that oral administration of oxytetracycline at a dose of 30-50 mg per kg body weight for 10-14 days was effective to control the disease. Clinical signs, susceptibility of various fishes to the pathogen and transmission electron-microscopy findings are described.
The inhibitory effects of chitosan on germination and production of zoospore, and growth of vegetative hyphae of Saprolegnia parasitica Coker were examined. Zoospore germination was completely inhibited by chitosan at the concentration of 0.06%. Mycelial growth was not observed on GY media containing 0.05% chitosan. Zoospore production from sporangium was also completely inhibited by 0.05% chitosan. The hyphae affected by chitosan were markedly shrunk and contracted.
Some hematological parameters and histopathological changes in the liver, spleen, kidney, gills, gut, skin, muscle and heart of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with virulent Vibrio anguillarum or with V. anguillarum extracellular products (ECPs) were compared. The number of circulating red blood cells, the hematocrit and hemoglobin values decreased in the fish injected with either bacteria or ECPs compared with controls. The erythrocyte sedimentation rate increased in both groups of injected fish; however, the serum protein levels were similar to those observed in control fish. Light and electron microscopic observations of samples obtained from fish injected with V. anguillarum cells or its ECPs revealed similar histopathological changes in most tissues. Both groups of fish showed degeneration and necrosis of hepatocytes, vasodilation of the glomerular capillaries, cloudy swelling and hyaline droplet degeneration of the epithelial tubular cells, edematous gills, desquamation of the intestinal mucosa and necrosis of the muscle fibers at the site of injection. However, the bacterium multiplied extensively in the kidney and spleen where it caused more acute alterations than ECPs. Based on these results, it was concluded that the extracellular products play an important role in the virulence mechanisms of V. anguillarum.
Experimental treatment by increasing temperature from 10°C to 16°C was carried out on juvenile coho salmon (Oncorhynchus kisutch) naturally infected with erythrocytic inclusion body syndrome (EIBS). Cumulative mortality of control fish kept at 10°C throughout the experimental period of 40 days was 57%. In contrast, that of experimental fish kept at 16°C for the first 23 days and then at 10°C for 17 days was 10%. The longer the period at which the fish were kept at the higher temperature (16°C), the lower the mortality. Experimental challenge with EIBS to the survivors of the temperature-increasing treatment revealed an increase in the resistance to reinfection of EIBS.
A disease due to lower fungus was found in pet fish, dwarf gourami (Colisa lalia). In almost all cases, swelling of the abdomen due to swollen internal organs without accumulation of ascitic fluid was characteristic external sign of infected fish. Histopathological examination showed fungal hyphae and many granulomas present in the internal organs and musculature. Fungi were isolated from the lesions and were identified as a member of the genus Aphanomyces. The fungus had high pathogenicity to goldfish (Carassius auratus). No oogonia were produced in hemp seed cultures. This fungal infection closely resembled in pathology the mycotic granulomatosis which is known in cultured ayu in Japan.
The serological characteristics of the strains of Cytophaga psychrophila isolated from different fishes in different areas were compared by the microtiter method. Included were 3 strains from coho salmon (Oncorhynchus kisutch) in Miyagi Prefecture, 4 from rainbow trout (Oncorhynchus mykiss) in Nagano and Tokyo Pref., 6 from ayu (Plecoglossus altivelis) in Tokushima and Wakayama Pref. In addition to these Japanese strains, 3 strains from coho salmon in USA were used as references. Three antisera were prepared with the type strain NCMB1947 from coho salmon in USA, FPC828 from coho salmon in Miyagi, and FPC840 from ayu in Tokushima. In the comparison of C. psychrophila strains by using these antisera, all were positively agglutinated by each of the antisera, indicating the presence of common antigen (s). On the basis of the absorption analysis with thermostable antigens, two distinct serotypes (O-1 and O-2) were recognized. The strains from coho salmon in Japan and USA belonged to serotype O-1. Serotype O-2 occurred only in Japan, and was distributed among rainbow trout as well as ayu.
Following severe outbreaks of a disease with dermal necrosis, several bacterial strains were isolated from sea bass (Dicentrarchus labrax) reared on French coasts. A phenotypic study revealed that the isolates closely resembled Flexibacter maritimus. Nine selected strains were compared to 8 F. maritimus reference strains from other geographical origins and to the type strain NCIMB 2154T. DNA base composition of the strains tested was in the G + C range of 30.6 to 32 mol%. A study on the DNA relatedness among the strains by the S1 nuclease DNA-DNA hybridization method showed that the 12 strains tested formed a tight genomic species with DNA homologies above 74%. The profiles obtained from API ZYM galleries, polyacrylamide gel electrophoresis of whole-cell proteins, and fatty acids analysis revealed that F. maritimus is a very homogeneous bacterial species. This is the first identification of this fish pathogen in France and its first isolation from sea bass.
A new rapid identification method of V. anguillarum from cultured fish was developed. A new selective and differential medium (V-1 medium) for Vibrio species was subjected to the primary isolation of Vibrio spp. from diseased cultured red seabream and yellowtail. Fifty strains, which were tentatively identified as the genus Vibrio, were examined on their species' levels by DNA homology values obtained from a quantitative colorimetric microdillution plate hybridization method. The identification of V. anguillarum was made when the homology values with type strain were higher than 75%. By using this method, 6 out of 50 strains of Vibrio spp. were identified as V. anguillarum. It was possible to identify V. anguillarum within 4 days from the primary isolation by this method consisting of selective isolation and DNA-DNA hybridization.
Beginning in the spring of 1993, a serious mortality among cultured kuruma shrimp, Penaeus japonicus occurred in Japan. The typical sign of this disease was white spots on the inside surface of the carapace. Challenge tests demonstrated that the causative agent was highly virulent. This was demonstrated by injection of a filtrered homogenate of the lymphoid organ obtained from diseased shrimp. A non-occluded bacilliform virus was found by electron microscopy in the lymphoid organ of both naturally and experimentally infected shrimps. The virion was bacilliform and surrounded by a virion envelope (a lipid bilayer membrane). The dimension of the virion was 83 nm in diameter and 275 nm in length. From these results, the bacilliform virus is considered to be the causative agent of the disease.
During the winter of 1988, a mass mortality occurred in the population of maricultured coho salmon, Oncorhynchus kisutch, in Shizugawa Bay, Miyagi Prefecture of Japan. Since the first outbreak, the disease was reported every winter at more than 10 coho salmon farms in the same area. Diseased fish were 100-1, 000 g in body weight and showed excoriations of fin, erosion and ulcers on the body surface and pale spots in the liver. Cumulative mortalities ranged from 10 to 31% in the epizootic periods. Necrosis of hepatocytes was the most evident histopathological change of the diseased fish. Numerous viral capsids having icosahedral shape were observed in the necrotic hepatocytes. A virus was isolated in CHSE-214 cells from spontaneously diseased fish which had the signs mentioned above. The isolant was identified as salmonid herpesvirus type 2 by morphological observation and serological tests. In infection trials, more than 90% of the coho salmon juveniles which received intraperitoneal injection of the isolant died. The virus was reisolated from all the dead fish showing signs similar to spontaneously affected fish. From these results, the virus was considered to be the causative agent of the disease.
From March to October in 1993, high mortality of more than 80% occurred among Kuruma shrimps, Penaeus japonicus (0.015-25.0 g in body weight) at farms in Hiroshima, Yamaguchi, Ohita, Kumamoto, Kagoshima and Okinawa prefectures in Japan. Abnormal red coloration or discoloration and white spot of the body were characteristic signs of the diseased shrimps.The high mortalities were noticed in all the shrimp farms that had imported juvenile Kuruma shrimps from China. This fact together with spreading pattern of the disease in shrimp farming areas suggested that the disease transmitted horizontally from the imported shrimps from China. No parasite or bacterium was found to be correlated with the disease. The disease was reproduced in healthy shrimps by injecting filtered (at 0.45μm) homogenate of lymphoid organ of spontaneously diseased shrimps, suggesting the disease was caused by a virus.
In 1993 very high mortalities of cultured kuruma shrimp, Penaeus japonicus, outbroke in Japan, forcing many shrimp farms to be temporarily closed. Moribund shrimp exhibited red coloration or discoloration and small white spots on the body. Degenerated cells characterized by hypertrophied nuclei, being stained homogeneously with hematoxylin, were observed in various tissues originated from meso- and ectoderm, such as cuticular epidermis (most frequently), connective tissue, lymphoid organ, antennal gland, hematopoietic tissue and nervous tissue. Hemocytic encapsulations, ranging from 20 to 50 μm in diameter, of necrotic cells were also frequently observed in many shrimp examined. No difference in histopathological characteristics between spontaneously diseased and experimentally infected shrimp was recognized. Moreover, the same histopathological changes were observed in a kuruma shrimp which was sampled from the imported package from China, sacrificed one day after arrival in Japan. Histopathological comparison of this disease with the already-known diseases of penaeid shrimp suggested that this is a new infectious disease in penaeid shrimp of which etiological agent might be a virus.
Mass mortalities presumably caused by an infectious disease of kuruma shrimp, Penaeus japonicus, occurred in many culture ponds in some western prefectures of Japan in 1993. The present study revealed the close resemblance in histological changes between spontaneously diseased and experimentally infected shrimp : degenerations of the cuticular epidermis, connective tissue and lymphoid organ. Electron microscopy revealed rod-shaped, enveloped, nonoccluded virus in nuclei in the cuticular epidermis of the stomach. The size of the nucleocapsid was 84±6×226±29nm. Empty capsid of 61nm in diameter and capsid originator of 52nm in diameter were also found in nucleoplasm. Although the virus shared certain morphological characteristics with various baculovirus species, it differed in that the nucleocapsid is not a true cylinder, which is also the case of the Polydnaviridae. The virus is temporarily named RV-PJ (rod-shaped nuclear virus of Penaeus japonicus) until its taxonomical position is ascertained by analyzing the structure of the genomic DNA.