Flounder Farms Research Articles

넙치 양식장 위해요소중점관리기준(HACCP) 적용모델 개발

We attempted to apply the HACCP system adopted in the food industry to a olive flounder (Paralichthys olivaceus) aquaculture farm to ensure the hygiene safety of farmed fish. In this study, HACCP system procedures, including HACCP team organization, critical point determination, establishment of standard limits etc., were established using Codex 12 procedures. To determine whether hazards were critical elements, we evaluated the likelihood and seriousness of each hazard element. The likelihood of residual medicine exceeding the acceptable limit in shipped fish products was demonstrated to be a critical hazard element. Management of the shipment stage was determined to be a critical control point (CCP). Checking the records for stock and release and maintaining a history of medicine use before shipping the fish were suggested as monitoring methods. The standards for acceptable residual medicine were set based on relevant laws and regulations, and then the limits were adapted and established for the CCP. An HACCP plan applicable to olive flounder farms was established.

Lactococcus lactis BFE920 activates the innate immune system of olive flounder (Paralichthys olivaceus), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large-scale field studies

The protective effect of a food-grade lactic acid bacterium Lactococcus lactis BFE920 against disease of olive flounder (Paralichthys olivaceus) cultivated on a large scale was studied. Initially, antimicrobial activity of L. lactis against several fish pathogens was evaluated in vitro; the probiotic showed strong antibacterial activity against Streptococcus iniae, Streptococcus parauberis and Enterococcus viikkiensis, and moderate activity against Lactococcus garviae. When olive flounders were fed for two weeks with experimental diets containing varying concentrations of L. lactis (1 × 106, 5 × 106, 2.5 × 107 and 1.25 × 108 CFU/g feed), all the experimental feed groups showed 68–77% survival upon challenge with S. iniae. A field-scale feeding trial with L. lactis dietary supplement was conducted in a local fish farm (n = 12,000) for three months, and disease resistance, innate immune parameters and growth performance were evaluated. The average weight gain and feed efficiency were increased up to 6.8% and 8.5%, respectively. At the end of the feeding trial, the olive flounders were challenged with S. iniae. The L. lactis-fed group was protected from S. iniae challenge with a 66% survival rate. This disease protection is due to the flounder's innate immunity activated by the L. lactis administration: increased lysosomal activities and production of IL-12 and IFN-γ. These data clearly indicated that L. lactis BFE920 may be developed as a functional feed additive for protection against diseases, and for enhancement of feed efficiency and weight gain in olive flounder farming.

한국 주요 양식종의 수산동물 폐사피해 모니터링

2012년 5월에서 11월 사이에 우리나라 넙치, 조피볼락, 무지개송어, 뱀장어, 흰다리새우, 전복 양식장에서 발생하는 폐사피해를 표본조사를 통해 모니터링하였다. 조사의 방식은 해당 양식장과 거래하고 있거나 관계를 맺고 있는 전국의 수산질병관리사 조사요원의 직접 현장 방문을 통한 면접과 표준 매뉴얼을 통한 진단을 통하여 실시하였다. 조사기간 중 넙치 양식장에서는 총 27.9%의 누적 피해율을 보여주었고, 이중 스쿠티카병, 연쇄구균병, VHS, 비감염성 원인에 의한 폐사, 비브리오병, 활주세균병의 순으로 폐사피해가 높은 것으로 나타났다. 조피볼락 양식장에서는 13.6%의 누적 피해율을 보여주었고, 아가미 흡충병, 연쇄구균병, 비브리오병, 활주세균병, 비감염성 원인에 의한 폐사 순의 폐사피해가 나타났다. 무지개송어의 경우 총 17.1%의 피해율을 기록하였고, 비감염성 원인에 의한 폐사와 백점병이 주된 폐사 원인으로 집계되었다. 뱀장어의 경우 비교적 낮은 0.6%의 피해율을 기록하였으며, 에드워드병, 백점병, 아가미흡충병이 주된 피해요인이었다. 흰다리새우는 71.2%의 피해율을 보였으며, 대부분 비감염성 원인에 의한 폐사으로 집계되었고, 바이러스성 흰반점병도 12.5%의 피해를 준 것으로 나타났다. 전복의 경우 10.7%의 피해율 모두가 비감염성 원인에 의한 폐사 피해인 것으로 조사되었다. The practical monitoring was performed to survey the mortalities in aquaculture farms of olive flounder, rockfish, rainbow trout, Japanese eel, white shrimp and abalone in South Korea from May to November, 2011. The aquatic organism disease inspectors, who have the national licenses for the diagnosis and prevention of aquatic disease and have close relationship with the farms, investigated the rates and causes of mortalities according to the standard manual. In 70 flounder farms, the cumulative mortalities rate was 27.9%, and the mortalities were caused by scuticociliatosis, streptococcosis, VHS, non-infectious loss, vibriosis and gliding bacterial disease. The moralities rate of 30 rockfish farms was 13.6%, and those were mainly contributed by gill flukes and streptococcosis. Most of mortalities of rainbow trout were caused by non-infectious loss and protozoan white spot disease. The mortalities rate of Japanese eel was 0.6% by edwardsiellosis, protozoan white spot disease and gill flukes. The loss rate of white shrimp was 71.2%, and most of them was related with non-infectious ones, such as carnivalization, transportation loss, and the rest was caused by viral white spot disease. The mortalities rate in the abalone farms was 10.7% and all of them were related with non-infectious loss.

Genetically similar VHSV isolates are differentially virulent in olive flounder Paralichthys olivaceus

Two viral hemorrhagic septicemia virus (VHSV) isolates, VHSV-KR-CJA and VHSV-KR-YGH, were isolated from viral hemorrhagic septicemia disease outbreaks in flounder farms in South Korea. The VHSV-KR-CJA isolate was isolated from a flounder farm with high mortality (80%), while the VHSV-KR-YGH isolate was isolated from a flounder farm with low mortality (15%), suggesting that these isolates differ in virulence. The virulence of these isolates was evaluated in juvenile flounder via intraperitoneal injection. Consistent with their virulence in the field, mortality data revealed that the VHSV-KR-CJA isolate was highly pathogenic (cumulative mortality of 80%), while the VHSV-KR-YGH isolate was less pathogenic in flounder (cumulative mortality of 20%). To characterize the genotypes of these viruses, the full open reading frames (ORFs) encoding nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L of these viruses were sequenced and analyzed. Sequence analysis revealed that both isolates are genetically very similar (identical amino acid sequences for P, M, NV, and L and >99.7 and 99.8% amino acid sequence identity for N and G, respectively). Phylogenetic analysis indicated that both of these viruses belong to the Genotype IVa group, suggesting that they originated from a common ancestral virus. The low pathogenicity VHSV strain may potentially evolve to become a more pathogenic strain through only a few nucleotide substitutions. Further functional analyses of mutations in VHSV genes are necessary to identify factors that determine VHSV pathogenicity in flounder.

Philasterides dicentrarchi, a histophagous ciliate causing scuticociliatosis in olive flounder, Philasterides dicentrarchi – Histopathology investigations

Philasterides dicentrarchi is a histophagous scuticociliate infecting Korean olive flounder farms in Jeju Island, South Korea, where it causes significant economic losses. However, the route of entry of these parasites in olive flounder is currently unknown. In the present study, we attempted experimental infection with different doses (concentrations) of P. dicentrarchi by intraperitoneal (IP) injection; maximum cumulative mortality of 95% was recorded on the administration of 2.1×108ciliates/ml. In small size group (3cm length, 24±2gwt) the ciliates were found in connective tissues of muscle, fins, and nervous tissue while in large size group (5cm length, 47±3gwt), the ciliates were predominantly occurred with associated symptoms of liquefaction in the central nervous system. The major clinico-pathological manifestations were loss of scales, appearance of bleached spots that coalesced to form brownish patches, hemorrhagic, and severe dermal necrotic lesion. In addition there was degeneration of muscle fibers, necrosis of the hepatic parenchyma, and severe edema of the intestinal walls, extensive fouling, necrotic degeneration and hyperplasia in the gill bronchial epithelium. In this study, many ciliates were found in the blood vessel, brain, dorsal fins, muscle, kidney, and vertebra of infected flounder. The histopathological investigations are of significant importance in view of possible routes of entry into the host and doses of ciliates that trigger fast infection for potential utility of drugs as a strategy for the control of P. dicentrarchi in farmed olive flounder.

Protection of olive flounder, Paralichthys olivaceus, against viral hemorrhagic septicemia virus (VHSV) by immunization with NV gene-knockout recombinant VHSV

The potential of the NV gene-knockout recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP) as a live attenuated vaccine was assessed. In an in vivo virulence experiment, olive flounder ( Paralichthys olivaceus) intramuscularly (i.m.) injected with 10 3 and 10 5 PFU of rVHSV-ΔNV-EGFP showed no mortality. The rVHSV-ΔNV-EGFP had ability to spread and replicate in the internal organs of olive flounder reared at either 15 °C or 20 °C. In the vaccination experiment, the cumulative mortality rates of olive flounder i.m. immunized with 10 3, 10 4, and 10 5 PFU of rVHSV-ΔNV-EGFP were 40–50%, 10–30%, and 0–10%, respectively, at 4 weeks after the primary immunization. In the second challenge experiment conducted at 4 weeks after the boost immunization, the cumulative mortality in fish immunized with 10 3 PFU of rVHSV-ΔNV-EGFP was 10–30%. No mortality was recorded in fish immunized with 10 4 and 10 5 PFU of the recombinant virus. While the cumulative mortality of fish in control group was 90–100%. The serum neutralization titers were also proportional to the survival rates of vaccine groups. The present results suggest that the rVHSV-ΔNV-EGFP has a high potential to be used as a live vaccine in olive flounder farms.

Detection of Japanese Flounder Antibody against Fimbrial Major Protein of Edwardsiella tarda

A recombinant fimbrial protein of Edwardsiella tarda was designed to detect the specific antibody in Japanese flounder Paralichthys olivaceus. The specific antibody was detected from the fish intraperitoneally injected with E. tarda expressing the fimbria on LB agar containing 3% NaCl, but not from those injected with E. tarda without the fimbria, Streptococcus iniae, S. parauberis or viral hemorrhagic septicemia virus. In a flounder farm suffered with edwardsiellosis, the antibody-positive fish increased in ration with the occurrence of mortality. These results suggested that E. tarda with the fimbria was important for epidemics of edwardsiellosis at flounder farms.

제주도 넙치 양식현장에서 배합사료 성장 평가

This study was conducted to evaluate extruded pellet (EP) diet compared with a raw fish moist pellet (MP) diet for flounder, Paralichthys olivaceus on field feeding experiments in Jeju area. The experimental EP diet (juvenile, growing and adult) was prepared based on the former studies on nutrient requirements and feed ingredient of flounder. Fish were distributed randomly to each aquarium as a group of 102,300 fish (initial mean weight 97 g) in field experiment I and reared randomly to each aquarium as a group of 40,000 fish (initial mean weight 120 g) in field experiment II. In field feeding experiment I conducted in commercial flounder farm, survival, wt. gain (94-1,090 g), feed efficiency and condition factor of fish fed experimental EP were comparable to those of fish fed MP, but considerably higher protein efficiency ratio were observed in fish fed experimental EP. In field feeding experiment II, no significant difference was observed in final mean weight of fish fed the experimental EP (1,234 g) and MP (1,286 g), and any noticeable problem caused by feeding the experimental EP was not found during the whole period of feeding trial. Survival of fish fed the experimental EP was lower than that of MP, and there was no significant difference in feed efficiency between the fish fed experimental EP and MP. Therefore, these results strongly suggest that diet EP could be developed to replace MP diet for the marketable size (1.3 kg) of production for flounder without adverse effects on growth performance. In particular, abdominal dropsy and reduction of condition factor, which are frequently occurred in commercial EP feeding farms, were not observed in flounder fed experimental EP.

Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40 ± 3.1% to 79.4 ± 1.7%) and it decreased from August to November (63 ± 2.3% to 30 ± 2.6%) in olive flounder farms at Jeju Island, South Korea during 2000–2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49 ± 7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50 ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100 ppm for 30 min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.

양식넙치에 경구투여한 Ciprofloxacin의 근육조직 잔류량을 이용한 휴약 기간 설정 연구

Ciprofloxacin is the most commonly used fluoroquinolones for treating bacterial disease in olive flounder (Paralichthys olivaceus) farming, but its withdrawal time for industrial-scale farming has not been established. The depletion of ciprofloxacin was investigated in the olive flounder under field conditions. Fish were kept in an inland fish farm and fed a commercial diet containing 5 mg/kg of ciprofloxacin for 13 days. Seven fishes per sampling time were examined during and after the treatment. Ciprofloxacin and its major metabolite, enrofloxacin, were analyzed using HPLC with a fluorescence detector. The concentrations of ciprofloxacin and enrofloxacin in muscle increased during the medication period, and then decreased rapidly. The ciprofloxacin and enrofloxacin concentration in the olive flounder peaked on days 11 and 13, respectively, with maximum concentrations in muscle of 0.58 and 0.73 mg/kg. Residual ciprofloxacin and enrofloxacin were eliminated rapidly; 6 days after treatment, the respective levels in muscle were 0.04 and 0.10 mg/kg and neither was detected 15 days post treatment. The level of ciprofloxacin accumulation at the beginning of oral administration was variable according to the farming conditions, but the overall exhaustion time was similar. In conclusion, an adequate withdrawal period for enrofloxacin is 15 days in the case of oral ciprofloxacin administration.

Transformation and expression of Paralichthys olivaceus growth hormone cDNA in Synechocystis sp. PCC6803

The cyanobacterium Synechocystis sp. PCC6803 was transformed with a constructed vector pZGH containing Paralichthys olivaceus (flounder) growth hormone (fGH) cDNA under the control of iron-stressed chlorophyll-binding protein ( isiA) promoter. Southern-blotting analysis was employed in verifying the stable integration of introduced DNA. Even after ten successions in medium without kanamycin, the fGH gene could still be detected in the transformed Synechocystis by PCR. The transcription of the foreign gene in induced transformed Synechocystis was tested by RT-PCR. Western-blotting analysis indicated that transformed Synechocystis could express fGH under induction and over 0.2 μg of fGH expression per mg wet transformed Synechocystis was estimated by ELISA–RA. In a bioactivity study, the growth of P. olivaceus was proved to be increased by transformed Synechocystis. At the end of the 49 day feeding trial, the growth rate of P. olivaceus fed with 0.5% transformed Synechocystis was 43% higher than that of the control and the conversion rate of feed declined from 2.0 to 1.6. Whereas Synechocystis at the dose of 2%, increased the growth rate of P. olivaceus by 55% over the control with a feed conversion rate of 1.2. Transformed Synechocystis in fish feed sounds an efficient way of enhancing growth and potentially will have positive impacts on flounder farming industry.

Vibrio harveyi and other bacterial pathogens in cultured summer flounder, Paralichthys dentatus

A monitoring program for Vibrio harveyi and other potential bacterial pathogens of summer flounder was conducted at two facilities in the Northeast United States. Bacterial samples were collected from larval and juvenile fish and live-feed, and identified using API 20E biochemical profiles and 16S rDNA sequencing. Histopathological examinations were conducted in order to relate histological changes with the presence of potential bacterial pathogens. V. harveyi, Vibrio ichthyoenteri, and Photobacterium damselae subsp. damselae, three known pathogens of flatfish, were isolated from diseased summer flounder. Although a high proportion of juvenile summer flounder showed microscopic signs of disease, the presence of these potential bacterial pathogens in fish was not associated with large-scale mortalities. An outbreak of flounder infectious necrotizing enteritis with 30% cumulative mortality occurred when juveniles were transported to a new facility. Isolates of V. harveyi from the disease outbreak were genetically different from the isolates from the commercial hatchery. V. harveyi isolates from both facilities were pathogenic to summer flounder by intraperitoneal injection. P. damselae subsp. damselae, V. ichthyoenteri, and Vibrio scophthalmi, also found in fish with gross lesions, were not pathogenic to juvenile summer flounder by intraperitoneal injection. Our research shows that several potential bacterial pathogens are associated with morbidity and mortality in summer flounder larvae and juveniles, especially in situations of stress. Increased knowledge about the environmental conditions that lead to disease, as well as the interactions of V. harveyi with other microbial species, could lead to the development of management strategies in summer flounder farms.

양식 넙치 (Paralichthys olivaceus)의 Enrofloxacin 휴약기간

Enrofloxacin (ENRO) is one of the most commonly used fluoroquinolones for treating bacterial disease in olive flounder (Paralichthys olivaceus) farming, but its withdrawal time for industrial-scale farming has not been well established. Withdrawal times of ENRO following oral administration were evaluated in olive flounder under field conditions. Fish were held in an inland fish tank and fed a commercial mediated diet containing 5 mg/kg of ENRO for 9 days. Seven fish per sampling point were examined during and after treatment. ENRO and its major metabolite, ciprofloxacin (CIP), were analyzed using high-performance liquid chromatography with a fluorescence detector. The concentration of ENRO and CIP in muscle increased during the medication period, and then decreased rapidly The sum of ENRO and CIP concentration in olive flounder peaked on day 6, with a maximal concentration in muscle of 4.30 mg/kg. ENRO residues were eliminated rapidly; at 10 days post treatment, the level in muscle was 0.10 mg/kg, but it took about 50 days to be reduced to below 0.1 mg/kg. After 60 days, the residual concentration was below 0.1 mg/kg in all samples. The level of ENRO accumulation at the beginning of oral administration was variable, according to the farming conditions, but the overall exhaustion time was almost the same. We concluded that an adequate withdrawal period of enrofloxacin is 60 days in the case of oral administration.

Effects of bile acids on proliferation and production of proteinase activity of Uronema marinum (Ciliophora: Scuticociliatida)

Little is known about the effects of bile acids in relation to infectivity on the biological characteristics of Uronema marinum, a serious opportunistic parasite of farmed olive flounder Paralichthys olivaceus. In this study, we examined the effects of bile acids on the proliferation of U. marinum and on proteinase production in vitro. Proliferation of U, marinum was significantly enhanced by lithocholic acid (LCA) at 30 and 60 pmol, and by chenodeoxycholic acid (CDCA) at 0.06 pmol. In contrast, a significant decrease in proliferation was observed with cholic acid (CA) at 30 and 60 micromol, and with deoxycholic acid (DCA) at all amounts used. Proteinase production from live U. marinum was significantly increased by LCA, whereas CA significantly decreased proteinase production. CDCA and DCA had no effect on proteinase production. Although the types and concentrations of bile acids in the faeces of olive flounder are not known, the present results suggest that bile acids in the culturing water might influence the proliferation and production of proteinases in U. marinum, resulting in an increased possibility of scuticociliatosis in olive flounder farms.

Ecological investigation of Streptococcus iniae in cultured Japanese flounder ( Paralichthys olivaceus) using selective isolation procedures

Streptococcus iniae is an important bacterial pathogen of Japanese flounder ( Paralichthys olivaceus). We investigated the prevalence of S. iniae in Japanese flounder and its cultural environment for two consecutive years in a flounder farm. Direct plating procedure using thallium acetate-oxolinic acid-blood (TAOAB) agar and enrichment culture in a selective broth (SB) followed by subculture on TAOAB agar were employed for detecting S. iniae from samples. Todd–Hewitt broth (1 l) containing 0.5 g thallium acetate, 10 mg colistin sulphate (CS), and 5 mg oxolinic acid (OA) was chosen as a selective broth. On most sampling dates except for those of low water temperature, S. iniae was detected from sediment and water samples of 1-year-old fish tanks. Detection rates of S. iniae for sediment and water samples from 0-year-old fish tanks were low from January, when rearing of fingerling was started in this farm, until the epizootic broke out. Detection rates of S. iniae for samples from 1-year-old fish tanks were always higher than those of 0-year-old fish tanks. S. iniae was detected also from water supply in high temperature months. A total of 15 out of 230 flounder sampled were S. iniae-infected as the pathogen was isolated from their brain and/or kidney. S. iniae was detected frequently from the gill and skin mucus of apparently healthy flounder. Broth enrichment procedure was suited for detection of S. iniae from samples of fish tissue and water supply. Direct plating procedure was suited for sediment samples. For water samples, both procedures should be employed.

Selective agars for the isolation of Streptococcus iniae from Japanese flounder, Paralichthys olivaceus, and its cultural environment.

Two kinds of selective agar were developed for the isolation of Streptococcus iniae, the causal agent of streptococcosis, from Japanese flounder (Paralichthys olivaceus) and from culture tanks in flounder farms. The selective agars were heart infusion agar with added thallium acetate and oxlinic acid (TAOA), and colistin sulphate and oxolinic acid (CSOA). For samples containing various bacterial flora, selective agars were supplemented with defibrinated horse blood in order to distinguish beta-haemolytic colonies of Strep. iniae. Streptococcus iniae was quantitatively isolated from the brain and kidney of diseased flounders in pure culture. Two-thirds of isolates picked up from selective blood agars inoculated with intestinal samples were identified as Strep. iniae. The bacterial colony numbers of deposits and water from culture tanks on selective blood agars were about 10-10(5) times smaller than those on control heart infusion agar; Strep. iniae was isolated from few deposit and water samples.

An Ecological Study on Edwardsiella tarda in Flounder Farms.

An ecological investigation on Edwardsiella tarda was carried out in two Japanese flounder (Paralichthys olivaceus) farms in Fukuyama City from April to December 1992. E. tarda was isolated from 86% of water, 44% of sediment and 14% of fish samples in one pond, and 22% of water, 0% of sediment and 2% of fish samples in the other pond, although epizootics caused by E. tarda were not observed in either pond. Serological studies proved that all the isolates of E. tarda from flounder and its environment shared a common heat stable O-antigen. Almost all the isolates tested exhibited pathogenicity to flounder by intramuscular injection. In survivability tests of selected isolates in sea water and freshwater, they perished within 7 days in sea water but survived longer period in freshwater.These results suggest that there is only one serotype of E. tarda which has pathogenicity to flounder in the environment of flounder farms.

ヒラメ養殖場におけるＥｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａの分布

ヒラメ養殖場におけるＥｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａの分布