Spleen Of Fish Research Articles

Molecular characterization, functional analysis, and defense mechanisms of two CC chemokines in Nile tilapia (Oreochromis niloticus) in response to severely pathogenic bacteria

Two full-length cDNAs encoding CC chemokine genes in Nile tilapia (Oreochromis niloticus) (On-CC1 and On-CC2) were cloned and characterized. On-CC1 and On-CC2 showed signature cysteine motifs consisting of four cysteines. The expression levels of On-CC1 and On-CC2 were analyzed by RT-PCR, which showed that low expression of these two genes was only observed in the peripheral blood leukocytes (PBLs) and spleen of normal fish. Expression levels of these two molecules were quantified in 13 tissues of fish infected with virulent strains of Streptococcus agalactiae and Flavobacterium columnare. Most tissues, especially PBLs, the spleen and the liver, expressed significantly higher mRNA levels than the controls, particularly at 12 and 24 h after infection (P < 0.05). The current study strongly indicates that CC chemokine genes in Nile tilapia are crucially involved in the early immune responses to pathogens. Functional analyses clearly demonstrated that 10 and 100 μg/ml of recombinant rOn-CC1 and rOn-CC2 proteins efficiently enhanced the phagocytic activity (in vitro) of Nile tilapia phagocytes. Finally, Southern blot analysis and searching in Ensembl databases demonstrated that two different functional CC chemokine genes and other pseudogene fragments were discovered in the Nile tilapia genome.

Starvation: An Alternate Measure to Improve Immunity and Physiology of Red Sea Bream During Edwardsiella Tarda Infection

Dietary restrictions during infectious challenges are quite common in animal kingdom. In the present investigation, we aimed to explore the positive implications of short-term starvation in Edwardsiella tarda infected red sea breams. Starvation resulted in depleted transcription of several iron binding protein (Hepcidin, Transferrin), which could have reduced the bacterial colonization in starved- infected fish. This was confirmed by the significantly (P<0.05) low bacterial load in the spleen and muscle of starved-infected fish. Gills showed mild damage to the secondary filaments architecture as well as elevated mucus production in the starved-infected fish compared to the fed ones. Massive mucus cell hyperplasia was observed in starved-placebo fish, which further increased after infection. Decreased activities of serum anti-oxidative enzymes and reduced total antioxidant capacity after starvation was suggestive of improved stress response and heightened stress withstanding capacity of these fish. Relatively higher haemoglobin and phagocytic activity along with the increased cytokines (TNFα, IL- 1β) level in starved-infected groups than their fed counterparts indicated the better immune condition of the former group. Additionally, our data also demonstrated that starvation enhanced the survivability and overall disease resistance index of infected fish, indicating that short period of starvation might be a beneficial measure to fight against infections.

Irradiation of rainbow trout at early life stages results in trans-generational effects including the induction of a bystander effect in non-irradiated fish

The bystander effect, a non-targeted effect (NTE) of radiation, which describes the response by non-irradiated organisms to signals emitted by irradiated organisms, has been documented in a number of fish species. However transgenerational effects of radiation (including NTE) have yet to be studied in fish. Therefore rainbow trout, which were irradiated as eggs at 48h after fertilisation, eyed eggs, yolk sac larvae or first feeders, were bred to generate a F1 generation and these F1 fish were bred to generate a F2 generation. F1 and F2 fish were swam with non-irradiated bystander fish. Media from explants of F1 eyed eggs, F1 one year old fish gill and F1 two year old fish gill and spleen samples, and F2 two year old gill and spleen samples, as well as from bystander eggs/fish, was used to treat a reporter cell line, which was then assayed for changes in cellular survival/growth. The results were complex and dependent on irradiation history, age (in the case of the F1 generation), and were tissue specific. For example, irradiation of one parent often resulted in effects not seen with irradiation of both parents. This suggests that, unlike mammals, in certain circumstances maternal and paternal irradiation may be equally important. This study also showed that trout can induce a bystander effect 2 generations after irradiation, which further emphasises the importance of the bystander effect in aquatic radiobiology. Given the complex community structure in aquatic ecosystems, these results may have significant implications for environmental radiological protection.

Validation of Methods Employing Fast Alkaline Solubilization to Determine Cadmium in Fish Liver, Spleen, Gills and Muscle by Graphite Furnace Atomic Absorption Spectrometry

The validations of analytical methods were performed to determine Cd in fish liver, spleen, gill, and muscle by graphite furnace atomic absorption spectrometry (GF AAS). The methods were based to alkaline solubilization of the samples with benzyltrimethylammonium hydroxide 40% v/v in water (Universol®), and when added to each sample, immediately solubilized the samples and formed an indefinitely stable and highly homogeneous solution, without a need for temperature, microwaves or any other apparatus. Ru was the best permanent modifier (500μg) for all samples except for liver, while permanent Ir (500μg) proved best for liver. The optimum pyrolysis temperatures were 450°C for liver and spleen, 500°C for gill, and 400°C for muscle, while for atomization, the optimum temperatures were 1800, 1100, 1600, and 900°C, respectively. No matrix effects were observed for liver and gill when external calibration was performed. For spleen and muscle, matrix effects were observed and matrix matching calibration was used. The limits of quantifications were 0.13, 1.20, 0.34, and 1.80μgg−1 for liver, spleen, gill, and muscle, respectively. The absorption signals obtained for each sample were symmetrical and returned to baseline in less than 5s. Recoveries levels (three levels of Cd for each matrix) ranged from 87.1 to 108.2. For the analysis of seven certified reference materials, the obtained values (n=5 samples of each material) were not statistically different from the certified values (95% of confidence). This reagent (Universol®) promoted an immediate solubilization of the samples, thus forming an indefinitely stable suspension which can be an alternative for fast solubilization of any biological sample, such as food and other organic material.

Effects of dietary linseed oil on innate immune system of Eurasian perch and disease resistance after exposure to Aeromonas salmonicida achromogen.

This study was designated to investigate the effects of dietary fish oil (FO diet) replacement by linseed oil (LO diet) on regulation of immune response and disease resistance in Eurasian perch (Perca fluviatilis). A control diet containing fish oil (FO = cod liver oil) and characterized by high levels of n-3 high LC-PUFA (6% EPA, 7.5% of total fatty acids (FAs)) was compared to linseed oil diet (LO diet) composed of low LC-PUFA contents (1% EPA, 2.3% DHA of total FAs) but high C18 fatty acids levels. The experiment was conducted in quadruplicate groups of 80 fish each. After 10 weeks of feeding, the innate immune status was evaluated in various organs (liver, spleen, and head-kidney) (feeding condition). Two days later, a bacterial challenge was performed on fish from 2 rearing conditions: fish infected with Aeromonas salmonicida (bacteria condition) and fish injected with sterile medium but maintained in the same flow system that fish challenged with bacteria (sentinel condition). Three days after injection of bacteria, a significant decrease of lymphocyte, thrombocyte and basophil populations was observed while neutrophils were not affected. In addition, plasma lysozyme activity and reactive oxygen species production in kidney significantly increased in fish challenged with A. salmonicida while the plasma alternative complement pathway activity was not affected. Increase of plasma lysozyme activity as well as reactive oxygen species production in spleen and kidney of sentinel fish suggest that these immune defenses can also be activated, but at lower bacteria concentration than infected fish. No differences in leucocyte populations, plasma lysozyme and alternative complement pathway activities were observed between dietary treatments. Similarly, expression of genes related to eicosanoid synthesis in liver were not affected by the dietary oil source but were strongly stimulated in fish challenged with A. salmonicida. These findings demonstrated that the use of linseed oil does not deplete the innate immune system of Eurasian perch juveniles.

Evaluation of lymphoid tissue structure in Sole (Euryglossa orientalis) and Yellowfin Seabream (Acanthopagus latus) affected by environmental contaminants in the Persian Gulf

This study sought to analyze structures of lymphatic tissues in two commercial fish species, e.g. Sole (Euryglossa orientalis) and Yellowfin Seabream (Acanthopagus latus), collected from five stations with varying levels of pollution in the Musa Creek near the Persian Gulf, e.g. Petro-chemical, Gaafari, Majidieh, Ghazaleh and Zangi Stations. Samples from Genaveh Station located outside Musa Creek were collected as controls. To correlate findings of changes in the studied tissues with local pollution status, levels of Hg, Pb, Zn, Cu and Cd were measured in sediments and water at each station. Fish were caught from the sampling stations; the spleen and head kidney were collected and sections prepared to permit histologic evaluation. The results indicated that, in both species, the most common changes were observed in fish collected near the Petrochemical station and included an increase in melano-macrophage aggregates, hemorrhage and damaged/dead red blood cells in the spleen; in the head kidney, the major findings were melano-macrophage aggregation, hemorrhage and lifting of the tubular basement membrane. No pathological alternations were noted in the spleen and head kidney of fish from the Zangi station. Samples of A. latus collected from Gaafari station and of E. orientalis from Majidieh station also had pathological changes. No significant differences were found in the tissue structures of fish recovered from the Zangi and Genaveh control stations. The concentrations for nearly all of the studied metals in sediment and water samples collected from the different stations followed the pattern: Petrochemical station ≈ Majidieh ≈ Gaafari >> Ghazaleh > Zangi Stations. From the data, it was concluded that changes in lymphoid tissues of the fish studied here “correlated” with geographical conditions and sources of pollution at the different test stations. What these changes mean to the long-term health of both species remains to be determined in ongoing studies.

Histopathology and ultrastructural pathology of cyprinid herpesvirus II (CyHV-2) infection in gibel carp, Carassius auratus gibelio

Cyprinid herpesvirus II (CyHV-2) infection is identified in cultured gibel carp, Carassius auratus gibelio, with high mortality in China in recent years. Histological pathology includes acute hepatocellular necrosis, splenic necrosis, kidney necrosis, hyperplasia of the secondary lamellae with focal necrosis. Acute necrotic myocarditis and granulocytes are prominent within the cardiac lumen in infected fish. In addition, necrosis is observed in the submucosa and mucosa epithelium of intestinal tract. Edemas are observed in renal glomerulus, submucosa and mucosa epithelium of intestinal tract, myocardial cells and neurons. Transmission electron microscopy indicates the cytoplasmic inclusions in splenocytes, glomerulus cells and hematopoietic tissue cells of kidney, epithelial cells of gills and brain cells. The histopathology and ultrastructural pathology in CyHV-2 infected gibel carp are characterized with extensive necrosis and cytoplasmic inclusions in spleen, kidney, gill and brain, which suggests that CyHV-2 may mainly infect the spleen, kidney, gill and brain of fish.

A key gene of the small RNA pathway in the flounder, Paralichthys olivaceus: identification and functional characterization of dicer.

Dicer is critical for producing mature microRNAs (miRNAs) from precursor molecules and small interfering RNAs and plays an important role in controlling development and metabolism. In the present study, we cloned the flounder dicer gene, which is 6585 nucleotides (nt), including a 5'-untranslated region (UTR) of 231 nt, a 3'-UTR of 663 nt and an open reading frame of 5691 nt encoding a polypeptide of 1897 amino acids, and analyzed the conservation and expression pattern of dicer. The tissue distribution analysis indicated that dicer is abundantly expressed in the brain, heart, liver, spleen, stomach, kidney, gill, muscle, intestine and gonad of adult fish. Temporal expression analysis indicated that dicer mRNA is highly expressed during the embryonic and early larval stages, and exhibits low expression during the metamorphic stages. Treatment with thyroid hormone (TH) or thiourea indirectly or directly up-regulated dicer mRNA levels at 17 and 23dph, whereas treatment with TH down-regulated dicer mRNA levels at 36dph. The dicer-specific siRNA significantly down-regulated dicer mRNA and pol-let-7d levels, while pol-let-7d precursor levels were not differentially changed compared with the control (NC). These results demonstrated that dicer plays a key role in development and metabolism through the production of mature miRNAs, providing basic information for further studies concerning the role of dicer in Paralichthys olivaceus development.

An oral DNA vaccine against infectious haematopoietic necrosis virus (IHNV) encapsulated in alginate microspheres induces dose-dependent immune responses and significant protection in rainbow trout (Oncorrhynchus mykiss).

Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7 d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10 μg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100 μg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also be observed. Although the protective effects of the oral pIRF1A-G vaccine after a challenge with IHNV were partial, significant differences in cumulative percent mortalities among the orally vaccinated fish and the unvaccinated or empty-plasmid vaccinated fish were observed. Similar levels of protection were obtained after the intramuscular administration of 5 μg of pIRF1A-G or after the oral administration of a high dose of pIRF1A-G vaccine (100 μg); with 70 and 56 relative percent survival values, respectively. When fish were vaccinated with alginate microspheres containing high doses of the pIRF1A-G vaccine (50 or 100 μg), a significant increase in the production of anti-IHNV antibodies was detected in serum samples of the vaccinated fish compared with that in unvaccinated fish. At 10 days post-challenge, IHNV N gene expression was nearly undetectable in kidney and spleen of orally vaccinated fish which suggested that the vaccine effectively reduced the amount of virus in tissues of vaccinated fish that survived the challenge. In conclusion, our results demonstrated a significant increase in fish immune responses and resistance to an IHNV infection after the oral administration of increasing concentrations of a DNA vaccine against IHNV encapsulated into alginate microspheres.

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria.

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

PP2A (PR65) in Silver Carp: cDNA Cloning and Expression Analysis

The full-length sequence of the protein phosphatase 2A subunit A (PP2A-A) cDNA from silver carp was cloned and sequenced. Additionally, the transcription of PP2A-A in the liver, kidney, and spleen of fish after microcystin (MC)-exposure were also determined in this study. The results reveal that PP2A-A cDNA is 2833 base pair (bp) long and contains an open reading frame of 1767bp encoding a protein of 589 amino acids. Sequence analysis demonstrates that PP2A is highly conserved in fish. Furthermore, the results of quantitative real-time PCR indicate that PP2A-A is constitutively expressed in all examined silver carp tissues and primarily in the brain and liver. Additionally, we found that 50 or 200μg/kg of crude MC exposure significantly downregulated PP2A-A transcription in fish liver, kidney, and spleen, suggesting that PP2A-A may be involved in MC toxicity in silver carp.

Susceptibility of farmed juvenile giant grouper Epinephelus lanceolatus to a newly isolated grouper iridovirus (genus Ranavirus)

A ranavirus was isolated from the diseased farmed groupers (Grouper iridovirus in genus Ranavirus, GIV-R), Epinephelus hybrids (blotchy rock cod, Epinephelus fuscoguttatus ♀×giant grouper, Epinephelus lanceolatus ♂), in Sanya, Hainan, in July 2013. In this study, susceptibility of farmed juvenile giant grouper E. lanceolatus to GIV-R was determined by intraperitoneally injection. The cumulative mortality reached to 81% at 5 day post infection. Histologically, severe degeneration with massive pycnotic nuclei in spleen and kidney tissues was observed, and some small-size inclusion body-bearing cells (IBCs) existed in spleen. Hemorrhage and infiltration of inflammatory cells were presented in gill, liver and heart along with tissue degeneration and necrosis of varying severity. The results of immunohistochemistry analysis showed that the strongest immunolabellings were obtained from the kidney and spleen tissues, while intermediate intensity signals were observed in the heart, stomach, gill and liver tissues, and the weakest signals were obtained from the intestine and brain, but no signal was obtained in eyes. Electron microscopy revealed that spleen of moribund fish contained many viral particles in cytoplasm. Interestingly, in surviving fish, abnormal hypertrophic cells were observed in both splenic corpuscle and renal corpuscle, while no hypertrophic cell was observed in the other parts of spleen and kidney tissues. Moreover, immunolabellings only stained the hypertrophic cells in splenic corpuscle and renal corpuscle. This indicated that splenic corpuscle and renal corpuscle play an important role in GIV-R infection and replication.

Alteration in the cytokine levels and histopathological damage in common carp induced by glyphosate

Glyphosate is one of the most frequently used herbicides, and it has been demonstrated to generate a series of toxicological problems in animals and humans. However, relatively little is known about the effects of glyphosate on the immune system of fish. In the present study, the acute toxicity of glyphosate on common carp was first determined; then, the contents of interferon-γ (IFN-γ), interleukin-1β (IL-1β), and tumor necrosis factor -α (TNF-α) and histopathological alterations in the liver, kidneys, and spleen of common carp exposed to 52.08 or 104.15mgL−1 of glyphosate for 168h were also determined and evaluated. The results of the acute toxicity tests showed that the 96h LC50 of glyphosate for common carp was 520.77mgL−1. Moreover, sub-acute exposure of glyphosate altered the contents of IFN-γ, IL-1β, and TNF-α in fish immune organs. For example, there was a remarkable increase in the IFN-γ content in the kidneys, while there was a decrease in the liver and spleen. The IL-1β content increased in liver and kidneys, but it decreased in the spleen, and TNF-α mainly increased in the fish liver, kidneys, and spleen. In addition, glyphosate-exposure also caused remarkable histopathological damage in the fish liver, kidneys, and spleen. These results suggest that glyphosate-caused cytokine alterations may result in an immune suppression or excessive activation in the treated common carp as well as may cause immune dysfunction or reduced immunity. In conclusion, glyphosate has immunotoxic effects on common carp.

Effect of dietary glutamine on growth performance, non-specific immunity, expression of cytokine genes, phosphorylation of target of rapamycin (TOR), and anti-oxidative system in spleen and head kidney of Jian carp (Cyprinus carpio var. Jian).

This study was designed to investigate the effects of dietary glutamine on the growth performance, cytokines, target of rapamycin (TOR), and antioxidant-related parameters in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed the basal (control) and glutamine-supplemented (12.0gglutaminekg(-1) diet) diets for 6weeks. Results indicated that the dietary glutamine supplementation improved the growth performance, spleen protein content, serum complement 3 content, and lysozyme activity in fish. In the spleen, glutamine down-regulated the expression of the interleukin 1 and interleukin 10 genes, and increased the level of phosphorylation of TOR protein. In the head kidney, glutamine down-regulated the tumor necrosis factor α and interleukin 10 gene expressions, phosphorylated and total TOR protein levels, while up-regulated the transforming growth factor β2 gene expression. Furthermore, the protein carbonyl content was decreased in the spleen of fish fed glutamine-supplemented diet; conversely, the anti-hydroxyl radical capacity and glutathione content in the spleen were increased by glutamine. However, diet supplemented with glutamine did not affect the lipid peroxidation, anti-superoxide anion capacity, and antioxidant enzyme activities in the spleen. Moreover, all of these antioxidant parameters in the head kidney were not affected by glutamine. Results from the present experiment showed the importance of dietary supplementation of glutamine in benefaction of the growth performance and several components of the innate immune system, and the deferential role in cytokine gene expression, TOR kinase activity, and antioxidant status between the spleen and head kidney of juvenile Jian carp.

Improvement in non-specific immunity and disease resistance of barramundi, Lates calcarifer (Bloch), by diets containing Daphnia similis meal

A 42-day study was conducted with barramundi, Lates calcarifer, to evaluate the effects of Daphnia meal derived from Daphnia similis on fish growth, immune response, and disease resistance to Aeromonas hydrophila. Three isonitrogenous (45%) and isolipid (10%) experimental diets were formulated to contain 0% (control), 5% (D5), and 10% (D10) Daphnia meal. Growth was depressed when fish were fed with the D10 diet for 42 days compared to control. However, the growth in fish fed with control and D5 diets for 42 days was not significantly different. By day 42, the leukocyte phagocytic activity and respiratory burst activity were significantly increased in D5 and D10 groups compared to control. Mx gene expression in the spleen and head kidney of fish after being injected with nerve necrosis virus was also significantly up-regulated in both groups compared to control. In an increased immune response, D5 and D10 fish had significantly higher survival rates than control after being challenged by A. hydrophila. Therefore, we suggest that a 5% Daphnia-meal diet could improve the barramundi immune response and disease resistance without a negative impact on growth.

Comprehensive and comparative transcription analyses of the complement pathway in rainbow trout

The complement system is one of the most ancient and most essential innate immune cascades throughout the animal kingdom. Survival of aquatic animals, such as rainbow trout, depends on this early inducible, efficient immune cascade. Despite increasing research on genes coding for complement components in bony fish, some complement-related genes are still unknown in salmonid fish. In the present study, we characterize the genes encoding complement factor D (CFD), CD93 molecule (CD93), and C-type lectin domain family 4, member M (CLEC4M) from rainbow trout (Oncorhynchus mykiss). Subsequently, we performed comprehensive and comparative expression analyses of 36 complement genes including CFD, CD93, and CLEC4M and further putative complement-associated genes to obtain general information about the functional gene interaction within the complement pathway in fish. These quantification analyses were conducted in liver, spleen and gills of healthy fish of two rainbow trout strains, selected for survival (strain BORN) and growth (Import strain), respectively. The present expression study clearly confirms for rainbow trout that liver represents the primary site of complement expression. Spleen and gills also express most complement genes, although the mean transcript levels were generally lower than in liver. The transcription data suggest a contribution of spleen and gills to complement activity. The comparison of the two rainbow trout strains revealed a generally similar complement gene expression. However, a significantly lower expression of numerous genes especially in spleen seems characteristic for the BORN strain. This suggests a strain-specific complement pathway regulation under the selected rearing conditions.

Transcription alteration of immunologic parameters and histopathological damage in common carp (Cyprinus carpio L.) caused by paraquat.

The toxic effects of paraquat (PQ) on the transcription of immunoglobulin M (IgM), complement C3 (C3), and lysozyme (LYZ) and the histopathological alteration of the liver, kidney, and spleen of common carp were evaluated by subacute exposure to 1.596 or 3.192 mg/L of PQ for 7 days. The results demonstrated that PQ exposure altered the transcription of IgM, C3, and LYZ. For example, IgM and C3 expression was generally downregulated, but LYZ was either down- or upregulated, suggesting that PQ may disturb the function of the fish immune system. The results of the histopathological examination revealed that the liver, kidney, and spleen of PQ-treated fish were injured. These injuries included cellular swelling, intracytoplasmic vacuolization, nucleus distortion, and pycnosis in the liver and spleen, and vacuolization of the renal parenchyma and intumescence of the renal tubule in the kidney. This finding indicates that PQ causes toxicity in common carp.

EFFECTS ON PATHOLOGICAL AND TOXICOLOGICAL ASPECTS OF NILE TILAPIA Oreochromis niloticus OF USING MALATHION TO ERADICATE Lethocerus niloticum INSECTS

Male tilapia fish (Oreochromis niloticus)and water bugs (Lethocerus niloticum) were studied in a presence of malathion (diethyldimethoxy thiophosphoryl thiosuccinate) of commercial grade 57%. Fish were fed during the experimental period once daily on a commercial dry pellet ration, while water bugs were fed on fish offales. They all were acclimatized to the same laboratory conditions for 2 weeks before the beginning of the experiment. Different malathion concentrations were experimented. The present study revealed that the minimum malathion concentration level that induced mortality of water bugs without any lethality on Oreochromis niloticus fish was 0.26 mg/L for 96 hrs exposure period. However, some toxic effects and histopathological alterations were observed at that condition. Gonadosomatic index, serum Testosterone, T3and T4concentration levels were determined. Brain AChE and LDH activities were also analyzed. Fish liver, spleen, gills, testes, brain and thyroid gland samples were histopathological studied.

Molecular cloning and expression analysis of immunoglobulin M heavy chain gene of blunt snout bream (Megalobrama amblycephala)

Immunoglobulins (Igs), which bind antigens with high specificity, are essential molecules in adaptive immune system of jawed vertebrates. In this study, cDNA encoding the secreted form of the immunoglobulin heavy chain of IgM (sIgM) was cloned from the mesonephros of blunt snout bream (Megalabrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of sIgM heavy chain gene has 1961 nucleotides encoding a putative protein of 569 amino acids, constant region shares high amino acid identity with that of Ctenopharyngodon idella (80%), Carassius auratus langsdorfii (65%) and Danio rerio (59%). Multiple protein sequence alignment revealed that blunt snout bream sIgM was clustered with the homologues of cyprinid fish and constructed one clade. Using quantitative real-time PCR (qRT-PCR) analysis, the level of sIgM mRNA was determined, with a V-shape change pattern: decreased initially from unfertilized egg stage to 4 cells stage and increased from 16 cells stage to prelarva. This sharp drop indicates that sIgM mRNA is maternally transferred, and was continuously degraded until 16 cells stage. The drastic rising in sIgM level from blastula stage to prelarva might be attributed to embryonic stem cell differentiation procedure. Compared with juvenile fish, the expression of sIgM was significantly higher in pronephros, liver, spleen, gill and muscle of adult fish. After the injection of Aeromonas hydrophila, the expression pattern of sIgM was found first down-regulated at 4 h, then up-regulated and reached the peak at 7 d and 21 d in mesonephros, spleen, liver and gill, respectively.

Design and evaluation of a tandemly arranged outer membrane protein U (OmpU) multi-epitope as a potential vaccine antigen against Vibrio mimicus in grass carps (Ctenopharyngodon idella)

Vibrio mimicus (V. mimicus) is an extracellular pathogen that causes ascites disease in aquatic animals. In our previous studies, the outer membrane protein U (OmpU) of V. mimicus has been proven to be a protective antigen, and several mimotopes of the protein were identified. Here, a tandemly arranged multi-epitope peptide (named 6EPIS) was designed with six mimotopes and heterologously expressed. Then, the immunoprotection efficacy of recombinant 6EPIS (r6EPIS) was evaluated in grass carps (Ctenopharyngodon idella) by determining relative percentage survival (RPS), specific immunoglobulin M (IgM) antibody titer, and transcriptional levels of immune-related genes of inoculated grass carps. Fish vaccinated with r6EPIS via intraperitoneal injection exhibited 85.71% RPS over the control, when challenged with V. mimicus. The enzyme-linked immunosorbent assay titer of specific IgM antibodies against r6EPIS reached 1:12,800 on Day 28 post the primary immunization. After 28 days post immunization, the transcriptional level of total IgM mRNA was significantly higher in the r6EPIS-vaccinated fish than in those vaccinated with recombinant OmpU, inactivated bacterin and rHis tag peptide (p<0.05). In addition, the transcription levels of interleukin-1β and tumor necrosis factor-α genes in the spleen and head kidney of r6EPIS-vaccinated fish were significantly increased during the period of immunization and early phase of infection, while the transcription level of interleukin-10 gene was significantly increased from Day 3 to 7 post challenge, compared to the control level. These results show that r6EPIS was highly immunogenic and could elicit strong protective immune responses. It may be an attractive vaccine candidate against V. mimicus infection.