Effects of lactic acid bacteria on the microflora in water and larvae guts of Japanese flounder (Paralichthys olivaceus )
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Japanese flounder (Paralichthys olivaceus) larvae were reared on feed supplement with Lactobacillus rhamnosus P15 alone and flavomycin. After 60 days of feeding, the total numbers of aerobes, vibrios, and lactic acid bacteria (LAB) were measured by plate colony count. The result showed that the numbers of LAB rose in both culture water and intestine of the flounder administrated with suspended and lyophilized bacteria. After 30 days, LAB colonized in the flounder gut with stable number. Meanwhile, the number of vibrios reduced, especially in the flounder gut, owing to the inhibitory effect of LAB. LAB had no effects on aerobes in culture water and Japanese flounder gut. The influences of LAB on the microflora in culture water and Japanese flounder gut were similar to those of flavomycin. It was suggested that LAB additive could be used in Japanese flounder culture as substitute for antibiotics.Keywords:
Olive flounder
Paralichthys
Lactobacillus rhamnosus
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Paralichthys
Olive flounder
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Two bacteria strains were isolated from emaciated olive flounder (Paralichthys olivaceus) in aquafarm and were identificated as Vibrio harveyi (JV1) and Edwardsiella tarda (JE1), respectively. In the challenge experiments, we found 100% cumulative mortalities in all of olive flounder injected with JV1, JE1 or JV1+JE1 within eleven days after the injection. Two bacteria strains were reisolated from dead fish and were analyzed using the PCR method. In the physilogical analysis, the hematocrit, AST, ALT and cholesterol levels in experimental groups were increased significantly compared to those in control group, but the glucose, total protein and triglyceride levels were significantly decreased. Additionally, the lysozyme activity in the blood serum was decreased. The histopathological observations of the intestine showed that all groups had detachment and destruction of epithelial tissues except for the control group.
Paralichthys
Olive flounder
Edwardsiella tarda
Vibrio harveyi
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Abstract Three bacterial isolates obtained from diseased olive flounder larvae, Paralichthys olivaceus , were identified as Vibrio ichthyoenteri based on the results of phenotypic characterization and 16S rRNA gene sequencing studies. Bacterial enteritis was reproduced in 16 and 22 days post‐hatch (dph) larvae by administering brine shrimp nauplii, Artemia salina , dosed with the environmental isolates and reference strains of V. ichthyoenteri . To investigate the effect of the disease on development of the stomach, a pepsin activity assay and reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of the expression of the pepsinogen gene were performed. Expression of olive flounder pepsinogen was detected from 30‐dph larvae and the increased level of pepsin activity coincided with reduced susceptibility to the disease. Growth rates of V. ichthyoenteri , V. anguillarum and Edwardsiella tarda were tested in artificial stomach conditions using HCl and porcine pepsin. All the strains of V. ichthyoenteri were inhibited by low pH conditions which corresponded with an increase in pepsin levels. This suggests that differentiation of the stomach in olive flounder larvae and juveniles, an essential physiological development, also provides the host with a non‐immunological defence mechanism.
Paralichthys
Olive flounder
Vibrio anguillarum
Pepsin
Brine shrimp
Edwardsiella tarda
Litopenaeus
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The diseased larvae of the flounder, Paralichthys olivaceus, were sampled several times from a fish hatchery located in Cheju Island between December, 1991 and April, 1992. They turned out to be infected with Vibrio sp. and diagnosed as intestinal necrosis of flounder larvae(INFL) based on morphological, biological, and biochemical examinations. INFL was known to be caused by the live food organism infected with Vibrio sp. The optimal growth conditions of the isolates for temperature, pH, and NaCl concentration were , 6~8, and 2~4%, respectively. In the drug sensitivity test, the isolates were sensitive to oxytetracycline, nalidixic acid, kanamycin, and novobiocin, but resistant to ampicillin, erythromycin, spiramycin and sulfa-drug.
Paralichthys
Olive flounder
Kanamycin
Vibrio anguillarum
Niclosamide
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Investigations on the aerobic bacterial flora in the intestine of larval and juvenile stages in Japanese flounder (Paralichthys olivaceus) were carried out at two different fisheries stations. The bacterial flora of ambient water and feeds were also analyzed. The number of bacteria in the intestine of larvae measuring 10 mm in total length was 105 CFU/fish, remained nearly constant until 14 mm, and decreased to 104 CFU/fish in 15-19 mm. The decrease in bacterial numbers was hypothesized to be due to the change in feeds from live diets (rotifer and brine shrimp) to artificial diets. The bacterial numbers on ZoBell's 2216e agar were 104, 108, 105, and 106 CFU/ml or g in water, live feeds, artificial feed and minced fish, respectively.The composition of intestinal flora was characterized by two predominating groups of Vibrio and Pseudomonas followed by Moraxella, Cytophaga and Alcaligenes. Similar generic composition was recovered in water and live diets, while those of artificial diets comprised of Acinetobacter and Gram-positive bacteria in addition to Moraxella. Vibrio alginolyticus was frequently isolated from fish examined in one station where no apparent fish mortality occurred.
Paralichthys
Cytophaga
Vibrio alginolyticus
Olive flounder
Brine shrimp
Moraxella (Branhamella) catarrhalis
Juvenile fish
Pagrus major
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Haliotis discus
Facultative
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Paralichthys
Olive flounder
Streptococcus iniae
Hatchery
Vibrio anguillarum
Fish hatchery
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The characteristics of a bacterium strain M3, isolated from cultured flounder Paralichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the species Vibrio anguillarum . Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic for Paralichthys olivacues . The LD 50 dose was 5.144×10 3 CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell free supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease.
Paralichthys
Olive flounder
Vibrio anguillarum
Asymptomatic carrier
Pathogenic bacteria
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Many bacteria producing anti-viral substances were isolated from the aquatic environment. Fish intestinal bacteria such as Aeromonas spp. and Vibrio spp. producing anti-viral substances were isolated from intestinal contents of masu salmon (Oncorhynchus masou), Japanese flounder (Paralichthys olivaceus) and barfin flounder (Verasper moseri). These Aeromonas strains produced anti-infectious hematopoietic necrosis virus (IHNV) substances and Vibrio strains showed anti-IHNV, Oncorhynchus masou virus (OMV) and barfin flounder nervous necrosis virus (BF-NNV) activities. When Aeromonas spp. strains M-26 and M-38 were mixed with food pellets and fed to rainbow trout (O. mykiss) and masu salmon, both bacteria became dominant in the intestinal microflora and anti-IHNV activity was observed in homogenates of intestinal contents. These rainbow trout and masu salmon fed the Aeromonas spp. showed more resistance to the artificial IHNV challenge test. Barfin flounder fed Vibrio sp. strain 2IF6a with Altemia salina showed the anti-OMV and BF-NNV activities in the intestinal contents. Larvae fed the Vibrio sp. showed a higher survival rate than the fish cultured using the virus free sea water.
Paralichthys
Olive flounder
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Turbot(Scophthatmus maximus) breeding,especially the larval breeding,has been seriously affected by the bacterial diseases that were caused by the interactions of fish,pathogens and the environment.The gastrointestinal flora is the dominant location for the occurrence of diseases.This study is to analyze the gastrointestinal microflora of turbot larvae and expound their formation and succession.Using conventional bacteriological culture techniques,we isolated and quantified the bacteria from the gastrointestinal tract of turbot larvae,tank inlet water and feed in a turbot breeding farm.The dominant strains were identified by sequencing their 16 S r DNA for alignment with NCBI 16 S r DNA sequences database.The results showed that the total bacteria count in turbot larvae GI tract was increased and then decreased from Day 5 to Day 36.From Day 17 to Day 26,the culturable bacteria in GI tract were approximately 105-106 CFU/g,which showed extremely significant difference compared to the other stages(P0.01).Similarly,vibrio counts were increased initially and then tended toward stable.Day 17 is the cutoff for the significant differences(P0.05).Ultimately,the numbers of total bacteria and vibrio remained stable at 104 CFU/g in GI tract.Naturally,Vibrios were the dominant strains in the GI tract of turbot larvae.These results indicated that the dominant bacterial strains in the GI tract changed significantly at different developmental stages of turbot larvae.The bacteria in biological feeding could obviously impact the microflora formation in GI tract in the early stage of turbot,and that Vibrio ichthyoenteri was the dominant strain in the GI tract.
Vibrio anguillarum
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