With the aim of evaluating the effect of a Mongolian dairy product derived Lactobacillus paracasei spp. paracasei (strain 06TCa22) (Lpp) on the cytokine-mediated immune responses to Vibrio harveyi infection, we examined 16 cytokine expressions in the Japanese pufferfish, Takifugu rubripes. Fish were orally treated with the heat-killed Lpp at 1 mg g(-1) body weight d(-1) for 3 days. At 24 h post treatment, fish were infected by an intramuscular injection of 0.1 mL V. harveyi bacterial suspension (10(8) cfu mL(-1)). Additionally, superoxide anion production (SAP) and phagocytic activity (PA) of head kidney cells were assessed during 120 h post infection period. Significant up-regulation of pro-inflammatory (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immune inducing (IL-12p35, IL-12p40 and IL-18), antiviral/ intra-cellular pathogen killing (I-IFN-1 and IFN-γ), anti-inflammatory (IL-10) and lymphocyte agonistic (IL-2, IL-7, IL-15, IL-21 and TGF-β1) cytokines was observed in the treated fish compared to control ones during the pathogen infection. Furthermore, significantly increased SAP and PA (P<0.01; 0.05) were recorded in the treated fish compared to untreated fish. These results suggest the beneficial role of Lpp in enhancement of cytokine-mediated immunity in the Japanese pufferfish against V. harveyi infection and application of this product as a potential fish immunostimulant.
The effectiveness of dietary beta-1,3-glucan (BG), derived from Schizophyllum commune, in modulating the non-specific immunity of the grass prawn Penaeus monodon and its resistance to white spot syndrome virus (WSSV) were investigated. Juvenile P. monodon (6.5+/-0.4 g) were fed for 20 days on a series of test diets containing graded levels of BG (0, 1, 2, 10, 20 g kg(-1)diet) and were then challenged by injection of WSSV. The haemolymph total haemocyte count (THC), phagocytosis (PI), phenoloxidase (PO), superoxide anion (O(2)(-)) and superoxide dismutase (SOD) production were measured at days 0, 1, 3, 6, 9, 12 and 24 after challenge, and shrimp survival rate was also recorded. All the shrimps fed on diets containing BG no more than 1 g kg(-1)died by day 12. Conversely, the survival rate of shrimp fed with the diet containing 10 g kg(-1)BG was significantly higher (P<0.05) by day 9 than that of the other groups. When screened by the WSSV PCR diagnostic procedure, the percentages of surviving juveniles of the BG 2, 10, 20 g kg(-1)groups that were 2-step WSSV negative, were 55, 65 and 65%, respectively. The haemolymph THC, PO, O(2)(-)and SOD production of the 2, 10 and 20 g kg(-1)BG diet groups dropped drastically immediately after the WSSV challenge but subsequently returned to normal. Therefore, oral administration of BG at an optimal level of 10 g kg(-1)diet for 20 days effectively enhanced the immune system and improved the survival of WSSV-infected P. monodon.
The immunostimulant beta-1,3 glucan was fed at 0.1% in feed for 7 days to healthy and aflatoxin B1 (AFB1)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB1, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB1 at 1.25 mg kg(-1) body weight) caused a significant (P < 0.05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB1-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB1 exposed fish. Although feeding of glucan was able to increase specific immunity, all measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.
Current knowledge on cis-regulatory elements of immune genes of shrimp is poor. In this study, the genomic sequence of the Fenneropenaeus chinensis anti-lipopolysaccharide factor (ALFFc) gene was obtained by using PCR and genome walking techniques, and the promoter was identified. The ALFFc gene contained three exons interrupted by two introns. Immune-related transcription factor binding sites recognized by nuclear factor-kappa B, octamer binding protein 1, GATA binding factor 1 and specificity protein 1 were identified in the regin from +1 to -702. The activity of ALFFc promoter was analyzed in insect sf9 cell lines. The putative promoter sequence of pALF-702 drive the expression of reporter EGFP gene successfully by adding lipopolysaccharide or (1,3)-β-D-glucan, but the shorter promoter sequence pALF-318 is only by (1,3)-β-D-glucan. The results pointed out that these transcription elements might contribute to the differences in promoter of ALFFc. Our results would provide supports for future studies to identify the functional transcription elements in the ALF promoter and to expand our knowledge on regulation of innate immune genes in Chinese shrimp.
Lipopolysaccharide (LPS)- and beta-1,3-glucan-binding protein (LGBP) complementary (c)DNA was cloned from the hepatopancreas of the giant freshwater prawn Macrobrachium rosenbergii using oligonucleotide primers and a reverse-transcription polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1389 bp encoding a protein of 378 amino acids (aa) including a 15-aa signal peptide. The calculated molecular mass of the mature protein (363 aa) was 41.2 kDa with an estimated pI of 4.73. The M. rosenbergii LGBP sequence contains (1) three putative integrin-binding motifs, (2) a glucanase motif, (3) a putative N-glycosylation site, (4) four protein kinase C phosphorylation sites, (5) four casein kinase II phosphorylation sites, and (6) a putative recognition motif. Sequence comparison showed that the deduced amino acids of LGBP of M. rosenbergii had overall similarities of 60-71% to those of known crustacean LGBPs and beta-1,3-glucan-binding proteins (BGBPs). The LGBP of M. rosenbergii was mainly expressed in the hepatopancreas. The LGBP transcript of M. rosenbergii was downregulated in haemocytes, but was upregulated in the hepatopancreas when injected with LPS and poly:IC after 12 h. The LGBP messenger (m)RNA expression of prawns in the postmolt stage was significantly upregulated in haemocytes, but downregulated in the hepatopancreas, which revealed a complementary relationship between haemocytes and the hepatopancreas in the molt cycle.
Six isonitrogenous and isocalorific diets supplemented with five different levels of beta-1,3-glucan (0.08%, 0.1%, 0.2%, 0.4% and 0.8%) were formulated and tested for marron (Cherax tenuimanus) growth, survival, organosomatic indices, osmoregulatory capacity and immunological parameters (total and differential haemocyte counts, haemolymph clotting time and bacteraemia). The sixth diet without any beta-1,3-glucan was used as a control. Each diet was provided to 18 marron (0.47 +/- 0.02 g initial weight) replicated 3 times in individual 250 L fiberglass cylindrical tanks. Each tank was provided with a biological filtration recirculating water system. After 84 days of culture, the survival and yield were higher in the marron fed 0.1% beta glucan supplemented diet. The different levels of beta glucan did not alter any of the physiological parameters of marron. However, dietary supplementation with beta glucan resulted in significantly higher (P < 0.05) total haemocyte count (THC) and granular cells. The bacteraemia rank was lower in all diets having beta glucan supplemented with more than or equal to 0.1% compared to the control and 0.08% beta glucan supplemented diets. Results suggest that dietary beta-1,3-glucan at a minimum concentration of 0.1-0.2% can improve the immune system of marron.
Time-series changes in transcript abundance of nine genes encoding important immune proteins in haemocytes or hepatopancreas of Pacific white shrimp Litopenaeus vannamei fed daily in a 1-week feeding trial diets containing three levels (0%, 0.2% or 1%) of beta-1,3-glucan from Schizophyllum commune were quantified by real-time PCR. As a whole, the immune modulation elicited by beta-glucan is bimodal, one swift reaction of up- or down-regulation occurred within 24h and a delayed regulation was commenced as late as 3-7days. Haemocyanin, crustin, prophenoloxidase (proPO) and transglutaminase (TGase) did not respond to the glucan treatment. While penaeidin 3 (Litvan PEN3) was swiftly down-regulated (0-24h), lysozyme and cytosolic manganese superoxide dismutase (cMnSOD) were swiftly up-regulated (0-24h). In contrast, the two pattern recognition proteins (PRPs), beta-glucan binding protein-high density lipoprotein (BGBP-HDL) and lipopolysaccharide/beta-glucan binding protein (LGBP), showed a delayed up-regulation. Their expressions were not maximized until as late as 72h or 7days, respectively, which coincide with the initiation of reported immune enhancement (6-24days) of PO and SOD activity, phagocytosis and superoxide anion production in penaeid shrimp receiving glucan-containing diet. These immune responses could be the downstream effects of the two PRP gene up-regulation that predispose the shrimp to a state of high immune responsiveness. Increased dosage of beta-glucan from 2 to 10gkg(-1) diet did not affect the expressions of the genes, indicating the sufficiency of beta-glucan supplementation at 2gkg(-1) diet.
A lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte and hepatopancreas of white shrimp Litopenaeus vannamei using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 1101 bp encoding a protein of 367 amino acids including a 17 amino acid signal peptide. The calculated molecular mass of the mature proteins (350 amino acids) is 39.92 kDa with an estimated pI of 4.37. Two putative integrin binding motifs (cell adhesion site), RGD (Arg-Gly-Asp) and a potential recognition motif for beta- (1-->3) linkage of polysaccharides were observed in the LGBP. Sequence comparison showed that LGBP deduced amino acid of L. vannamei has an overall similarity of 95%, 92% and 61% to that of blue shrimp Litopenaeus stylirostris LGBP, tiger shrimp Penaeus monodon BGBP and crayfish Pacifastacus leniusculus LGBP, respectively. Quantitative real-time RT-PCR analysis showed that LGBP transcript in haemocyte of L. vannamei increased in 3- and 6-h post Vibrio alginolyticus injection.
Shrimp BGBP was purified as a 100 kDa glycoprotein by affinity chromatography using immobilised heparin. BGBP bound simple carbohydrates, glycosaminoglycans like heparin sulphate and glycoproteins, but it was unable to agglutinate erythrocytes. Using an ELISA-based microplate assay, it was shown that simple carbohydrates such as n-glucose and D-mannose are competitive inhibitors of heparin sulphate binding to BGBP. Based on these properties BGBP is considered as a new type of heparin binding protein.
Pattern recognition proteins play an important role in the innate immune response of invertebrates. Herein we report the evolutionary relationships among Gram-negative bacteria binding proteins (GNBPs) that were previously identified and characterized from a wide array of invertebrates. Our results, together with those obtained in previous studies, indicate that decapod lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP/BGBP) has retained the crucial components for glucanase activity, and shares a common ancestor with GNBPs, as well as with the glucanase proteins of a wide range of invertebrates, rather than with GNBPs of some arthropods. However, experimental evidence of earlier studies suggested a lack of glucanase activity by these proteins, thus implying that during evolutionary time these proteins might have lost their glucan binding protein, but retained their glucan binding activity. The present results have also revealed that although a vast majority of the decapod LGBP/BGBP codons are constrained to purifying selection, certain codons are shown to have a higher rate of nonsynonymous substitutions per nonsynonymous site (dN) than synonymous substitutions per synonymous site (dS), indicating these codons have evolved adaptively (dN/dS>1). Although purifying selection (dN/dS<1) appears to be the major driving force in the evolution of a vast majority of LGBP/BGBP codons in decapods, the findings of several hotspots for nonsynonymous substitutions in this protein indicate host immune selection might play an important role in maintaining diversity among these ecologically diversified decapod species.
A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.
The aim of the study was to evaluate the effect of micronized β-1.3/1.6-D-glucan (BG) derived from the oyster mushroom Pleurotus ostreatus Hiratake and tetracycline antibiotic oxytetracycline (OTC) on biometrical, haematological, biochemical, and immunological indices, and histopathological changes in tissues of one- to two-year-old common carp (Cyprinus carpio L.). The fish tested were divided into five experimental groups and one control. Carp in the control group were fed commercial carp feed pellets. Fish in the five experimental groups were fed the same pellets supplemented with either OTC, a combination of OTC and BG, or BG as follows: 75 mg oxytetracycline kg(-1) bw (OTC group), 75 mg oxytetracycline kg(-1) bw and 0.5% β-glucan (OTC + 0.5% BG group), 75 mg oxytetracycline kg(-1) bw and 2.0% β-glucan (OTC + 2.0% BG group), 0.5% β-glucan (0.5% BG group), and 2.0% β-glucan (2.0 % BG group). OTC- and BG-supplemented diets and the control diet were administered to experimental and control carp for 50 days (i.e. samplings 1 - 3, the exposure period); for the following 14 days, fish were fed only control feed pellets with no OTC or BG supplementation (i.e. sampling 4, the recovery period). Blood and tissue samples were collected both during, and at the end of the study. No significant changes in biometrical indices (i.e. total length, standard length, total weight, hepatosomatic and spleen somatic index, and Fulton´s condition factor) were found in experimental carp compared to control in any sampling. In haematological indices, significant changes were found only in sampling 2, in which shifts in PCV (P < 0.01), Hb (P < 0.01), and WBC (P < 0.01), and in the counts of lymphocytes (P < 0.01), monocytes (P < 0.01), and neutrophil granulocytes-segments (P < 0.05) were revealed. As for biochemical profiling, plasma concentrations of glucose, albumins, cholesterol, natrium, and chlorides (all P < 0.01), and total proteins, lactate, phosphorus, and potassium (all P < 0.05) as well as the catalytic activity of ALP (P < 0.05) were altered in common carp. A significant change in induced (opsonized-zymosan particles, OZP) chemiluminescence (P < 0.05) in sampling 3 and no shifts in serum immunoglobilins concentration were found in the immunological analysis. Histopathological examination of skin, gills, liver, spleen, and cranial and caudal kidneys revealed no obvious specific changes in any tissue analysed. The use of β-glucans in clinically healthy aquaculture remains an issue. Nevertheless, their use in breeding endangered by stress stimuli, infectious disease, or adverse environmental factors is defensible.
Freshwater prawn Macrobrachium rosenbergii inoculated with 100 μl novel pathogen spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for alkaline phosphatase (AKP) activity, acid phosphatase (ACP) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as expressions of 7 immune related genes in hepatopancreas after 1-28 d. Hematoxylin-eosin (HE) staining showed obvious pathological features in hepatopancreas connective and epithelial tissue. Enzyme activity analyse showed that hepatopancreas AKP and ACP activity increased markedly (P < 0.05) when inoculated with spiroplasma MR-1008 after 5 d and 10 d, respectively. SOD enzyme activity changed less obviously and slightly increased at 1 day post- inoculation, but CAT activity decreased significantly after 5 d inoculation. The expression levels of lipopolysaccharide-and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglobulin (α2M), AKP, ACP, CAT, and copper/zinc SOD (Cu, Zn-SOD) genes in the hepatopancreas were examined by Real Time-PCR (qRT-PCR) and the results demonstrated that these immune-related genes were induced by challenge with spiroplasma MR-1008. The results suggested that the prawn immune responses could be activated or inhibited by spiroplasma MR-1008, and that the hepatopancreas also plays key roles in innate immunity for defense against the pathogen.
Lectins play important roles in crustacean innate immunity through recognition of foreign pathogens. In this study, 20 lectins including C-type lectins [dual-carbohydrate recognition domain (CRD) type and single-CRD type], L-type lectin, and lectin with low-density lipoprotein class A (LDLa) domain were identified from the freshwater prawn Macrobrachium rosenbergii. The tissue distribution and expression patterns of these lectins under spiroplasma strain MR-1008 challenge were investigated. Most of the lectins were found to be mainly distributed in the hepatopancreas. Lectin5, Lectin14, Lectin17, and Lectin18 exhibited the highest expression level in the hemocytes, nerve, intestine, and heart, respectively. MrLec1 to MrLec6 (dual-CRD lectins) in the hepatopancreas were up-regulated by spiroplasma challenge. Single-CRD lectins reached the highest level at 72 h after spiroplasma challenge. Lectin9 and Lectin15 both belong to L-type lectins. At post-spiroplasma challenge, Lectin9 expression was up-regulated, whereas Lectin15 expression was down-regulated. Lectin11 with LDLa domain showed the highest level after 12 h. Lectin18 and Lectin20, namely, CD209, were also up-regulated by spiroplasma challenge. Lectin14, a C-type lectin, quickly reached the highest level after 2 h. Lectin16 showed the highest level after 72 h. Lectin5 reached the highest level in cultured hemocytes after 6 h. Lectin17 in the intestine and Lectin14 in the nerve were slightly up-regulated after 6 and 2 h, respectively. Our research results indicate that lectins may play important roles in early or late immune responses against spiroplasma challenge.
Flow cytometry provides rapid and reproducible methods for analyzing crustacean cellular immune responses to pathogens. We used this method to investigate the hemocytes sub-populations of freshwater prawn Macrobrachium rosenbergii and their immune responses to a novel pathogen spiroplasma MR-1008. M. rosenbergii inoculated with 100 μl spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for total hemocytes count (THC) and changes in differential involvement of hemocytes sub-populations during 1-28 d after inoculation. The results showed that THC was dramatically lowered 1 d after inoculation, and it obviously increased at the 5 d after inoculation; thereafter, a high level of THC was maintained to 15 d. Three morphologically distinct hemocytes sub-populations including granular cells (GC), semigranular cells (SGC) and hyaline cells (HC) could be identified by flow cytometry, and the proportions of the 3 kinds of cell categories varied obviously during the infection of spiroplasma suggesting differential involvement according to the pathogen. The flow cytometry used in this study confirmed that the semigranular cells were the main hemocytes involved in the cellular defense against spiroplasma in the M. rosenbergii.
Occurrence of widespread epizootics among cultured stock of shrimp has put research programmes on preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study two bacteria, Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 were selected as candidate probionts from a pool of bacteria isolated from gut of farm reared sub-adult shrimp and tested for their effect on the immunity indicators of tiger shrimp. Sub-adult shrimp, weighing 14 to 22 g were treated in separate experiments with Pseudomonas sp. PM 11 and V. fluvialis PM 17 at 10(3) bacterial cells ml(-1) in the experimental shrimp culture tanks. One set of experimental animals was treated every 3 days and another set of animals every 7 days with each of the candidate probionts. Estimation of immunological indicators such as haemocyte counts, phenol oxidase and antibacterial activity showed declining trends. The haemocyte counts dropped from 31 x 10(3) to 65 x 10(3) ml(-1) on the first day to 4-16 x 10(3) ml(-1) on the 45th day. Similarly, the phenol oxidase activity declined from 12-32 units on the first day to 11-14 units on 45th day of the experiment. Antibacterial activity of haemolymph reduced to 46-67 percent on the 45th day of the experiment. The results of the study suggest that, the criteria used for the selection of putative probiotic strains in the present study, such as predominant growth on primary isolation media, ability to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Hence, new protocols have to be evolved for selection of microbe(s) as putative probiotics and that, detailed understanding of proven probiotics, employed presently on empirical basis may provide a clue on the selection procedure.
The immunotoxicity of chemical combinations commonly encountered by the lake trout (Salvelinus namaycush) immune system was the focus of this study. It was hypothesised that combinations of an environmental contaminant (mercuric chloride or Aroclor 1254) and an immunomodulatory agent (bacterial endotoxin or cortisol) might interact to produce a greater toxicity than that of the environmental contaminant alone at concentrations typically encountered in piscine blood and other tissues. Thus lake trout thymocytes were isolated and treated with mercuric chloride or Aroclor 1254 in the presence and absence of cortisol or lipopolysaccharide. Incubations were performed for 6 or 20 h at 4 degrees C or 10 degrees C. Lipopolysaccharide did not affect the toxicity of either contaminant. In contrast, cortisol enhanced the toxicity of both environmental contaminants. Hence, stressors that lead to increased cortisol production, but not lipopolysaccharide directly, may increase the toxicity of mercury and Aroclor 1254 to lake trout thymocytes.
The toxic effects of Aroclor 1254 (0.05, 0.5, 5 and 50 microg l(-1)) on scallop (Chlamys farreri) immune system in vivo were studied. The results showed that Aroclor 1254 had significant toxic effect on the parameters tested in this paper (P<0.05). The total number of haemocytes, the proportion of granulocytes, phagocytosis in all groups as well as the lysosomal membrane stability (LMS) in 5, 50 microg l(-1) and bacteriolytic activity 0.5, 5, 50 microg l(-1) treatments decreased significantly, while the proportion of hyalinocytes and the production of O2(-) in all treatments remarkably increased during the sampling time and tended to be stable gradually after 6-15 d. The bacteriolytic activity in 0.05 microg l(-1) treatments, LMS in 0.05, 0.5 microg l(-1) groups and the DNA damage (comet ratios and arbitrary values) in all treatments increased at the beginning of exposure and reached their peaks on day 1, day 1, day 6 and day 3, following that they all decreased gradually and became stable after 9-15 d. When the indices reached stability, except for DNA damage was higher than controls, the others were all significantly lower than those of controls (P<0.05). Thus, Aroclor 1254 has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms. Also it supports the speculation that the PCBs pollution is one of the important reasons of the mass mortality of the C. farreri.
The growth of the lymphoid organs, such as head kidney, spleen and thymus were studied in flounder, Paralichthys olivaceus Temminck & Schlegel, from hatching to 13 months of age. Except for the thymus, all organs grew as the fish grew. By 2 months of age the lymphoid organs attained their maximum relative weight. The organ weight showed a closer correlation to body weight than they did to age. The total number of leucocytes in the lymphoid organs increased with age, but the number per milligram of lymphoid organ remained constant. A micro and ultrastructural study of the lymphoid organs showed that the full development of the lymphoid organs was not achieved until the juvenile stage. The spleen and head kidney had mixed populations of "red" and "white" cells. The head kidney was more lymphoid than the spleen. The thymus involuted quickly during the first 6 months. The blood components had no obvious relationship with age or season during the period studied.
The efficacy of immersion vaccination Yersinia ruckeri bacterin containing Montanide(TM) IMS 1312 VG was evaluated in 100-120 g rainbow trout against yersiniosis. Healthy fish were vaccinated by immersion vaccination with inactivated whole cells (1 × 10(8) cells/ml) of a virulent strain of Y. ruckeri biotype I with and without Montanide (1:1; Montanide/antigen) for 2 min at 12-14°C. Control group was immersed in sterile PBS. Leukocyte counts, serum lysozyme assay, alternative hemolytic complement (ACH50) assay, antibody titration and relative percent survival (RPS) were measured on 2-10 weeks post-immunization. No significant difference was seen in leucocyte population of trout immunized either with Y. ruckeri antigen or Y. ruckeri antigen containing Montanide (P>0.05), while leucocyte and heterophil populations in control group were significantly lower and higher, respectively, than both immunized groups (P<0.05). In addition, there was no significant difference in lymphocyte population of trout immunized either with Y. ruckeri antigen or Y. ruckeri antigen containing Montanide (P>0.05), while lymphocyte population in control group was significantly lower than both immunized groups (P<0.05). Lysozyme activity in immunized fish with Y. ruckeri containing Montanide was higher than the immunized fish with Y. ruckeri antigen alone during 8 weeks post-immunization ((P<0.0.5).Also, level of lysozyme in control fish was generally lower than both immunized groups (P<0.05). The level of ACH50 between both immunized groups was insignificant (P>0.05) but these were significantly higher than control group through the experiment (P<0.05). The lowest anti-Y .ruckeri antibody titers in both immersion vaccination groups were significantly higher through 2-8 weeks post-vaccination compared to the control group (P<0.05). In the group immersion vaccinated with Y. ruckeri bacterin plus Montanide the titers 2-8 weeks post-vaccination were significantly higher the titer in the immersion vaccinated with Y. ruckeri bacterin (P<0.05).Fish vaccinated with antigen without Montanide resulted in RPS of 80-82% on 2-10 weeks post-vaccination, while those for antigen containing montanide gave RPSs of 93.8-100% 2-10 weeks post-immunization (P<0.05).
Ceruloplasmin is an acute phase protein found to be activated by the host immune system during stress conditions. The ceruloplasmin gene has been reported in several teleosts and here we characterize the gene and test its association with resistance to Aeromonas hydrophila in rohu, Labeo rohita. A ceruloplasmin mRNA sequence of 3355 base pairs was derived (GenBank ID: JX010736). The complete coding sequence (CDS) comprised of 3276 bp nucleotides that coded for 1092 amino acids. Alignment results showed the greatest similarity with zebrafish followed by channel catfish sequences, and a phylogenetic tree constructed on the basis of amino acid sequences showed that rohu shares a common clade with these two species. In the ontogeny study, the expression of ceruloplasmin was detected at 9 h post-fertilization onwards, and a strong level of expression was detected at 24 h (38-fold) and 15 days (34-fold) post-fertilization. The ceruloplasmin transcripts were evident in liver, spleen, stomach and heart. Expression was undetectable in gill, brain, eye, skin, muscle, intestine, anterior and posterior kidney tissues. Expression of ceruloplasmin after A. hydrophila infection was up-regulated 6 h post-challenge that was modulated until 15 days post-challenge. The level of ceruloplasmin was also compared in rohu selectively bred for higher growth and disease resistance. The gene showed a 3.7-fold higher level of expression in resistant line over susceptible line rohu selected based on family challenge test survival to A. hydrophila. Serum ceruloplasmin levels in three year classes of rohu selected for higher growth showed a positive correlation (0.49 ± 1.11) with survival against challenge with A. hydrophila. The estimated heritability was also found to be quite high (0.50 ± 0.22) for this parameter. Thus, ceruloplasmin could be one of the useful marker traits for selection against A. hydrophila resistance in fish.
Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.
Thioredoxin (abbreviated as Trx) is an important ubiquitous disulfide reductase, which can protect organisms against various oxidative stresses. In the present study, a thioredoxin-related protein of 14 kDa (named as Ec-TRP14) was identified from the marine fish grouper, Epinephelus coioides by RACE PCR. The full-length cDNA of Ec-TRP14 was comprised of 1066 bp with a 372 bp open reading frame that encodes a putative protein of 123 amino acids. Similar to most TRP14s, Ec-TRP14 contained the conserved motif C-P-D-C. Ec-TRP14 mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-TRP14 was up-regulated in the liver of grouper challenged with SGIV. Ec-TRP14 was recombined and expressed in E. coli BL21 (DE3), and the rEc-Ec-TRP14 fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-TRP14 (8.3 μg/ml), which significantly enhanced cells viability under damage caused by viral infection. These results together indicated that Ec-TRP14 could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge.
Effects of dietary administration of Bacillus amyloliquefaciens FPTB16 on systemic and mucosal immunity and disease resistance of catla (Catla catla) against Edwardsiella tarda infection were evaluated in the present study. The laboratory maintained B. amyloliquefaciens was used to study antagonistic activity against fish pathogenic bacteria by agar well diffusion assay. Healthy catla were challenged by this bacterium for determination of its safety. For preparation of probiotic supplemented diet, the bacteria were added to the basal diet (control) at three different inclusion levels i.e., 1×10(9), 1×10(8) and 1×10(7) CFU/g diet. Fish (weight 25-30 g) were fed with these diets and various immune parameters and disease resistance study were conducted at 4 weeks and 8 weeks post-feeding. The bacterial antagonism study showed inhibition zone against E. tarda, Aeromonas hydrophila, Vibrio parahaemolyticus and V. harveyi. B. amyloliquefaciens was harmless to catla as neither mortalities nor morbidities were observed after the challenge. Study of different systemic and mucosal immunological parameters viz. superoxide anion production and nitric oxide production, myeloperoxidase content, lysozyme activity and total protein content showed significant enhancement (p < 0.05) in fish fed with 10(8) and 10(9) CFU/g B. amyloliquefaciens at both time points with the highest values observed in case of 10(9) CFU/g. For fish fed with 10(7) CFU/g B. amyloliquefaciens, all the parameters showed significant enhancement (p < 0.05) at both time points except the lysozyme activity of serum at 8 weeks. Diet containing 10(8) and 10(9) CFU/g B. amyloliquefaciens significantly enhanced (p < 0.05) the resistance of catla against bacterial challenge at both time points. These results collectively suggest that B. amyloliquefaciens is a potential probiotic species and can be used in aquaculture to improve health status and disease resistance with an optimal dietary supplementation of 10(9) CFU/g.
In fish, interleukin (IL)-2, IL-21 and IL-15 genes have recently been identified by in-silico cloning. Fish IL-15 gene is similar to counterparts from mammals and other vertebrates. A zebrafish genomic database-search initiated to find IL-2 and IL-21 genes from zebrafish (Danio rerio) led to the identification of an IL-15 like gene (IL-15L). This gene was cloned by prediction and the transcripts were subsequently cloned by PCR. The predicted translation yielded a 162 amino acid protein with a 42 amino acid-long signal peptide. This protein shared identities of 28.4% to 31.5% with other mammalian and vertebrate IL-2, IL-15 and IL-21 genes. The gene occupies 7.7 kb of the genomic DNA and the coding region spans into four exons and is interrupted by three introns, which is similar to the genomic structure of IL-2 gene. The chromosomal synteny and phylogenetic analyses support our view that this IL-15L gene is specific to teleosts. Furthermore, two alternatively spliced forms have been identified with differential exon usage translating for proteins of 108 and 120 amino acids in length. The analysis of the alternative splicing suggests it may play an important role in regulating the function of this novel gene. Analyses by RT-PCR and in situ hybridization show gene expression in lymphoid tissues like intestine, gills, spleen, pancreas and kidney suggestive of a role in immunity of fish.
The alpha-chain of IL-15 receptor complex serves as a high-affinity, specific subunit for IL-15 binding. It shares functional and structural features with IL-2 receptor alpha-chain. Here we report for the first time that the molecular cloning, characterization and sequence analysis of the teleostean IL-15R alpha from Oncorhynchus mykiss. The full-length cDNA comprises of a 5' untranslated region (UTR) of 167 bp, an open reading frame of 732 bp and a large 3'UTR of 630 bp, constitutively expressed in all the tissues examined. Another two variants are found derived from alternative splicing. Two copies of rainbow trout IL-15R alpha (rtIL-15R alpha) were detected in the genome by Southern blot hybridization. Moreover, EST evidence is also traced from other fishes. The deduced rtIL-15R alpha of 243 amino acids contains a 17 aa signal peptide at N-terminus and a transmembrane region at C-terminus, as well as a typical sushi domain included in the extracellular region. Phylogenetic tree analysis groups rtIL-15R alpha with other IL-15R alphas but separates it from IL-2R alphas. In addition, a putative sequence was found in the sushi domain which is conserved and versatile among all species and this might relate to cytokine recognition.
IL-17 from pearl oyster Pinctada fucata, one of mollusk, was identified and characterized, and its genomic structure and promoter were analyzed. The full-length cDNA of P. fucata IL-17 (PfIL-17) is 907 bp with an open reading frame of 585 bp encoding a putative protein of 194 amino acids. The deduced PfIL-17 contains a 19 amino acid signal peptide and a conserved IL-17 domain. Multiple sequence alignments and phylogenetic analysis revealed that PfIL-17 has lower similarity with other invertebrate IL-17 and was clustered with CgIL-17, but not clustered with other invertebrate IL-17. Gene expression analysis indicated that PfIL-17 took part in the immune response to LPS and poly (I:C) stimulation, and dual-luciferase reporter assays showed that PfIL-17 could active vertebrate target genes containing the NF-κB binding site and involve NF-κB signal pathway in HEK293 cells. Combined with the results mentioned above, it is suggested that PfIL-17 might involve and activate NF-κB signal pathway against extracellular pathogens.
Hepcidin is a highly conserved antimicrobial peptide and iron-regulatory hormone. Here, we identify two hepcidin genes (hep-1 and hep-2) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Hepcidin-1 contains a putative ATCUN metal-binding site in the amino-terminus that is missing in hepcidin-2, suggesting that hepcidin-1 may function as an iron-regulatory hormone. Both hepcidins are predominately expressed in the liver of largemouth bass, similar to other fish and mammals. Experimental exposure of pond-raised largemouth bass to 17beta-estradiol and/or the bacteria Edwardsiella ictaluri led to distinct changes in expression of hep-1 and hep-2. Estradiol reduced the constitutive expression of hep-1 in the liver. Bacterial exposure induced expression of hep-2, suggesting that hepcidin-2 may have an antimicrobial function, and this induction was abolished by estradiol. To our knowledge, this is the first report of the regulation of hepcidin expression by estradiol in either fish or mammals.
Ubiquitin-conjugating enzymes (UBE2s or E2s) are characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain, which predominantly determines the type of ubiquitin chains and directly controls the cellular fate of the substrate. In this study, an E2 homologue was identified and functionally characterized in abalone, which we named ab-UBE2D. The full-length cDNA consists of 1005 bp with an ORF encoding a protein of 147 amino acids. The deduced amino acid sequence shows ab-UBE2D shares conserved UBC domain with other E2 proteins and belongs to classⅠE2 enzyme family, which are further confirmed by phylogenetic tree analysis. Real-time PCR and western blot analyses showed that ab-UBE2D was ubiquitously expressed in abalone and the expression level of ab-UBE2d was significantly induced by LPS and Poly (I:C). Immunofluorescence microscopy staining demonstrated that native ab-UBE2D was mainly distributed in the cytoplast. Ubiquitination assay showed that ab-UBE2D had ubiquitin conjugating activity to form the enzyme-(Ub)n conjugates. Taken together, these results strongly suggest that ab-UBE2D is an E2 homologue and it may be involved in the immune response of abalone, H. diversicolor supertexta.
Cytokines are one of the major signaling molecules involved in immunity. Many of these cytokines have been isolated in vertebrates and found to play a significant role in host defense mechanism. Interleukin-17 (IL-17) family of genes are known to have pro-inflammatory actions and associated with specific disease conditions, these genes are conserved across vertebrate evolution. In this study, computational screening for the zebrafish (Danio rerio) genome resulted in identification of five contigs harboring IL-17 genes. Zebrafish cDNA encoding five IL-17 genes exhibited percentage identities of 19.3%-61.9% with that of human homologs. The molecules show conservation of cysteines, important for disulphide bonds for IL-17 molecules. The structural composition of these genes shows two introns and three exons except for IL-17D gene that has only one intron and two exons. Phylogenetic analysis using maximum parsimony algorithm showed that zebrafish IL-17 genes clustered well with other IL-17 homologs further proving the structural similarity with IL-17 genes from other organisms. Expression analysis by RT-PCR revealed expression of IL-17 genes in normal and stimulated tissues of kidney, spleen, gills and intestine. The expression of IL-17 in un-stimulated tissues indicates that these genes may play important roles in normal conditions as well.
In humans, the IL-17 family is composed of six members (A-F). The A, E and F forms have been extensively studied in numerous mammalian species. However, there are few reports regarding IL-17 expression in teleost. In this study, IL-17 family genes were isolated from the Japanese pufferfish (Fugu) and their structure and expression profile were analyzed. Screening of the Fugu genome database revealed the existence of five scaffolds containing IL-17 family homologous genes. Scaffold_1 contained three IL-17 family homologues including IL-17A/F1, 2 and C2, and IL-17A/F1 and two located in tandem. This was similar to the IL-17A/F1 and two genes in zebrafish and to human IL-17A and F. Other scaffolds 38, 143 and 430, contained IL-17 family homologous genes that were identical to IL-17D, A/F3 and C1 in Fugu, respectively. Moreover, IL-17 family homologues on scaffold_264 included a novel type of IL-17 family genes in teleost. These isolates contained four cysteine residues that were involved in the formation of a typical cysteine knot consisting of two disulphide linkages. However, IL-17A/F2 did not demonstrate any conservation at the second and fourth cysteine residues. The tissue distribution of the Fugu IL-17 family genes was also found to differ. In particular, IL-17 family genes were highly expressed in the head kidney and gill. Moreover, expression of IL-17 family genes was significantly up-regulated in the lipopolysaccharide-stimulated head kidney. These results suggested that Fugu IL-17 family members were involved in inflammatory responses.
Interleukin-17 (IL-17) is a cytokine family composed of six ligands (A-F). Especially, the IL-17A and IL-17F are best characterized cytokines of IL-17 family cytokine. These are produced by Th17 cells and induce the expression of many mediators of inflammation properties. In addition, the five member of IL-17 receptor family (RA-RE) have been identified in mammals. Although the research on fish IL-17 is a little to date, this review discusses some of the recent advances in research on IL-17 ligand and receptor genes in fish. IL-17 family member was chosen from the fish genome database, and its structure and phylogeny is analyzed in detail. Moreover, invertebrate IL-17 genes are also discussed, and the isolation and current status of fish IL-17 receptor genes are summarized. Comparative genomic analysis of the IL-17 family among mammals, teleost and invertebrates provided new insights. Novel IL-17 ligand (IL-17N) was identified from teleost, moreover it was suggested that IL-17N may be a teleost specific ligand by synteny and phylogenetic analysis. On the other hand, IL-17 receptors are well conserved between mammal and teleost, the five member of IL-17 receptor family: IL-17RA-RE were found on the teleost genome. In addition, the IL-17RA gene was duplicated in tandem on the stickleback and medaka genome. Knowledge about the IL-17 ligand/receptor in fish is very limited. Therefore this review will hopefully encourage future studies of IL-17 in fish.
The candidate genes interleukin-1 receptor associated kinase 4 (IRAK-4), Interleukin 17 (IL-17) and Inhibitor of NF-κB (I-κB) were cloned and evaluated in Californian abalone (Haliotis rufescens) hemocytes in response to Vibrio anguillarum. Molecular characterization evidenced that HrI-κB has a full cDNA sequence of 3,027 bp with an encoding region of 401 amino acids (aa), HrIRAK-4 comprised 1,969 bp that encoded for 516 aa, and Hr-IL17 had a full sequence of 806 bp encoding for 165 aa. qPCR analysis showed the higher constitutive expression level of Hr-IL17 in hemocytes; meanwhile Hr-IκB and Hr-IRAK4 gene expression levels were higher in gills and mantle. The assessment of gene expression in hemocytes after infection with V. anguillarum evidences the immune responses of Hr-IκB, Hr-IRAK4, and Hr-IL17 and their relationships through the NF-κB signaling pathway.
Immunoglobulin of the torafugu, Takifugu rubripes, was purified by a combination of precipitation by low ionic strength dialysis and gel filtration. The Ig was used to immunise mice for the production of monoclonal antibody (MAb). Supernatants of hybridoma cultures were screened by enzyme-linked immunosorbent assay using purified-torafugu Ig-coated plates, and two stable hybridomas producing MAbs against torafugu Ig were obtained. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting indicated that one MAb (16F3) was specific for the deglycosylated heavy chain of torafugu, and the other MAb (4H5) did not bind to the reduced Ig, suggesting that 4H5 recognised the higher-order structure of Ig. Under non-reduced conditions, both MAbs recognised mainly a 750 kDa band and also minor bands of 672, 410 and 205 kDa. MAb 16F3- and 4H5-primed magnetic beads (Dynabeads) adsorbed 84.9+/-3.3% and 63.6+/-4.4% of the torafugu Ig, respectively. The Ig adsorbed by MAb 16F3-primed Dynabeads was reactive to 4H5 on immunoblotting, and vice versa, indicating that the epitopes for both MAbs are held on the same Ig molecule. Both of these MAbs cross-reacted extensively with the Ig of other Takifugu species, but not with other genus. The MAbs were used to identify surface Ig-positive lymphocytes in the spleen, pronephros, peripheral blood and thymocytes of torafugu by flow cytometry. Flow cytometric analysis of the cells in the lymphocyte-enriched fraction revealed that 50.2+/-6.9% in the PBL, 11.8+/-1.7% in the mesonephros, 13.3+/-2.1% in the pronephros, 42.5+/-4.3% in the spleen and 3.2+/-0.6% in thymus were reactive to 4H5 or 16F3.
A novel member of Cystatin superfamily was identified from Indian catfish, Clarias batrachus, in response to oxidation stress induced by environmental hypoxia. Integrated genomic approaches, expression profiling and computational techniques showed that CbCystatin had putative cystatin/monelin like domain and might be a transmembrane and/or intermediate protein in signaling pathways. CbCystatin was found to be clustered into family 2 Cystatins. At transcriptional level, its expression was significantly up-regulated in response to short as well as long periods (more than 20 fold) of hypoxia, suggesting its positive association with oxygen concentrations lower than physiological concentrations.
The innate immune system is a fundamental defense weapon of fish, especially during early stages of development when acquired immunity is still far from being completely developed. The present study aims at looking into ontogeny of innate immune system in the brown trout, Salmo trutta, using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs and hatchlings at 0, 1 h and 1, 2, 3, 4, 5, 6, 7 weeks post-fertilization was subjected to RT-PCR using self-designed primers to amplify some innate immune relevant genes (TNF-α, IL-1β, TGF-β and lysozyme c-type). The constitutive expression of β-actin was detected in all developmental stages. IL-1β and TNF-α transcripts were detected from 4 week post-fertilization onwards, whereas TGF-β transcript was detected only from 7 week post-fertilization onwards. Lysozyme c-type transcript was detected early from unfertilized egg stage onwards. Similarly, tissues such as muscle, ovary, heart, brain, gill, testis, liver, intestine, spleen, skin, posterior kidney, anterior kidney and blood collected from adult brown trout were subjected to detection of all selected genes by RT-PCR. TNF-α and lysozyme c-type transcripts were expressed in all tissues. IL-1β and TGF-β transcripts were expressed in all tissues except for the brain and liver, respectively. Taken together, our results show a spatial-temporal expression of some key innate immune-related genes, improving the basic knowledge of the function of innate immune system at early stage of brown trout.
The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.
Antimicrobial peptides (AMPs) are a pivotal component of innate immunity in lower vertebrates. The aim of this study was to develop an immunological method for quantifying AMPs in Salmo salar skin mucus. A known antimicrobial peptide derived from histone H1 previously purified and described from S. salar skin mucus (SAMP H1) was chemically synthesized and used to obtain antibodies for the quantification of the molecule via ELISA. Using skin mucus samples, a correlation of bacterial growth inhibition versus SAMP H1 concentration (ELISA) was established. The results provide the first evidence for quantifying the presence of active AMPs in the skin mucus of S. salar through the use of an immunological method.
The grafting process used for pearl production in pearl oysters triggers a significant haemocyte response which has an influence on the quality of pearls formed. One hundred and ten selected healthy adult Pinctada margaritifera were grafted for pearl production. Beginning two days after grafting, oysters were sacrificed regularly until the 48th day and the pearl-sacs of sampled oysters were sectioned for histological analysis. The level of haemocytes present in the pearl-sacs decreased overtime with the samples from day 2 showing the highest levels. Haemocyte levels also varied between samples from a particular day. The exact cause(s) of varying levels of haemocyte accumulation during pearl-sac development in P. margaritifera is not known. However, it is reasonable to assume that haemocyte production is positively related to the degree of damage caused to host oyster tissues during the grafting procedure. While haemocytes have an important wound healing role in pearl oysters, excessive haemocyte presence may be detrimental to maximizing pearl quality.
The high export value of the Indian spiny lobster Panulirus homarus increasingly attracts the aquaculturists for farming and fattening. However, lack of knowledge on the effect of environmental parameters on the immune system of this animal could result in high mortality, which ultimately may cause major loss to the industry. Here, we report the effect of salinity (20, 25, 35, 40, and 45 per thousand), pH (5.0, 8.0, and 9.5), dissolved oxygen (DO) (1 and 5 mg L(-1)), and ammonia-N concentration (0, 0.5, 1.5 and 3 mg L(-1)) on the immune response of P. homarus measured in the haemolymph in terms of Total Haemocyte Count (THC), phenoloxidase (PO) activity, and NBT-reduction. Our data showed significant reduction (P<0.05) in THC, and NBT-reduction at lower (20 per thousand) and higher (45 per thousand) salinities. However, PO activity showed significant disparity, showing an increasing trend from 20 to 45 per thousand. Significant reduction (P<0.05) in THC and PO activity under acidic and alkaline conditions, under hypoxic condition (1 mg L(-1)), and at the higher ammonia-N concentrations than their respective optimal conditions were observed. Thus, suggesting that extreme environmental parameters can induce modifications in the immune system of the spiny lobster P. homarus, which may enhance their susceptibility to opportunistic pathogens. The humoral parameters such as THC, PO activity, and NBT-reduction can be used as potential stress indicators for healthy management of spiny lobsters.