Anemia of unknown etiology has been frequently observed in Japanese flounder Paralichthys olivaceus among both wild and cultured populations in recent years. In the present study, an epizootiological research on the anemia was conducted in wild flounder (416 fish) captured in 10 different coastal waters from Hokkaido to Kyushu from February 1999 to December 2000. As a result, anemia was observed in the fish captured in all coastal waters except for Hokkaido, beeing characterized by low hemoglobin levels, low erythrocyte numbers and a frequent appearance of immature erythrocytes. In these anemic flounder (130 fish), the blood-feeding monogenean Neoheterobothrium hirame and/or its vestiges were observed at a high prevalence rate (117/130=90%) on the buccal cavity wall. There was a negative correlation between the number of adult parasites and hemoglobin levels. These results suggest that the anemia was caused by the hematophagia of N. hirame.
Heavy mortalities due to luminescent vibriosis have been observed among pond-cultured Penaeus monodon shrimp in the Philippines. The species composition of luminescent Vibrio associated with mortalities was determined. A total of 189 luminescent bacteria isolated from the hepatopancreas of affected shrimps, rearing water and seawater from different shrimp farms in 11 provinces of the Philippines were examined for their morphological, physiological and biochemical characteristics. Results revealed a varied composition of Vibrio species. The most dominant luminescent Vibrio species was V. harveyi (65.5%) followed by V. logei (7%), Photobacterium sp. (6%) and V. orientalis (1%). Some isolates, based on their characteristics, were identified as V. campbellii (16%), V. mediterranei (3%), V. fluvialis (0.5%), V. cholerae (0.5%) and V. splendidus II (0.5%), which are known as non-luminescent Vibrios. V. harveyi is thought to be the major etiological agent associated with the luminescent vibriosis in pond-cultured P. monodon and its pathogenicity was confirmed through intramuscular injection to shrimp. Experimental infection showed that V. campbellii was also pathogenic to P. monodon.
Blood and kidney leukocytes were purified from two-year-old rainbow trout carrying infectious pancreatic necrosis virus (IPNV) and the cells were processed to detect the virus by two methods : 1) indirect immunofluorescence stain followed by flow cytometry analysis, and 2) RT-PCR or nested-PCR amplification. These methods were compared with separate homogenization of visceral samples, followed by inoculation on cell cultures and seroneutralization, a procedure routinely employed by most disease diagnostic laboratories. IPNV was isolated by homogenization of samples in all seven specimens examined. The assay took between 7 and 28 days required for the appearance of cytopathic effects, which is usual in subclinical samples. From purified leukocytes, IPNV was detected by RT-PCR in five out of the seven specimens. The nested-PCR improved the sensitivity of the assay and gave positive results for all the fish. In addition, flow cytometry demonstrated the presence of IPNV in all the fish in 8-24 h. Examination of leukocytes is a practical way of detecting IPNV and much less time-consuming than the homogenization technique.
A new disease with high mortalities has been occurring in summer scince 1998 in different ages of carp Cyprinus carpio cultured in Korea. The clinical signs of the disease included erratic swimming of moribund fish at the water surface, respiratory distress, presence of thick and foggy mucus covering the body and gills. Dermal ulcers were also noticed in a few cases. Microbiological, parasitological and virological investigations were carried out on the fish samples collected from various localities. Although bacteria and a protozoan parasite were recovered from some fish, their occurrences were not consistent. Histologically, the kidney and spleen showed discernible changes. Inoculation of tissue filtrates from affected fish to various fish cell lines showed cytopathic effects only in FHM. Ultra-thin sections of the infected FHM cells under an electron microscope revealed distinct cytoplasmic virus-like particles with a diameter of 70-80 nm. Immersion infection trials, using a viral suspension of 103.0TCID50/mL obtained from the infected FHM cells, produced 100 and 80% mortalities in Israel carp and common carp, respectively. This is the first epizootic of viral disease reported among cultured carp in Korea.
For a survey on fish pathogenic viruses among wild Japanese flounder Paralichthys olivaceus, 274 fish (body weight : 29-2240 g) were collected in 9 coastal areas of Japan in 1999 and 2000. Aquabirnaviruses were isolated, using RTG-2 and/or SSN-1 cells, from 111 fish (40.5%) caught in almost all the coastal areas and the representative isolate was identified as YTAV (yellowtail ascites virus) by cross-neutralization tests. Besides aquabirnaviruses, VHSV (viral hemorrhagic septicemia virus) was isolated, using EPC, SSN-1 and/or FHM cells, from 18 fish (6.6%) collected in 2 areas mainly in winter and spring. In a pathogenicity test, 4 isolates of aquabirnaviruses did not cause any mortality or morbidity in young Japanese flounder (average body weight 8.7 g) by intramuscular injection at 103.0-107.6 TCID50/ fish. Inoculation of an isolate of VHSV at 102.0 and 106.0TCID50 /fish produced mortalities of 60% and 100%, respectively, in flounder (24.8 g). Common external signs of flounder experimentally infected with VHSV were dark coloration of the body with hemorrhagic fins and abdominal swelling, and the internal signs included ascites and extensive hemorrhage in the muscle and/or viscera.
We report green fluorescent protein (GFP) as a useful reporter molecule to track the occurrence of Pseudomonas plecoglossicida in ayu Plecoglossue altivelis. The gfp gene was placed in a shuttle vector pME4510 and then three kinds of GFP vectors, pSKL01, pSKT03 and pSKN04 were constructed, in which lac, tac, and neophosphotransferase II (npt2) promoters drove the expression of GFP, respectively. A promoterless GFP vector was designated as pSKP02. P. plecoglossicida carrying pSKT03 gave the highest expression of GFP. In addition, pSKT03 was relatively stable under non-selective pressure and there was no significant changes in the in vitro growth and pathogenicity of the cells carrying this plasmid. The cells of fluorescent P. plecoglossicida attaching to the body surface of ayu were easily detected under a fluorescence microscope. It was revealed that they adhered predominantly to the microscopic injuries in the skin and fins.
To investigate the course of Streptococcus iniae infection in Japanese flounder Paralichthys olivaceus, fish (average weight 118±14 g) were experimentally infected by oral and bath methods, and the distribution and multiplication of S. iniae in the fish were monitored by bacteriological and immunohistochemical examinations. S. iniae was detected first in relatively high numbers in the kidney and spleen. Viable counts of S. iniae in the blood, brain, liver, stomach, intestine, gill, skin mucus and nares were high only when those in the kidney and spleen were high. S. iniae-laden phagocytic cells were observed in the lumen of the blood vessels distributing in the organs and tissues in the initial stage of infection. Many fish showed hemorrhagic lesions on the fins, and the extracellular multiplication of S. iniae in the hemorrhagic fins was observed in the initial stage of infection. These observations were common among the fish challenged by either method. There was no evidence of the entrance of S. iniae through the stomach, intestine, gill, eye or olfactory pouches of nares. It was presumed that S. iniae entered directly from the water through the abrasive sites of fins and was disseminated by the blood circulation to cause systemic infection.
For clarification of the etiological agent of the anemia recently prevailing in wild and cultured populations of the Japanese flounder Paralichthys olivaceus, flounder were challenged with the monogenean Neoheterobothrium hirame. In the challenged flounder, the hematological characteristics similar to those in the recent anemia were induced. Moreover, when N. hirame were artificially removed from the anemic flounder produced by the challenge, the hematological conditions recovered to normal levels. These results suggest that the parasite itself can induce anemia in Japanese flounder and refute a possibility that the parasite acts as a vector of other pathogens. They also indicate that the removal of N. hirame from anemic flounder is effective as a treatment of the anemia.
In orderto develop a vaccine for bacterial hemorrhagic ascites caused by Pseudomonas plecoglossicida in cultured ayu Plecoglossus altivelis, the efficacy of oiladjuvanted vaccines was examined. Formalin-killed P. plecoglossicida bacterin emulsified with an oil-adjuvant (MONTANIDE-ISA711 or MONTANIDE-ISA763A) or with saline was intraperitoneally injected into fish. Intraperitoneal injection with virulent P. plecoglossicida was carried out 22 days and 52 days after vaccination. The relative percent survivals of vaccinated fish were 17-58% without any adjuvants, 57-92% with ISA711 and 65-86% with ISA763A. The adjuvants remained in the body cavity of vaccinated fish for at least 65 days.
The viral etiology of mass mortalities of groupers, Epinephelus coioides and E. akaara, cultured in the People's Republic of China was examined. Disease outbreaks occurred in 7 to 45 day-old fish with erratic swimming motion and marked vacuolation was observed in the brain and retina of the affected fish. The piscine nodavirus (the Betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues by electron microscopy, indirect fluorescent antibody test and reverse transcription-polymerase chain reaction. This paper is the first record of the agent in the People's Republic of China.