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

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.     

Probiotics and herbal mixtures enhance the growth, blood constituents, and nonspecific immune response in Paralichthys olivaceus against Streptococcus parauberis

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 × 10? 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.     

Inonotus obliquus containing diet enhances the innate immune mechanism and disease resistance in olive flounder Paralichythys olivaceus against Uronema marinum

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.     

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

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 (2.47 × 10(8) CFU ml(-1)) 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.     

Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections

The probiotic activity of two bacteria (Bacillus subtilis and Lactobacillus acidophilus) was evaluated by its effect on the immune response of Nile tilapia (Oreochromis niloticus), beside its protective effect against challenge infections. Furthermore, their in-vitro inhibitory activity was evaluated. The in-vitro antimicrobial assay showed that Bacillus subtilis and Lactobacillus acidophilus inhibited the growth of A. hydrophila. The B. subtilis inhibited the development of P. fluorescens while L. acidophilus inhibited the growth of Strept. iniae. The B. subtilis and L. acidophilus proved harmless when injected in the O. niloticus. The feed, containing a mixture of B. subtilis and L. acidophilus or B. subtilis alone, showed significantly greater numbers of viable cells than feed containing L. acidophilus only after 1, 2, 3 and 4 weeks of storage at 4 degrees C and 25 degrees C. The survival rate and the body-weight gain were significantly increased in the fish given B. subtilis and L. acidophilus for one and two months after application. The hematocrit values showed a significant increase in the group that received the mixture of B. subtilis and L. acidophilus compared with the control group. The nitroblue tetrazolium (NBT) assay, neutrophil adherence and lysozyme activity, showed a significant increase in all the probiotic-treated groups after 1 and 2 months of feeding, when compared with the untreated control group. The serum bactericidal activity was high in the group that was given a mixture of the two bacteria. The relative level of protection (RLP) was significantly higher against A. hydrophila, in the bacterial mixture treated group and against P. fluorescens in the L. acidophilus treated group, after one month of the feeding trial. A significantly higher RLP, against A. hydrophila or P. fluorescens, was noticed after 2 months of the feeding trial in the group given a mixture of the two bacteria, and against Strept. iniae in the group fed a diet containing L. acidophilus.     

Immunomodulatory effect of sodium alginate enriched diet in kelp grouper Epinephelus brneus against Streptococcus iniae

The effect of diets containing sodium alginate at 0, 0.5, 1.0, and 2.0 g kg^?1 following challenge with Streptococcus iniae in kelp grouper Epinephelus bruneus were assessed with reference to survival rate and innate immune parameters such as alternative complement, lysozyme, natural haemagglutination, respiratory burst, superoxide dismutase, and phagocytic activities on week 1, 2, and 4. Fish fed with sodium alginate containing diet at 1.0 and 2.0 g kg^?1 after being challenged with S. iniae had higher survival rates of 75% and 60%, respectively than those fed with control diet (0 g kg^?1). With any enriched diet the percentage of macrophages significantly decreased from week 1–4, while the percentage of neutrophils and lymphocytes significantly increased. The alternate complement activity, natural haemagglutination, and phagocytic activities of infected fish fed with sodium alginate containing diet at 1.0 g kg^?1 on week 2 and 1.0 and 2.0 g kg^?1 diets on week 4 were significantly higher when compared to the control. The lysozyme, respiratory bursts, and superoxide dismutase activities of fish fed with enriched diets at 1.0 and 2.0 g kg^?1 were significantly increased on week 2 and 4. We therefore recommend that at 1.0 or 2.0 g kg^?1 dietary administration of sodium alginate can enhance innate immunity and disease resistance in kelp grouper against S. iniae.     

嗜酸乳杆菌对斜带石斑鱼幼鱼生长、消化性能和抗病力的影响

研究嗜酸乳杆菌(Lactobacillus acidophilus, GIM:1.730)对斜带石斑鱼(Epinephelus coioides)幼鱼生长、消化酶活性、抗病力和相关酶mRNA表达的影响。选取720尾初始均重为(5.60±0.05) g的斜带石斑鱼幼鱼为研究对象, 共设8个处理, 每个处理3个重复。投喂不同添加比例(0、0.1%、0.2%、0.4%、0.6%、0.8%、1.0%和1.2%)嗜酸乳杆菌的饲料, 养殖期8周。结果表明: 增重率(Weight gain rate, WGR)与特定生长率(Specific growth rate, SGR)实验组均显著高于对照组(P<0.05)。当嗜酸乳杆菌添加0.6%时, 饲料系数(Feed conversion ratio, FCR）最低, 其他各组显著低于对照组(P<0.05), 而蛋白质效率(Protein efficiency ratio, PER)变化规律则与FCR相反。肝脏和肠蛋白酶(Trypsin, TRY)、淀粉酶(Amylase, AMY)活性实验组均显著高于对照组(P<0.05)。肝脏TRY、AMY mRNA表达, 实验组均显著高于对照组(P<0.05)。其中, 肝脏淀粉酶、蛋白酶活性在添加量为0.6%达到最大值, 肠蛋白酶、淀粉酶活性分别在添加量0.2%和0.4%达到最大值。以哈维弧菌(Vibrio harveyi)攻毒7d, 石斑鱼攻毒后成活率(Survival rate, SR)随着嗜酸乳杆菌添加量的增加显著升高(P<0.05)。以WGR为判断依据得出, 在饲料中添加0.42%嗜酸乳杆菌(0.72×108 cfu/g)可显著促进斜带石斑鱼幼鱼的生长, 并提高消化酶活性和抗病力。     

Growth response of African catfish,Clarias gariepinus (B.), larvae and fingerlings fed protease‐incorporated diets

African catfish, Clarias gariepinus (B.), is one of the promising freshwater fish species in African aquaculture but the expansion of its farming needs more production of its larvae. The use of live food organisms at first feeding for larvae is still obligatory. That increases the cost of larvae production. Hence, the incorporating of exogenous enzymes especially protease in artificial microdiets may provide affordable alternatives for enhancing the larvae performance. The present study was carried out to evaluate the growth and survival of larvae or fingerlings of African catfish fed artificial diets incorporated with different protease levels. Four artificial diets were formulated and enriched with protease enzyme at levels of 0.0, 750, 1,000, and 1,250 unit/kg diet; after that diets were made into crumbles (100–200 µm diameter). After absorption of the yolk sac, diets were offered to fish larvae (3.6 ± 0.2 mg) in triplicates as a starter feed up to apparent satiation every two hours for 30 days. In another treatment, fish larvae were fed on newly hatched Artemia nauplii (2,500 Artemia/L) as a starter food. In another experiment, African catfish fingerlings (10.1 ± 1.6 g) were fed on the same diets up to satiation twice a day for 2 months. It was noticed that the dietary protease improved larval growth and survival but not as Artemia nauplii did where fish larvae fed on Artemia nauplii showed highest growth and survival followed by those fed a diet enriched with 1,250 unit/kg diet of protease. The mortality of larvae fed protease‐enriched diets as well as the control diet was occurred mostly at the first week reaching its maximum at the third week. The poor growth was observed with fish larvae fed the control diet. Meanwhile, catfish fingerlings fed protease‐enriched diets showed higher growth over those fed the control diet. The larvae survival (11.0%–41.7%) was enhanced by increasing protease levels and it was lower than that of fingerlings (95.6%–100.0%). Furthermore, protein retention and digestibility were significantly improved with protease supplementation over the control diet especially at a level of 1,000 unit/kg diet. As compared with the previous studies, live food should be used in larvae rearing for the first week after that a starter diet enriched with protease at levels of 1,250 unit/kg diet should be used. In case of fish fingerlings, the dry diets should be enriched with 1,100 unit/kg diet to improve diet digestibility and subsequently enhance their growth.     

Non-specific immune response and disease resistance induced by Siegesbeckia glabrescens against Vibrio parahaemolyticus in Epinephelus bruneus

The immunomodulatory effect of Siegesbeckia glabrescens extract-supplementation diets on innate immune response and disease resistance of kelp grouper, Epinephelus bruneus against Vibrio parahaemolyticus at weeks 1, 2, and 4 is reported. The serum lysozyme activity was significantly enhanced with any enriched diet from weeks 1-4 when compared to control diet (0%). The alternative complement haemolytic activities significantly were enhanced with all enriched diets on weeks 2 and 4 whereas the cellular reactive oxygen species (ROS) was significantly enhanced only with 1.0% and 2.0% diets. The reactive nitrogen intermediate (RNI) value was significantly enhanced with any enriched diet on weeks 2 and 4, but on first week it did not differ from control. The myeloperoxidase (MPO) production significantly increased with 1.0% and 2.0% diets from second week onwards; with other enriched diets the increase manifested on fourth week; but during first week it did not vary from that of the control with any enriched diet. The protection in terms of cumulative mortality was the least being 25% and 20% when fed with 1.0% and 2.0% diets. The present results indicate that feeding kelp grouper with S. glabrescens extract enriched diet at 1.0% and 2.0% levels significantly enhance the immunological parameters, increase the disease resistance and minimize the cumulative mortality in E. bruneus against V. parahaemolyticus.     

Morphology and biology of parasite responsible for scuticociliatosis of cultured olive flounder Paralichthys olivaceus.

Based on the arrangement and shape of the buccal structure, scuticociliates isolated from cultured olive flounder Paralichthys olivaceus belonged to the family Uronematidae and showed many characteristics of Uronema marinum. There was variation in the morphometry of clinical isolates taken from different organs of infected flounder. However, the isolates did not show any significant difference in morphometry under cultured conditions. The ciliates were easily maintained in in vitro medium to which antibiotic agents had been added and which had been enriched with the raw brain tissue of a healthy olive flounder. The ciliates propagated in a wide range of both temperature (6 to 30 degrees C) and salinity (10 to 35 ppt).     

益生乳酸菌Lactobacillus casei Zhang和Lactobacillus plantarum P8对全价饲料pH及微生物类群变化的研究

研究了益生乳酸菌干酪乳杆菌Zhang(Lactobacillus casei Zhang)和植物乳杆菌P8(Lactobacillus planta-rum P8)对全价饲料pH及微生物类群变化的影响。分别将L.casei Zhang、L.plantarum P8单一菌种及复合菌种(11)以6.30 lg cfu/g的接种总量发酵全价饲料,测定25℃10 d发酵期间全价饲料pH和微生物类群的变化,应用选择培养基测定发酵饲料中的乳酸菌及杂菌(酵母菌、霉菌、大肠菌群、芽胞杆菌和梭状芽胞杆菌)的动态变化,应用RT-PCR技术测定试验组中的L.casei Zhang和L.plantarum P8的动态变化。结果显示,试验组pH下降显著,发酵10 d时,L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料的pH分别为4.23、4.24和4.22,显著低于对照组(P0.05);L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料中的L.casei Zhang、L.plantarum P8活菌数分别为8.91、8.89、6.58和8.69 lg cfu/g。发酵期间,试验组中酵母菌、霉菌、大肠菌群、芽胞杆菌及梭状芽胞杆菌活菌数显著低于对照组(P0.05),其中L.plantarum P8单一菌种发酵和复合菌种发酵对杂菌抑制效果显著优于L.casei Zhang单一菌种发酵(P0.05)。结果表明,全价饲料经L.casei Zhang、L.plantarum P8发酵可以显著降低其pH,抑制其中杂菌的生长,同时L.casei Zhang、L.plantarum P8在饲料中具有良好的稳定性。     

Effect of dietary inclusion of Lactobacillus acidophilus ATCC 43121 on cholesterol metabolism in rats

This study examined the effects of Lactobacillus acidophilus ATCC 43121 (LAB) on cholesterol metabolism in hypercholesterolemia-induced rats. Four treatment groups of rats (n = 9) were fed experimental diets: normal diet, normal diet+LAB (2 x 10(6) CFU/day), hypercholesterol diet (0.5% cholesterol, w/w), and hypercholesterol diet + LAB. Body weight, feed intake, and feed efficiency did not differ among the four groups. Supplementation with LAB reduced total serum cholesterol (25%) and VLDL + IDL + LDL cholesterol (42%) in hypercholesterol diet groups, although hepatic tissue cholesterol and lipid contents were not changed. In the normal diet group, cholesterol synthesis (HMG-CoA reductase expression), absorption (LDL receptor expression), and excretion via bile acids (cholesterol 7 alpha-hydroxylase expression) were increased by supplementation with LAB, and increased cholesterol absorption and decreased excretion were found in the hypercholesterol diet group. Total fecal acid sterols excretion was increased by supplementation with LAB. With proportional changes in both normal and hypercholesterol diet groups, primary bile acids (cholic and chenodeoxycholic acids) were reduced, and secondary bile acids (deoxycholic and lithocholic acids) were increased. Fecal neutral sterol excretion was not changed by LAB. In this experiment, the increase in insoluble bile acid (lithocholic acid) reduced blood cholesterol level in rats fed hypercholesterol diets supplemented with LAB. Thus, in the rat, L. acidophilus ATCC 43121 is more likely to affect deconjugation and dehydroxylation during cholesterol metabolism than the assimilation of cholesterol into cell membranes.     

饲料糖对彭泽鲫生长和生理机能的影响

用分别含20%(低)、40%(高)葡萄糖和糊精的等氮(粗蛋白为35%风干物质)等能(16.4kJ/g)饲料饲养平均尾重为(2.40±0.16)g澎泽鲫Carassius auratus var. Pengze 16周,研究饲料糖种类和水平对鲫鱼生长、抗氧化力、非特异性免疫力和镉耐受性的影响,探讨鲫鱼对糖的利用能力及糖的生理功效。另用相同的饲料在相似的条件下饲养初重为(7.14±0.85)g的鲫鱼8周后胸腔注射12.5mg/kg体重的氯化镉溶液,观察其24h的存活率。实验结果表明饲料糖种类和水平都没有对鲫鱼的生长、脾指数和肝指数造成显著影响。高糖组糖化血红蛋白GHb值较高(P<0.05)。肝胰脏超氧化物歧化酶SOD活性、血浆SOD活性和总抗氧化力T-AOC各组差异不显著,肝胰脏的T-AOC为葡萄糖组显著高于糊精组(P<0.05)。补体溶血活性、血浆溶菌酶活性各组差异不显著。红细胞脆性高糖组显著低于低糖组(P<0.05)。镉染毒存活率各组差异不显著。上述结果提示40%饲料糖对鲫鱼生理功能没有不良影响,一定量的饲料糖有利于提高鲫鱼的抗氧化力。       

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.     

Enhancement of Eriobotrya japonica extracts on non-specific immune response and disease resistance in kelp grouper Epinephelus bruneus against Vibrio carchariae

The present study investigated the effect of Eriobotrya japonica extracts at 0%, 0.1%, 1.0%, and 2.0% doses supplementation with feed on non-specific immune response, hematological and biochemical profile, and disease resistance against Vibrio carchariae in kelp grouper Epinephelus bruneus at weeks 1, 2, and 4. The white blood cell (WBC) significantly increased in fish fed with 0.1%, 1.0%, and 2.0% diets on weeks 1 and 2 when compared to the control. However, the glucose always decreased from the control except on week 2 against pathogen. The serum total protein, albumin, and globulin significantly increased at week 2 but they did not changed significantly at weeks 1 and 4. The superoxide anion, lymphokines production index, and phogocytosis did not significantly increased in any diet on the first week whereas it was significantly enhanced in 1.0% and 2.0% supplementation diets on weeks 2 and 4 against V. carchariae when compared to control. All diets significantly enhanced the serum lysozyme activity, bactericidal activity, and haemolytic complement activity from weeks 1-4 as compared to control. The serum agglutinating antibody titre did not significantly enhance on the first week whereas it was significantly enhanced on weeks 2 and 4. Fish fed with 1.0% and 2.0% doses diets was found lower mortality than 0.1% diet. Thus, this study suggested that 1.0% and 2.0% doses supplementation diets could be advocated to enhance the immune response and production disease from V. carchariae in E. bruneus.     

Pseudocohnilembus persalinus (Ciliophora: Scuticociitida) is an additional species causing scuticociliatosis in olive flounder Paralichthys olivaceus

In the present study, Pseudocohnilembus persalinus was first reported as a species causing scuticociliatosis in olive flounder Paralichthys olivaceus. Based on the stained specimens, P. persalinus was clearly differentiated from Uronema marinum, which has been shown to be a cause of scuticociliatosis in farmed olive flounder in Korea from its characteristic oral infraciliature structure. The 1754 bp small subunit ribosomal RNA (SS rRNA) gene sequence of P. persalinus showed 95% homology with the partial sequence of P. hargisi SS rRNA. Moreover, multiplex PCR based on the species-specific amplification of the SS rRNA gene sequence enabled us to distinguish P. persalinus from U. marinum in a simple and rapid manner. P. persalinus was clearly differentiated from U. marinum even when the host was infected simultaneously with both species. These data suggest that the multiplex PCR procedure would make it possible to avoid the cumbersome and time-consuming procedures of morphological analysis for the definitive identification of ciliates.     

Dietary administration of the probiotic,Lactobacillus plantarum,enhanced the growth,innate immune responses,and disease resistance of the grouper Epinephelus coioides

The percent weight gain (PWG) and feed efficiency (FE) of Epinephelus coioides were calculated, and the lactobacilli and total microbiota in the posterior intestines, and non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus were determined when the fish were fed diets containing Lactobacillus plantarum at 0 (control), 10⁶, 10⁸, or 10¹⁰ colony-forming units (cfu) kg^?1 for 4 weeks. Results showed that grouper fed a diet containing L. plantarum at the levels of 10⁶, 10⁸, and 10¹⁰ cfu kg^?1 had significantly increased PGW and FE especially at 10⁸ cfu kg^?1 group which were 404.6% and 1.26, respectively. L. plantarum significantly increased in the fish posterior intestines during the L. plantarum feeding period, but decreased rapidly from the intestine within 1 week after changing to the control diet (without L. plantarum). Fish fed a diet containing L. plantarum at 10⁶ and 10⁸ cfu kg^?1 had significantly higher survival rates than those fed the control diet after challenge with Streptococcus sp., as well as those fed 10⁸ cfu kg^?1 after challenge with an iridovirus, causing increases in the survival rates of 23.3%, 20.0%, and 36.7%, respectively, compared to the control group. The alternative complement activity (ACH₅₀) level of fish fed diets containing L. plantarum after 4 weeks was significantly higher than that of fish fed the control diet, and that of the 10⁸ cfu kg^?1 group was significantly higher than those of the 10⁶ and 10¹⁰ cfu kg^?1 groups, which increased by 83.4% compared to the control group. The lysozyme activity and glutathione peroxidase (GPx) activity of fish fed the L. plantarum-containing diets at 10⁸ and 10¹⁰ cfu kg^?1 significantly increased compared to those fed the 10⁶ cfu kg^?1 L. plantarum diet and control diet, and had increased by 76.3% and 136.6%, and 57.1% and 113.3%, respectively, compared to those fed the control diet. The phagocytic activity (PA), phagocytic index (PI), and respiratory bursts of head kidney leucocytes of fish fed 10⁶, 10⁸, and 10¹⁰ cfu kg^?1 L. plantarum diets were significantly higher than those of fish fed the control diet after 4 weeks of feeding, and increased 2.2-, 2.2-, and 2.3-fold; 1.8-, 1.8-, and 2.0-fold; and 1.4-, 1.4-, and 1.4-fold, respectively, compared to the control group. We therefore recommend dietary L. plantarum administration at 10⁸ cfu kg^?1 to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus of E. coioides.     

Survival, growth and persistence under farm conditions of a Lactobacillus plantarum strain inoculated into liquid pig feed

AIMS: To investigate the survival and persistence of Lactobacillus plantarum REB1 in the fermentation process of liquid pig feed on a working farm in standard production conditions. METHODS AND RESULTS: Two feed types, a control diet [nonfermented liquid feed (NFLF)] and a fermented diet [fermented liquid feed (FLF)], were compared. A rifampicin-resistant mutant L. plantarum REB1-Rif was used to initiate the fermentation of the feed. Inoculation with the experimental strain was repeated one or two times per week throughout the three month growing period. Four microbial groups were followed using standard microbiological techniques, as well as pH. Lactic acid bacteria (LAB) counts were high already at the beginning in FLF, while it took nine days to reach the corresponding LAB levels in NFLF. Yeasts were stable in FLF, whereas in NFLF there were occasional high counts during the first week. The numbers of Enterobacteriaceae were low, although in NFLF they were variable. The average pH in NFLF was 4.5 and 4 in FLF. CONCLUSIONS: The inoculation of L. plantarum REB1-Rif provided a LAB population of log 9 colony forming units (CFU) ml(-1) from the first feeding day, stable numbers of yeast and pH, and a drastic reduction of Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF THE STUDY: The inoculation by L. plantarum REB1-Rif offered a FLF microbiologically stable from the first week in actual production conditions.     

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10? CFU g?1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.