Ramasamy Harikrishnan

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg(-1) body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 x 10(7) cells ml(-1)). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.

Effect of probiotics enriched diet on Paralichthys olivaceus infected with lymphocystis disease virus (LCDV).

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.

Lactobacillus sakei BK19 enriched diet enhances the immunity status and disease resistance to streptococcosis infection in kelp grouper, Epinephelus bruneus.

The effect of Lactobacillus sakei BK19 (10(8) cells g(-1)) supplemented diet fed to kelp grouper, Epinephelus bruneus against streptococcosis caused by Streptococcus iniae and Streptococcus parauberis with reference to the innate immune response and disease resistance was evaluated at 1, 2, and 4 weeks. Maximum reduction in mortalities was observed in kelper feeding the probiotic diet for two weeks after challenged with the pathogens when compared to the infected group fed with basal diet; similarly the cellular and humoral immune responses such as head kidney macrophage phagocytic and peroxidase activities, serum lysozyme activity, and total protein levels increased significantly. The results reveal that, in streptococcosis infected kelp grouper feeding L. sakei BK19 enriched diet affords a higher level of disease protection due to stimulation of immune system.

Herbal supplementation diets on hematology and innate immunity in goldfish against Aeromonas hydrophila.

Goldfish, Carassius auratus (47 + or - 3 g, n = 300) were inoculated intramuscularly (50 microl) with Aeromonas hydrophila (1.8 x 10(6) cells ml(-1)). On the 6th day of post-infection the fishes were divided into i) control, without infection fed with normal diet (C), ii) infected fish, fed with normal diet (IU), and infected fishes treated with different doses of iii) 100 mg kg(-1), iv) 200 mg kg(-1), iv) 400 mg kg(-1) and vi) 800 mg kg(-1) mixed herbal extracts supplementation diets. Hematological and immunological parameters were determined on week 1, 2 and 4. In infected goldfish were fed diets containing 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds, the white blood cell (WBC) levels significantly increased (P < 0.05) throughout the experimental trial compared to the control. During the experimental period, the red blood cell (RBC) and haemoglobin (Hb) level in goldfish significantly decreased (P < 0.05) when fish fed with 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds while it was restored near control when infected fish fed with 400 or 800 mg kg(-1) of herbal extracts supplementation feeds. On the other hand, the haematocrit (Ht) values decline significantly (P < 0.05) in 100, 200 and 400 mg kg(-1) of mixed herbal supplementation feeding groups on weeks 2 and 4 when compared to control group. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) values almost significantly differ from the control values. The infected goldfish and treated with 100 or 200 mg kg(-1) of herbal supplementation feeds exhibited significantly decline (P < 0.05) in total protein (TP), glucose (GLU) and cholesterol (CHO) levels on week 1-4 whereas it was restored when infected fish fed with 400 or 800 mg kg(-1) of herbal supplementation feeds on week 4. In comparison to untreated control goldfish, the respiratory burst activity and phagocytic activity of blood cells was significantly enhanced in infected fish feeding with 200, 400 and 800 mg kg(-1) of herbal supplementation feeds compared to the control. On the other hand, infected fish fed with all the doses of mixed herbal supplementation feeds, the lysozyme activity was significantly enhanced throughout the experimental period. This study shows that the infected goldfish treated with 400 and 800 mg kg(-1) of herbal supplementation feeds preceding the challenge with live A. hydrophila had 30% and 25% mortality. However, 100 and 200 mg kg(-1) of herbal supplementation feeds treated groups were found the percentage mortality 50% and 45%, respectively. Our results indicate that 400 or 800 mg kg(-1) of mixed herbal supplementation feeds were restored the altered hematological parameters and triggering the innate immune system of goldfish against A. hydrophila.

Effect of chemotherapy, vaccines and immunostimulants on innate immunity of goldfish infected with Aeromonas hydrophila

We report the effect of various chemicals, vaccines and immunostimulants on innate immune mechanisms such as phagocytosis activity, superoxide anion production of blood leukocytes, complement activity and plasma lysozyme activity and disease resistance in goldfish Carassius auratus (23 +/- 2 g) against Aeromonas hydrophila on Days 15 and 30 post-infection. In infected fish, the administration of diets supplemented with either probiotics, tri-herbal extract or azadirachtin for 30 d significantly enhanced phagocytic activity; administration for 15 d had no effect. In fish treated with heat-killed and formalin-killed vaccines, and probiotics-, tri-herbal- and azadirachtin-supplemented diets, superoxide anion production was enhanced on Day 30. However, there was no superoxide anion production in fish treated with tetracycline, furanace, formalin or hydrogen peroxide. Complement activity was also significantly enhanced on Days 15 and 30 in fish groups treated with vaccine and probiotics-, tri-herbal- and azadirachtin-supplemented diets. Other groups had no complement activity. Lysozyme activity was significantly enhanced after 30 d in fish treated with heat-killed vaccine and probiotics-, tri-herbal- and azadirachtin-supplemented diets. The probiotics- and tri-herbal-containing diet administration groups had no mortality (0%) preceding the challenge with live A. hydrophila. Groups treated with heat- or formalin-killed vaccines or azadirachtin-supplemented diet suffered mortalities of 30, 35 and 10%, respectively, which represents an improvement of survival rate compared to untreated infected control groups. We conclude that probiotics-, tri-herbal- and azadirachtin-supplemented diets act as immunostimulants enhancing the goldfish innate immune response and disease resistance against A. hydrophila.

Effect of Punica granatum solvent extracts on immune system and disease resistance in Paralichthys olivaceus against lymphocystis disease virus (LDV).

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.

Diet enriched with mushroom Phellinus linteus extract enhances the growth, innate immune response, and disease resistance of kelp grouper, Epinephelus bruneus against vibriosis

The effect of diet supplemented with Phellinus linteus fed for 30 days was investigated in grouper Epinephelus bruneus challenged with Vibrio anguillarum, Vibrio harveyi, Vibrio alginolyticus, and Vibrio carchariae; infected and treated fish had a significantly higher percent weight gain and feed efficiency. In groups fed with enriched diet and challenged with V. anguillarum and V. harveyi the mortality rate declined with a consequent rise in survival rate than with other pathogens. On the other hand, in groups fed with P. linteus enriched diet and challenged with V. anguillarum, V. harveyi, and V. alginolyticus the cellular and humoral immune responses, such as the alternative complement activity (ACH(50)), serum lysozyme activity, phagocytic activity (PA), phagocytic index (PI) significantly higher than in the control group. The respiratory bursts (RB), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were found significantly enhanced when the groups fed with enriched diet against V. anguillarum and V. harveyi. The results reveal that kelp grouper fed for 30 days with P. linteus enriched diet had higher cellular and humoral immune response and disease protection from vibriosis than the group fed on basal diet with the protection linked to stimulation of immune system.

Protective effect of herbal and probiotics enriched diet on haematological and immunity status of Oplegnathus fasciatus (Temminck & Schlegel) against Edwardsiella tarda.

This study determines the effect of diet enriched with the herb Baical skullcap Scutellaria baicalensis, and/or probiotics Lactobacillus sakei BK19 in rock bream, Oplegnathus fasciatus (32 ± 3 g) against Edwardsiella tarda. The changes in haematological parameters, innate immune response, and disease resistance were investigated after 1, 3, and 6 weeks. The white blood cell count (WBC: 10(4) mm(-3)), red blood cell count (RBC: 10(6) mm(-3)), and haemoglobin (Hb: g dl(-1)) levels significant increased (P < 0.05) with mixed diet on 3rd and 6th week and probiotics enriched diet on 6th week. The haematocrit (Ht: %) level significantly increased (P < 0.05) when fed with mixed diet on weeks 1-6. Interestingly, in mixed diet group the lymphocytes (LYM), monocytes (MON), and neutrophils (NEU) significantly increased from week 1-6. The eosinophils (EOS) significantly increased in all the treated groups. In the probiotics or mixed diet groups the total protein (TP: g dl(-1)) increased significantly on weeks 3 and 6. The serum lysozyme activity significantly was enhanced in all the treated groups indicating an increase in the innate immunity level. Serum complement, antiprotease activities, reactive oxygen species (ROS) and reactive nitrogen species (RNS) production significantly increased from week 1-6 with mixed diet. The maximum protection against E. tarda was recorded in mixed diet group with a minimum cumulative mortality of 20% and a high relative percent survival (RPS) of 72.84. In the probiotics and herbal diet groups the cumulative mortality was 25% and 35% and RPS was 68.63 and 59.42, respectively. This study indicates that administration of probiotics or mixed diets can effectively minimize the mortality and restore the altered hematological parameters and enhancing the innate immunity in O. fasciatus against E. tarda.

Effect of traditional Korean medicinal (TKM) triherbal extract on the innate immune system and disease resistance in Paralichthys olivaceus against Uronema marinum

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.

Scuticociliatosis and its recent prophylactic measures in aquaculture with special reference to South Korea. Taxonomy, diversity and diagnosis of scuticociliatosis: Part I. Control strategies of scuticociliatosis: Part II.

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.