Phytotherapy of experimentally induced gill inflammation with Aeromonas hydrophila infection in goldfish, Carassius auratus

Harikrishnan, Ramasamy,Kim, Ju-Sang,Balasundaram, Chellam,Heo, Moon-Soo The Korean Society of Fish Pathology 2008 한국어병학회지 Vol.21 No.2

Goldfish, Carassius auratus (wt 13 g) was intramuscularly infected with Aeromonas hydrophila (4.3x106 cfu / ml). Infected gills showed edematous lamellae with bacterial invasion into the capillaries and gill congestion on 12th day. By 24th day post-infection, histological analysis revealed irregular aggregates of macrophages in gill lamellae, large amount of mucus cells, gill lamellae edematous with bacterial invasion into capillaries, gill congestion and damaged gill epithelium with hyperplasia. Inflammation of the gill filament and hemorrhage globe was associated with the development of severe necrosis on the 36th day in the infected fishes. In infected and herbal treated fish the regenerative responses like fibrosis and infiltration of the leucocytes (neutrophils and monocytes) occurred on 12th day; moderate hypertrophy in the gills was noticed on the 36th day. These results suggest that phytotherapy ensures better protection and regenerative response against A. hydrophila infection in goldfish, C. auratus.

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

Harikrishnan, R.,Heo, J.,Balasundaram, C.,Kim, M.C.,Kim, J.S.,Han, Y.J.,Heo, M.S. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.29 No.4

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<SUP>-1</SUP> body weight of solvent extracts. In groups treated with 50 and 100 mg kg<SUP>-1</SUP> body weight, the chosen innate immune parameters significantly increased after 8 weeks when compared to 0 mg kg<SUP>-1</SUP> dose, but not with 5 mg kg<SUP>-1</SUP>. 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<SUP>-1</SUP> dose clearly enhance the innate immune responses and disease resistance after 8 weeks in P. olivaceus against natural LDV infection.

Immune response and expression analysis of cathepsin K in goldfish during Aeromonas hydrophila infection

Harikrishnan, R.,Kim, M.C.,Kim, J.S.,Han, Y.J.,Jang, I.S.,Balasundaram, C.,Heo, M.S. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.28 No.4

The innate immunity and expression profiles of cathepsins D were determined in the goldfish (Carassius auratus) tissues after challenge with a fish pathogen Aeromonas hydrophila. The innate immunity of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were determined by peripheral blood leucocytes. Blood and tissue samples of the muscle, gills, liver, kidney, heart, spleen, and intestine were sampled at 1, 3, 6 and 12 h post-infection for cathepsin D expression by semi-quantitative RT-PCR. The ROS and RNS production did not significantly increase at 1 h post-challenged goldfish. However, the ROS and RNS production was significantly increased after 3 h post-challenged fish compared to the control. The cathepsin D expression was found very low in muscle and kidney of the control fish, other tissues was not found the expression. A similar pattern was found in goldfish at 1 h post-challenge with A. hydrophila. However, at 3 h post-challenge goldfish, the cathepsin D expression was high only in the heart. At 6 h post-challenge goldfish, the cathepsin D expression was seen high all the tissues, except in the spleen. However, the expression was decreased at 12 h post-infection samples. This result was suggested that the goldfish infected with A. hydrophila decreased the innate immunity level in peripheral blood and expressed the cathepsin D in tissues.

Influence of diet enriched with green tea on innate humoral and cellular immune response of kelp grouper (Epinephelus bruneus) to Vibrio carchariae infection

Harikrishnan, R.,Balasundaram, C.,Heo, M.S. Academic Press 2011 FISH AND SHELLFISH IMMUNOLOGY Vol.30 No.3

Effect of diet enriched with green tea at 0, 0.01, 0.1 or 1.0% levels on immune responses such as non-specific humoral (lysozyme, antiprotease and complement) and cellular (myeloperoxidase content, production of reactive oxygen, and nitrogen species) and disease resistance on week 1, 2 or 4 in kelp grouper Epinephelus bruneus challenged with Vibrio carchariae (2.47x10<SUP>8</SUP>CFUml<SUP>-1</SUP>) was quantified. At all doses green tea supplementation significantly enhanced the serum lysozyme activity from weeks 1 to 4. On the other hand, after week 2 the serum hemolytic complement activity, leucocyte myeloperoxidase content and reactive nitrogen species protection significantly increased in groups fed with 0.01 and 0.1% green tea supplementation diets. The serum antiprotease activity significantly increased in group fed with at 1.0% green tea from week 1 to 4. However, all diets except at 0.01% level resulted in a significant decrease in reactive oxygen species protection during the experimental period. Challenged groups fed with green tea enriched diet at 0.01 and 0.1% level had a higher relative percent survival than with 1.0% diet on week 1, 2 or 4. The results suggest that dietary administration of green tea supplementation at a concentration of 0.01 and 0.1% level positively enhances the non-specific humoral and cellular immune responses and disease resistance of kelp grouper E. bruneus to V. carchariae.

Korean mistletoe enriched diet enhances innate immune response in kelp grouper, Epinephelus bruneus against Philasterides dicentrarchi

Harikrishnan, R.,Balasundaram, C.,Heo, M.S. Elsevier Scientific Pub. Co 2011 Veterinary parasitology Vol.183 No.1

The present study investigated the immunostimulatory effect of Korean mistletoe Viscum album extract (KM-E) on innate immune response in kelp grouper Epinephelus bruneus against Philasterides dicentrarchi. Kelp grouper were divided into four groups of 25 each and fed with 0 (control), 0.5, 1.0, and 2.0% enriched diets with Korean mistletoe extract (KM-E). After feeding for 30 days, the fish were injected intraperitoneally (i.p.) with 100μl of P. dicentrarchi (4.2x10<SUP>7</SUP>ciliates/ml) to study the immune responses at weeks 1, 2, and 4. The respiratory burst activity did not significantly enhance when fed with 0.5% and 1.0% supplementation diets on week 1 when compared to control diet. On weeks 2 and 4, the respiratory burst activity significantly increased with 1.0% and 2.0% diets. The phagocytic activity significantly enhanced with 1.0% and 2.0% diets, but not with 0.5% diet at any time. When fed with 1.0% KM-E-diet the lysozyme activity did not significantly vary at any week whereas with 1.0% and 2.0% diets it was significantly enhanced. The total protein level significantly increased with 1.0% and 2.0% KM-E-diets from weeks 1 to 4 as compared to control. The present study suggests that 1.0% or 2.0% KM-E-supplementation diet positively enhances the innate immune response in E. bruneus against P. dicentrarchi infection.

Effect of dietary supplementation with <i>Suaeda maritima</i> on blood physiology, innate immune response, and disease resistance in olive flounder against <i>Miamiensis avidus</i>

Harikrishnan, Ramasamy,Kim, Ju-Sang,Kim, Man-Chul,Dharaneedharan, Subramanian,Kim, Dong-Hwi,Hong, Seung-Hyun,Song, Chang-Young,Balasundaram, Chellam,Heo, Moon-Soo Elsevier 2012 Experimental parasitology Vol.131 No.2

<P><B>Graphical abstract</B></P><P>The cumulative mortality in groups fed control (C) and <I>S. maritima</I> extract supplementation diets at 0%, 0.01%, 0.1%, and 1.0% and challenged with <I>M. avidus</I>.<ce:figure id='f0050'></ce:figure></P><P><B>Highlights</B></P><P>► RBC significantly increased with 0.1% and 1.0% enriched diets on week 4. ► Hb and Ht levels increased when with 0.1% and 1.0% diets on weeks 2 and 4. ► Lym, Mon, Neu, TP, GLU, and CAL levels increased in 0.1% and 1.0% diets on weeks 2 and 4. ► Lysozyme activity enhanced in 0.1% and 1.0% diets from weeks 1 to 4. ► Scuticocidal and respiratory burst activities enhanced in 0.1% and 1.0% diets on weeks 2 and 4.</P> <P><B>Abstract</B></P><P>The effect of <I>Suaeda maritima</I> enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder <I>Paralichythys olivaceus</I> against <I>Miamiensis avidus</I> on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with <I>M. avidus</I> 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% <I>S. maritime</I>-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from <I>M. avidus</I> infection in olive flounder.</P>

Immunomodulatory effect of probiotics enriched diets on Uronema marinum infected olive flounder

Harikrishnan, R.,Kim, M.C.,Kim, J.S.,Balasundaram, C.,Heo, M.S. Academic Press 2011 FISH AND SHELLFISH IMMUNOLOGY Vol.30 No.3

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.

Styrax japonica supplementation diet enhances the innate immune response in Epinephelus bruneus against bacterial and protozoan infections

Harikrishnan, R.,Kim, J.S.,Kim, M.C.,Balasundaram, C.,Heo, M.S. Academic Press 2011 Experimental parasitology Vol.129 No.3

Kelp grouper, Epinephelus bruneus, fed for 30days with 0% (control), 0.1%, 1.0%, and 2.0% of Styrax japonica supplementation diets, led to reductions in mortality after being challenged with a bacterium (Vibrio harveyi) and a ciliate protozoan (Uronema marinum). The enriched diets significantly increased the survival rate as compared to the controls. The phagocytic and respiratory activities were significantly increased in kelp groupers given 1.0% and 2.0% enriched diets. The complement activity, lysozyme activity, serum bactericidal activity, and total protein level significantly increased with any enriched diet against the pathogens; however antiprotease activity and myeloperoxidase levels significantly increased only with 1.0% and 2.0% enriched diets while the α2-macroglobulin level was significantly enhanced with 1.0% enriched diet. The study suggests that incorporation of S. japonica at 1.0% and 2.0% level in the diet significantly enhances the immune responses in the kelp grouper E. bruneus against V. harveyi and U. marinum.

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

Harikrishnan, R.,Balasundaram, C.,Heo, M.S. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.29 No.5

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.

Kalopanax pictus as feed additive controls bacterial and parasitic infections in kelp grouper, Epinephelus bruneus

Harikrishnan, R.,Kim, J.S.,Kim, M.C.,Balasundaram, C.,Heo, M.S. Academic Press 2011 FISH AND SHELLFISH IMMUNOLOGY Vol.31 No.6

Feeding kelp grouper, Epinephelus bruneus (26.1 +/- 1.4), with 0%, 0.1%, 1.0%, and 2.0% Kalopanax pictus extract-supplementation diets, for 30 days reduced mortality on being challenged intraperitoneally 100 μl with bacterium Vibrio alginolyticus (2.1 x 10<SUP>7</SUP> cfu ml<SUP>-1</SUP>) and ciliate parasite Philasterides dicentrarchi (2.3 x 10<SUP>7</SUP> ciliates ml<SUP>-1</SUP>). The red blood cells (RBC), white blood cells (WBC), haemoglobin, haematocrit, lymphocytes, and monocytes levels significantly increased in kelp grouper fed with all doses of K. pictus-supplementation diets and challenged with bacterium and parasite when compared to control. However, the levels of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), neutrophils, and thrombocytes did not significantly. The phagocytic activity, complement activity, and antiprotease activity did not significantly change in kelp grouper fed with 0.1% K. pictus-supplementation diets and challenged with bacterium and parasite. The respiratory activity, lysozyme activity, bactericidal activity, total protein level, and myeloperoxidase levels significantly increased in kelp grouper fed with all the doses of K. pictus-supplementation diet and challenged with bacterium and parasite. However, α2-macroglobulin level significantly increased with 1.0% diet, but not with 0.1% and 2.0% diets. Therefore this study suggests that 1.0% and 2.0% K. pictus-supplementation diets positively protected and enhanced the immune system in kelp grouper E. bruneus against V. alginolyticus and P. dicentrarchi infection.