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Olive flounder, Paralichythys olivaceus fed with pomegranate enriched diet and challenged with or without Philasterides dicentrarchi had a significantly higher white blood cell (WBC) count on weeks 2 and 4 than the infected group fed with non enriched diet (standard diet). Similarly the red blood cell (RBC) counts did not significantly change in control and treated fish on weeks 1 and 2. It was significantly increased in treated fish on week 2 when compared to the control. In both the groups the hemoglobin (Hb) and hematocrit (Ht) levels significantly increased on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly change at any time in both groups whereas mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) increased significantly on week 4 in the treated group. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu), and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in treated groups on week 2 or 4 as compared to the control. The scuticocidal activity and respiratory burst activity were significantly enhanced in treated groups with or without parasite on weeks 2-4. However, the serum lysozyme activity was significantly enhanced from weeks 1 to 4. The protective response in terms of cumulative mortality was low in groups fed with enriched diet against parasite when compared to control. Therefore, we suggest that pomegranate enriched diet following challenge with P. dicentrarchi restores the altered hematological and biochemical parameters, and improves the innate immune system in olive flounder against P. dicentrarchi., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study describes the effect of diet supplementation with Chaga mushroom, Inonotus obliquus extract at 0%, 0.01%, 0.1%, and 1.0% levels on the innate humoral (lysozyme, antiprotease, and complement), cellular responses (production of reactive oxygen and nitrogen species and myeloperoxidase), and disease resistance in olive flounder, Paralichythys olivaceus against Uronema marinum. The lysozyme activity and complement activity significantly increased in each diet on weeks 2 and 4 against pathogen. The serum antiprotease activity and reactive nitrogen intermediates production significantly increased in fish fed with 0.1% and 1.0% diets from weeks 1-4. However, reactive oxygen species production and myeloperoxidase activity significantly increased in 1.0% and 2.0% diets on weeks 2 and 4. In fish fed with 0.1% and 1.0% diets and challenged with U. marinum the cumulative mortality was 50% and 40% while in 0% and 0.01% diets the mortality was 85% and 55%. The results clearly indicate that supplementation diet with I. obliquus at 0.1% and 1.0% level positively enhance the immune system and confer disease resistance which may be potentially used as an immunoprophylactic in finfish culture., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 2.1×10(8) ciliates/ml. In small size group (3 cm length, 24±2 gwt) the ciliates were found in connective tissues of muscle, fins, and nervous tissue while in large size group (5 cm length, 47±3 gwt), 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present study was reported the effect of probiotics and herbals mixture supplementation diet on growth, blood constituents, and nonspecific immune response in olive flounder Paralichthys olivaceus against Streptococcus parauberis on weeks 1, 2, 4, 6, 8, 10, and 12 after injected intraperitoneally (i.p.) with 50 μl of PBS (phosphate buffer saline) containing S. parauberis (2.1 × 10⁷ CFU ml⁻¹). The initial weight did not significantly increased in supplementation diet group from 1 to 4 weeks, whereas it was significantly increased from weeks 6 to 12 as compared to fish fed without supplementation diet. The serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) activities significantly increased from weeks 4 to 12 in infected fish fed with supplementation diet compared to fish fed without supplementation diet. However, the total protein (TP) and glucose (GLU) levels were significantly increased in infected fish fed with supplementation diet after 6 weeks. The phagocytic, respiratory burst, complement, and lysozyme activities significantly enhanced in infected fish fed with supplementation diet from weeks 4 to 12 as compared to fish fed without supplementation diet. These results suggested that different probiotics and herbals mixture supplementation diet enhanced the growth, blood biochemical constituents, and nonspecific immunity in olive flounder against S. parauberis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effect of five probiotics, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus sakei, Bacillus subtilis, and Saccharomyces cerevisiae as individual and mixed enriched diet on the seasonal prevalence, activity and intensity of Uronema marinum infection in olive flounder Paralichthys olivaceus is reported. The growth performance, feed efficiency, blood biochemistry, survival rate, and non-specific immune response of U. marinum infected olive flounder on week 0, 1, 2, 4, 6, and 8 were quantified. The prevalence and infection intensity reached a peak from June to December and then it declined from December to March. The scuticocidal activity in the serum was significantly higher when fed with L. plantarum, L. acidophilus, and S. cerevisiae diets on weeks 2-8. All enriched diets significantly enhanced the weight gain significantly between week 6 and 8; the feed efficiency registered a significantly increase from week 4 to 8 when compared to infected fish fed with control diet. Infected fish fed with L. plantarum-supplemented diet had higher survival rate than with other enriched diets. The serum aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) levels significantly increased when fed with L. plantarum, L. acidophilus or S. cerevisiae-supplemented diet. Total protein (TP) and glucose (GLU) level significantly increased with any enriched diet from week 4 to 8. The superoxide anion production and serum lysozyme activity registered a significant increase when fed with L. plantarum, L. acidophilus, and S. cerevisiae-supplemented diet from week 4-8. The present study concludes that L. plantarum, L. acidophilus, and S. cerevisiae-supplemented diets act as immunostimulants enhancing the growth, feed efficiency, blood biochemistry, survival rate, and non-specific immune response in U. marinum infected olive flounder., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We investigated the effect of a mixed herb-enriched diet obtained from pomegranate Punica granatum, Dalmatian chrysanthemum Chrysanthemum cinerariaefolium, and mastic-leaved prickly-ash Zanthoxylum schinifolium on innate immune mechanisms (e.g., phagocytosis activity, respiratory burst activity, alternative complement activity, lysozyme activity, and disease resistance) of olive flounder Paralichthys olivaceus against the scuticociliate Philasterides dicentrarchi. All experimental groups were challenged with P. dicentrarchi (1 x 10(5) ciliates/mL) through intraperitoneal administration of the pathogen (50 microL) on day 1. On day 7, the infected groups were fed 0, 5, 50, and 100 mg/kg of the enriched diets. The innate immune parameters, cumulative mortality, and relative percent survival (RPS) were assessed at weeks 1, 2, 3, and 4. Administration of 50 or 100 mg/kg of the herbal-enriched diet enhanced immunity throughout the experimental period. However, at the 5-mg/kg dosage, the enriched diet did not enhance the innate immune estimates at any time. At doses of 50 and 100 mg/kg, administration of the diet preceding the challenge with P. dicentrarchi decreased the percentage cumulative mortality in the experimental groups and thereby increased RPS values. This study reports that administration of 50 or 100 mg/kg mixed herbal-enriched diet could positively influence the innate immune response to P. dicentrarchi and enhance the health status of olive flounder with respect to this microbe.

We report the effect of two probiotics, Lactobacil and Sporolac as separate or mixed diets on innate immune mechanisms, such as phagocytosis activity, superoxide anion production of blood leukocytes, complement activity and plasma lysozyme activity, and disease resistance in lymphocystis disease virus (LCDV) infected olive flounder, Paralichthys olivaceus (523.5 +/- 18.3 g) on week 1, 2, and 4. In infected fish, administration of diet supplemented with Lactobacil individually or mixed with Sporolac significantly enhanced the immune parameters like phagocytic activity superoxide anion production, complement activity, and plasma lysozyme. However administration of supplemented diet with Sporolac alone, all the chosen immune parameters did not enhance when compared to control group; this diets resulted in lower mortality (30% and 25%) than Sporolac diet group (45%) in 30 days. We conclude that Lactobacil individually or mixed with Sporolac act as immunostimulants that enhance the innate immune response and disease resistance in lymphocystis disease virus (LCDV) infected olive flounder., (Published by Elsevier Ltd.)

In olive flounder, Paralichthys olivaceus infected with lymphocystis disease virus (LDV) bath treatment with formalin, hydrogen peroxide or Jenoclean at 50, 100, and 200 ppm daily 10 min for 60 days enhanced the innate immune response and disease resistance. Jenoclean enhanced the immune parameters at the lowest concentration of 50 ppm; on the other hand, hydrogen peroxide bath treatment enhanced the immunity level at 100 ppm, while formalin was effective only at 200 ppm. A low cumulative mortality and high relative percent survival was noted in Jenoclean treated group followed by formalin and hydrogen peroxide treated groups. In olive flounder at low concentrations of 50 ppm or 100 ppm hydrogen peroxide and Jenoclean effectively prevent LDV infection., (Published by Elsevier Ltd.)

We report the effect of aqueous, ethanol, and methanol solvent leaf extracts of Punica granatum on innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity, lysozyme activity and functional immunity in terms of percentage cumulative mortality and Relative Percent Survival (RPS) in olive flounder Paralichthys olivaceus naturally infected with lymphocystis disease virus (LDV) after 8 weeks. Infected fish were intraperitoneally administered with 0, 5, 50, and 100 mg kg(-1) body weight of solvent extracts. In groups treated with 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters significantly increased after 8 weeks when compared to 0 mg kg(-1) dose, but not with 5 mg kg(-1). Administration of P. granatum solvent extracts for 8 weeks significantly reduced the percentage mortality with the consequent increase in RPS. The results suggest that intraperitoneal administration of the leaf extracts of P. granatum at 50 or 100 mg kg(-1) dose clearly enhance the innate immune responses and disease resistance after 8 weeks in P. olivaceus against natural LDV infection., (Published by Elsevier Ltd.)

We report the growth, innate immune response, and disease resistance in olive flounder (Paralichthys olivaceus) challenged with Streptococcus iniae after feeding with diet enriched with Zooshikella sp. strain JE-34 three different concentration i.e. Low (3.4 x 10(4), n = 50), medium (3.5 x 10(6), n = 50), and high (3.4 x 10(8), n = 50) cfu ml(-1) supplemented diets, the changes were monitored on weeks 1, 2, 4, 8, 12, and 16. With all diets the innate immune parameters, such as superoxide anion production, phagocytic and lysozyme activity were not enhanced on week 1 and 4. On the other hand, all tested immune parameters in the treated groups significantly enhanced after 8th week; the weight gain significantly increased after 4th week in fish fed with enriched diets. The mortality in olive flounder after administration with high concentration diet showed 25%. With low and medium enriched diets the mortality was 40% and 35%, respectively. In the infected untreated group mortality was 85% while there was no mortality in the control group. The results suggested that Zooshikella sp. strain JE-34 enriched diets could be used to enhance the innate immune response and disease resistance of P. olivaceus against S. iniae., (2010 Elsevier Ltd. All rights reserved.)

Scuticociliatosis caused by about 20 species belonging to the Phylum Ciliophora has been recognized as an emerging problem inflicting significant economic loss in aquaculture industry in the world. Among these Philasterides dicentrarchi, Miamiensis avidus, and Uronema marinum are the three species responsible for scuticociliatosis in olive flounder farms of South Korea. Some of the parasites living or scavenger ciliates also have become parasites of aquaculture fish. The major clinico-pathological manifestations of scuticociliatosis infected fishes are anemia, weight loss, dark coloration, enteritis, excessive body mucus, yellowish intestinal mucus, loss of scales, hemorrhagic and/or bleached spots on the skin, and dermal necrotic lesions that finally destroy tissues lead to high mortalities. Affected fish exhibit organ-specific pathological changes in the brain, eyes, muscle, gills, liver, kidney, intestine, and stomach that lead to severe mortality. At present, farmers in South Korea manage scuticociliatosis by using therapeutic measures, such as application of antibiotics like oxytetracycline, gentamycine, tetracycline, amoxycililin, and cefazolin and chemicals, such as formalin, hydrogen peroxide, malachite green, and jenoclean at a concentration of 350 +/- 150 ppm. However till date, no systematic scientific study has been conducted under field condition on the efficacy of these management measures. Under laboratory condition the ciliate can be effectively controlled with the antibiotics and chemicals while on the host, but on entering the host no systemic chemotherapeutic treatment has been yet proven effective. Furthermore the indiscriminate uses of harmful chemicals in aquaculture are increasingly becoming a cause of concern. Recently formalin and malachite green, the most widely used chemicals have been banned in food fish production by FDA as not consumer friendly and being carcinogenic respectively. Vaccines and immunostimulants can induce good immune response and protect against scuticociliatosis as it has been proved in the case of freshwater Ich. Now a days a number of probiotics and herbal formulations are in use against freshwater bacterial and fungal diseases, while, little information is available regarding the different prophylactic measures against marine scuticociliatosis. This review attempts to provide information on the various prophylaxic measures practiced against scuticociliatosis with special reference to olive flounder farms in South Korea., (2010 Elsevier Ltd. All rights reserved.)

We report the effect of aqueous-, ethanol- and methanol-solvent-derived extracts of three traditional Korean herbs, Punica granatum, Chrysanthemum cinerariaefolium and Zanthoxylum schinifolium, by monitoring the innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and the functional immunity in terms of percentage mortality and relative percent survival (RPS) in olive flounder (Paralichthys olivaceus) against Uronema marinum (1 x 10(5)ciliates ml(-1)) for 30 days. Fish were intraperitoneally administered with 5, 50 and 100 mg kg(-1) body weight of each traditional Korean medicinal (TKM) solvent extract except the control and infected untreated groups. In all the treated groups at concentrations of 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters were found significantly enhanced when compared to 0 mg kg(-1) dose. However, at 5 mg kg(-1) the tested immune parameters did not vary. Administration of TKM solvent extracts preceding the challenge with U. marinum for 30 days significantly reduced the percentage mortality with the consequent increase in RPS. Administration of 50 and 100 mg kg(-1) TKM solvent extracts clearly enhanced the innate immune responses and disease resistance in P. olivaceus against U. marinum., ((c) 2010 Elsevier B.V. All rights reserved.)

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 50ppm 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 100ppm for 30min 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., (Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.)

The histophagous scuticociliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis with P. dicentrarchi infections was very high from May to July (40+/-3 to 79+/-2%) in olive flounder at farms of Jeju Island, South Korea, from 2000 to 2004. The prevalence of mixed infection along with Vibrio spp. infection was higher (49+/-8%) than that of scuticociliatosis alone. To date no effective control measure for P. dicentrarchi infection has been described and large economic losses continue. In the present study 3 chemotheraputants (formalin, hydrogen peroxide and Jenoclean) were used. Among these, bath treatment with Jenoclean at a low concentration of 50ppm proved effective; the results were confirmed with in vitro motility assessments and morphological changes in P. dicentrarchi. A similar trend was noted following hydrogen peroxide treatment. However, formalin was only moderately effective at this concentration. Therefore, hydrogen peroxide and Jenoclean are the promising compounds effective at low concentrations with short application time.

Recently, mass mortality due to histophagous scuticociliate, Philasterides dicentrarachi in olive flounder, Paralichthys olivaceus occurred in aquaculture farms of Jeju Island, South Korea. We have artificially induced infection through immersion on day 12, the density of ciliates slowly decreased in the experimental tanks; on day 28 to 2300cells/ml in 3cm group and 1800cells/ml in 5cm group. The survival rate of P. olivaceus on days 24 and 28 of infection in the 3cm group was decreased to 4 and 6, respectively; however, no mortality was recorded in the 5cm group. In the control group 100% survival in the chosen period of both groups occurred except in the 3cm group whereas on days 24 and 28 the death rate was 1 and 2, respectively. Many ciliates with ingested red blood cells were observed in the gills, skeletal muscle, skin, fins and brains of infected flounder. The parasites were also observed in the lamina propria of the digestive tract, pharynx, peritoneal fluid, cerebrospinal fluid and cornea. Histopathological and inflammatory changes were characterized by swelling and generalized erythema and muscle tissue necrosis. Affected fish showed varying levels of tissue damage including severe epidermal and dermal necrotic lesions. Loss of scales, appearance of bleached spots that coalesced to form brownish patches, haemorrhagic and severe dermal necrotic lesions were the major clinico-pathological manifestations. The parasite was also seen distributed extensively in the entire brain causing widespread nerve necrosis after a prolonged period of infection. These results appear to support the hypothesis that the P. dicentrarchi may be penetrating via the gills or the skin, and then travel via the bloodstream to other parts of the body, including the body cavity.