Trunk Kidney Research Articles

Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses.

Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus.

Molecular Identification and Dual Functions of Two Different CXC Chemokines in Nile Tilapia (Oreochromis niloticus) against Streptococcus agalactiae and Flavobacterium columnare.

Two CXC chemokines in Nile tilapia (On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1–10 μg strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro.

Reactivation of cyprinid herpesvirus 2 (CyHV-2) in asymptomatic surviving goldfish Carassius auratus (L.) under immunosuppression

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen of goldfish (Carassius auratus) and Prussian carp (Carassius auratus gibelio) causing herpesviral hematopoietic necrosis. Our previous study revealed that CyHV-2 can persistently infect the kidney and spleen of goldfish that recovered from a primary infection. In this study, we tried to identify the cells persistently infected with the virus in surviving fish and investigated virus reactivation in the survivors injected with immunosuppressants, namely dexamethasone (Dex) and cyclosporine A (CsA). Virus DNA was detected from the monocytes that were isolated from the trunk kidney of the asymptomatic survivors, suggesting that monocytes/macrophages are major cells that may be persistently infected with CyHV-2. A significant increase of virus DNA levels was detected in the group injected with Dex at 10 and 21 days post-injection (dpi). In the fish group injected with CsA, the virus DNA level was the same as that in the control group at 10 dpi but increased in some organs at 21 dpi. Compared with Dex-injected fish at 10 dpi, the group injected with both Dex and CsA showed a greater increase in virus DNA levels. The gene expression of phagocytosis-associated genes, major histocompatibility complex (MHC) class II and p47phox, and anti-virus antibody levels increased in the CsA group due to virus reactivation in the infected cells but not in the Dex and Dex & CsA groups, indicating that Dex effectively suppressed monocyte/macrophage function and antibody production. In addition, recombinant interferon γ (IFNγ) supplementation in the kidney leukocyte culture that was isolated from survivors showed a reduction of virus DNA. CsA may inhibit T-helper 1 (Th1) cells and consequently IFNγ production, causing a synergetic effect with Dex on virus reactivation. The results suggest that the activity of monocytes/macrophages stimulated by IFNγ can relate to virus latency and reactivation in asymptomatic virus carriers.

CCR7, CD80/86 and CD83 in yellow catfish (Pelteobagrus fulvidraco): Molecular characteristics and expression patterns with bacterial infection

Dendritic cells (DCs) have a strong ability to stimulate naive T lymphocyte proliferation, so DCs play an important regulatory role in the initiation of the specific immune response. DCs cannot play the role of antigen presentation without the expression of surface molecules. The chemokine receptor CCR7 and the costimulatory molecules CD80/86 and CD83 are not only markers of DC maturation but also important functional molecules in the immune response of DC-T cells. In this study, partial cDNA sequences of CCR7, CD80/86 and CD83 were obtained by rapid amplification of cDNA ends (RACE) technology from yellow catfish. Bioinformatics analysis of deduced amino acid sequences of these three genes showed that CCR7, CD80/86 and CD83 genes in yellow catfish have similar functional domains to the homologs in other vertebrates, which indicated that the functions of these genes may be somewhat conserved during the evolution process. Afterward, the expression characteristics of these three genes in different tissues were detected by q-PCR. This result indicated that CCR7, CD80/86 and CD83 were expressed in all examined tissues, and the highest expression levels of CCR7 and CD80/86 and CD83 were detected in the trunk kidney, muscle and midgut, respectively. Meanwhile, the expression levels of CCR7 and CD80/86 were lowest in the gill, and the expression of CD83 was lowest in the stomach. Finally, healthy yellow catfish were infected with A.hydrophila (1.0 × 107 CFU/mL) or E.ictaluri (1.0 × 106 CFU/mL), q-PCR results indicated that both pathogenic bacteria can induce significant upregulation of CCR7, CD80/86 and CD83 in immune organs, and the expression levels of these genes in the intestine were higher than those in the skin and gill. Our results in this study provide a molecular basis for exploring the role of CCR7, CD80/86 and CD83 in the immune responses induced by bacteria, and can help us to understand the difference of immune responses induced by extracellular and intracellular bacteria.

Protective effects of metallothionein and vitamin E in the trunk kidney and blood of cadmium poisoned Ctenopharyngodon idellus.

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl2 on the first day and then VE, MT, or PBS was administered 4days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Immune responses induced by oil-adjuvanted inactivated vaccine against Flavobacterium psychrophilum in ayu Plecoglossus altivelis

Oil-adjuvant formulated formalin killed cells of Flavobacterium psychrophilum (FKC + Adj) is strongly effective against bacterial cold-water disease (BCWD) in ayu Plecoglossus altivelis. In this study, we aimed to understand mechanisms underlying the strong protection by the vaccine in ayu. Antibody titer of FKC + Adj and formalin-killed cells (FKC) group was significantly higher than those of modified cytophaga broth injected (MCY) group and MCY with the adjuvant (MCY + Adj) group. The highest antibody titer was observed in FKC + Adj group. Granulomatous inflammation without lymphocyte cuff was observed in the spleen and trunk kidney of FKC + Adj and MCY + Adj group, while the size of the granuloma was bigger in FKC + Adj than in MCY + Adj group. Gene expression level for IL-8 was significantly up-regulated in FKC + Adj group at 4 weeks after the vaccination. In contrast, IL-10 gene expression level was significantly suppressed in FKC + Adj at 4 weeks after the vaccination. F. psychrophilum was almost cleared in the spleen and trunk kidney of FKC + Adj group within 2 days after the challenge. Fluorescent immunohistochemistry showed that a lot of bacterial signals were detected in the spleen and trunk kidney of challenged fish in MCY, FKC and MCY + Adj group. However, the fluorescent signal was not detected in the organs of FKC + Adj group after the challenge. These data suggest that F. psychrophilum is immediately cleared in FKC + Adj vaccinated fish and both specific antibody and activation of phagocytes are essential to clear F. psychrophilum in ayu.

Molecular characterization and expression pattern of inositol-requiring enzyme 1 (IRE1) in blunt snout bream (Megalobrama amblycephala): its role of IRE1 involved in inflammatory response induced by lipopolysaccharide.

This study aimed to characterize the full-length cDNA of IRE1 from fish Megalobrama amblycephala and investigate its role in the pro-inflammatory response. A full-length cDNA coding IRE1 was cloned from blunt snout bream by RT-PCR and RACE approaches. The cDNA obtained covered 3665bp with an open reading frame of 3096bp encoding 1031 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (74-92%) among various species, retaining one signal peptide, one luminal domain, one serine/threonine kinase domain, one RNase domain, one activation loop, two N-linked glycosylation sites, and several phosphorylation sites. The highest IRE1 expression was observed in the trunk kidney followed by the brain and spleen, whereas relatively low expression levels were detected in the liver, intestine, adipose, skin, and heart. After lipopolysaccharide (LPS) challenge, the expressions of glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1 (IRE1), spliced X-box binding protein 1 (XBP1s), C/EBP homologous protein (CHOP), nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6) all increased remarkably in the spleen and brain at different sampling time points, while LPS also upregulated all the genes tested in the intestine except C/EBP homologous protein. Overall, the results indicated that the IRE1 gene of Megalobrama amblycephala shared a high similarity compared with other vertebrates including several bony fish species. Its expression in three tissues was induced remarkably by the LPS challenge, which indicated that IRE1 played a vital role in LPS-induced inflammation on fish.

Antibacterial and immunoregulatory activity of a novel hepcidin homologue in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var ♂)

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae, reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation.

Two Distinct C-Type Lysozymes in Goldfish: Molecular Characterization, Antimicrobial Potential, and Transcriptional Regulation in Response to Opposing Effects of Bacteria/Lipopolysaccharide and Dexamethasone/Leptin.

Lysozymes are key antimicrobial peptides in the host innate immune system that protect against pathogen infection. In this study, the full-length cDNAs of two c-type lysozymes (gfLyz-C1 and gfLyz-C2) were cloned from goldfish (Carassius auratus). The structural domains, three-dimensional structures, and amino acid sequences of gfLyz-C1 and gfLyz-C2 were highly comparable, as the two proteins shared 89.7% sequence identity. The gfLyz-C1 and gfLyz-C2 recombinant proteins were generated in the insoluble fractions of an Escherichia coli system. Based on the results of lysoplate and turbidimetric assays, gfLyz-C1 and gfLyz-C2 showed broad-spectrum antimicrobial properties with high levels of activity against Micrococcus lysodeikticus, Vibrio parahemolyticus, and Edwardsiella tarda, and relatively low activity against E. coli. Both gfLyz-C1 and gfLyz-C2 mRNAs were mainly expressed in the trunk kidney and head kidney, and gfLyz-C1 was expressed at much higher levels than gfLyz-C2 in the corresponding tissues. The expression of the gfLyz-C1 and gfLyz-C2 transcripts in the trunk kidney and head kidney was induced in these tissues by challenge with heat-inactivated E. coli and lipopolysaccharides (LPS), and the transcriptional responses of gfLyz-C1 were more intense. In goldfish primary trunk kidney cells, the levels of the gfLyz-C1 and gfLyz-C2 transcripts were upregulated by heat-inactivated E. coli, V. parahemolyticus, and E. tarda, as well as LPS, and downregulated by treatment with dexamethasone and leptins. Overall, this study may provide new insights that will improve our understanding of the roles of c-type lysozymes in the innate immunity of cyprinid fish, including the structural and phylogenetic characteristics, antimicrobial effects, and regulatory mechanism.

Molecular characterization and function analysis of Epinephelus coioides Hsp22 response to SGIV and Vribro alginolyticus infection

Heat shock protein 22 (Hsp22) is an important regulatory factor response to various stresses in mammals. In this study, the full length cDNA of Epinephelus coioides Hsp22, which was 1680bp in length, with a 289 bp 5′ UTR, a 725 bp 3′UTR, and a 666 bp open reading frame encoding 221 amino acids, was obtained. E. coioides Hsp22 contains a highly conserved α-crystallin domain. E. coioides Hsp22 mRNA was detected in all tissues examined by quantitative real-time PCR, with the highest expression in blood, followed by the spleen, skin, gill, head kidney, muscle, heart, liver, trunk kidney, stomach, pyloric caeca, intestine, brain and thymus. The expression patterns of E. coioides Hsp22 response to infection with Singapore grouper iridovirus (SGIV) and Vribro alginolyticus, the important pathogens of E. coioides, were studied. The expression levels of the gene were up-regulated in the tissues examined. Subcellular localization analysis demonstrated that E. coioides Hsp22 was distributed in both the cytoplasm and nucleus. In addition, E. coioides Hsp22 significantly inhibited the SGIV-induced cell apoptosis. In summary, the E. coioides Hsp22 might play a critical role in pathogenic stimulation.

IκBα phosphorylation and associated NF-κB activation are essential events in lymphocyte activation, proliferation, and anti-bacterial adaptive immune response of Nile tilapia

Inhibitory protein IκBα plays a crucial role in the inflammatory process and immune response by regulating the activity of transcription factor NF-κB. In teleost, great progress has been achieved regarding NF-κB signaling for innate immunity, but whether this pathway modulates adaptive immunity, and how, remains largely unclear. In this study, after characterizing the sequence, structure, and phylogeny of Nile tilapia Oreochromis niloticus IκBα (defined as On-IκBα), we investigated the association between IκBα-regulated NF-κB activation and the lymphocyte-mediated adaptive immune response in Nile tilapia. We found that On-IκBα was evolutionarily conserved, and its mRNA was expressed widely in various tissues, with most abundance in the trunk kidney. mRNA expression of On-IκBα was significantly upregulated in spleen at both innate and adaptive immune stages after Aeromonas hydrophila infection. Moreover, phosphorylation of On-IκBα and the downstream On–NF–κB p65 was obviously elevated in spleen leukocytes at 3, 5, or 8 days after A. hydrophila infection, indicating the activation of NF-κB signaling. Correlating with the augmented protein phosphorylation, leukocyte proliferation was enhanced during the same immune stage, suggesting the potential association of IκBα and IκBα-regulated NF-κB signaling in the primary adaptive immune response. Although lymphocyte activation by the T cell-specific mitogen PHA did not alter On-IκBα mRNA expression significantly, lymphocyte activation by the agonist PMA obviously elevated On-IκBα and OnNF–κB p65 phosphorylation in spleen leukocytes. Together, the results suggest that IκBα phosphorylation and its regulated NF-κB activation are essential events associated with lymphocyte activation, proliferation, and anti-bacterial adaptive immune response in Nile tilapia. Our study aids to understand the regulatory mechanism of adaptive immunity in teleost.

Chemokine CXCb1 stimulates formation of NETs in trunk kidney neutrophils of common carp

Both in mammals and in fish, CXC chemokines activate leukocytes and regulate their migration both under normal physiological and inflammatory conditions. Moreover, in mammalian neutrophils CXC chemokines also stimulate the formation of neutrophil extracellular traps (NETs). Here, we investigated the effects of recombinant carp CXCL8s and CXCb1 on NET formation in neutrophils from the head (HK) and trunk (TK) kidney of carp.We found that neither recombinant CXCL8s nor CXCb1 stimulated DNA release in HK-derived neutrophils, while in TK-derived cells rcCXCb1 stimulated the release of NETs, composed of extracellular DNA co-localized with citrulline H3 histone and neutrophil elastase. Furthermore, CXCb1-induced NET release required NADPH oxidase activity, while it did not change upon treatment with CXCR inhibitors.In conclusion, we demonstrated, for the first time in fish, that CXCb1 chemokine induces formation of NETs in TK-derived neutrophils and this process is ROS-dependent. The difference between HK and TK-derived neutrophils is probably related to differences in the maturation state of these cells.

РАЗВИТИЕ И ФУНКЦИОНАЛЬНАЯ ОРГАНИЗАЦИЯ ЦЕНТРАЛЬНЫХ ОРГАНОВ ГЕМОПОЭЗА ЛИЧИНОК ЛЕЩА ОБЫКНОВЕННОГО (ABRAMIS BRAMA (L.))

The article dwells upon physiological and morphological studies of fish carried out on a large scale, due to the need to control their reproduction and assess the impact of living conditions on the body of fish. Currently, a large amount of data on hematopoietic organs and the formation of blood cells in fish have been accumulated, but the facts are quite contradictory. At the same time, the state of the hematopoietic system can be justified only according to histogenesis of hematopoietic organs. From the results of examining reticular intercanal tissue of the head kidney (pronephros) of bream, a typical representative of bony fish, it can be inferred that pronephros is one of the main hemopoietic organs, in which the process of developing red blood cells and leukocytes takes place, with the majority of cells accounted for blood cells of erythropoietic series. Pronephros loses its excretory function in the early stages of ontogenesis. Throughout its development pronephros is presented as a dense formation, in the intercanal tissue of which there is reticular tissue with numerous hematopoietic elements. Active hematopoiesis occurs in mesonephros (trunk kidney) of bream larvae. The study of the morphophysiology of mesonephros in ontogenesis showed that this organ forms blood elements of all series, with more than half of the hematopoietic elements accounted for erythropoietic cells. The excretory function of the trunk kidney is maintained throughout the whole period of the organ developing. Thus, mesonephros of bream is also one of the main organs of hematopoiesis represented by a paired organ which stretches along the spine.

Effects of stress and cortisol on the polarization of carp macrophages

In teleost fish, myelopoiesis is maintained both in the head (HK) and trunk kidney (TK), but only the HK holds the endocrine cells that produce the stress hormone cortisol. We now compared the effects of prolonged restraint stress (in vivo) and cortisol (in vitro) on the polarization of HK and TK-derived carp macrophages.Monocytes/macrophages from both sources were treated in vitro with cortisol, lipopolysaccharide or with both factors combined. In vivo, fish were challenged by a prolonged restraint stress. Gene expression of several markers typical for classical M1 and alternative M2 macrophage polarization, as well as glucocorticoid receptors, were measured.Cells from both sources did not differ in the constitutive gene expression of glucocorticoid receptors, whereas they significantly differed in their response to cortisol and stress. In the LPS-stimulated HK monocytes/macrophages, cortisol in vitro counteracted the action of LPS while the effects of cortisol on the activity of TK monocytes/macrophages were less explicit.In vivo, restraint stress up-regulated gene expression of M2 markers in freshly isolated HK monocytes/macrophages, while at the same time it did not affect TK monocytes/macrophages. Moreover, LPS-stimulated HK monocytes/macrophages from stressed animals showed only minor differences in the gene expression of M1 and M2 markers, compared to LPS-treated monocytes/macrophages from control fish. In contrast, stress-induced changes in TK-derived LPS-treated cells were more pronounced. However, these changes did not clearly indicate whether in TK monocytes/macrophages stress will stimulate classical or alternative polarization.Altogether, our results imply that cortisol in vitro and stress in vivo direct HK, but not TK, monocytes/macrophages to the path of alternative polarization. These findings reveal that like in mammals, also in fish the glucocorticoids form important stimulators of alternative macrophage polarization.

Molecular, biochemical and histopathological studies on the ameliorative effect of vitamin C on the renal and muscle tissues of Nile tilapia fish ( Oreochromis niloticus ) affected by the usage of engine oil

The present study was performed to evaluate the effect of vitamin C on the performance of Nile tilapia (Oreochromis niloticus) affected by the usage of engine oil. A total of 120 tilapia fish were randomly divided into four equal groups according to dietary treatments applied for 3 weeks: group I (control); group II was exposed to engine oil; group III was exposed to engine oil and supplemented with vitamin C: and group IV was fed only a diet supplemented with vitamin C. According to our results, the group exposed to engine oil (II) showed an induced elevation of kidney function parameters, an upregulation of CYP1A1 mRNA expression level, a reduction in antioxidant status (SOD, GSH) and severe abnormal histological and ultra-structural changes in the head kidney, trunk kidney and muscle tissue. Supplementation of group III with vitamin C ameliorated the toxic effect induced by engine oil through improvement of kidney function parameters, elevation of the antioxidant status of tissue and improvement of the histological images of renal and muscle tissues. In conclusion, vitamin C has the ability to ameliorate engine oil oxidative stress toxicity in Nile tilapia.

Interleukin-2 (IL-2) in flounder (Paralichthys olivaceus): Molecular cloning, characterization and bioactivity analysis

Interleukin-2 (IL-2) is mainly produced by CD4+ T helper lymphocytes, which is an important immunomodulatory cytokine that primarily promotes activation, proliferation and differentiation of T cells. In the present study, flounder (Paralichthys olivaceus) interleukin 2 homologue (poIL-2) was identified for the first time, and its expression patterns were characterized in healthy, virus- or bacteria-infected flounder. The full-length cDNA sequences of poIL-2 was 989 bp with an open reading frame of 423 bp coding a polypeptide of 140 amino acids (aa). The deduced aa sequences shared low similarities (<53%) with other known fish IL-2s. Multiple alignment of aa sequences revealed that poIL-2 own the classical IL-2 family signature of “C-X(3)-EL-X(2)-(T/V)-(V/M/L)-(K/T/R)-X-EC” and “DS-X-(F/L)Y(A/T/S)P”. In healthy flounder, IL-2 mRNA was highly expressed in PBLs, spleen and hindgut, and moderately expressed in gill, trunk kidney and stomach. PHA, LPS and Con-A could effectively induce poIL-2 expression in primary cultured peripheral blood leukocytes in vitro. poIL-2 transcripts were significantly up-regulated in spleen, kidney, gill and hindgut post infections with Edwardsiella tarda and Hirame novirhabdovirus (HIRRV). The eukaryotic expression vector encoding poIL-2 (pcIL-2) was constructed and intramuscularly injected, which could be successfully expressed in flounders and induced significantly higher expressions of six immune related genes including poIL-2, β-defensin, CD4-1, CD8α, IFN-γ and TNF-α compared with the injection with control plasmid. Moreover, pretreatment with pcIL-2 could markedly increase the survival rate of flounder challenged with HIRRV. Our results demonstrated that poIL-2 plays an important role in the induction of immune responses and immune defense against bacterial and virus infection, which indicated its potential use as an immunopotentiator to prevent diseases in flounder.

Assessment of gentamicin effect on oxidoreductive balance and microstructure of trunk kidney in Prussian carp (Carassius gibelio)

Assessment of gentamicin effect on oxidoreductive balance and microstructure of trunk kidney in Prussian carp (Carassius gibelio)

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection.

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C.auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV-2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non-permissive temperature for 4days, initiated at three different time points post-infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51days post-infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5days. Following inoculation with the homogenate of PCR-positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV-2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.

ClpV is a key virulence gene during in vivo Pseudomonas plecoglossicida infection.

Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one-sided bacterial pathogen in tissue has not been reported so far across different stages of infectiousdisease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV-RNAi strain, no deaths were observed up to 20dpi. The spleens, kidneys and livers of all the E.coioides that received clpV-RNAi strain failed to developvisible nodules at 5-8dpi, with the swelling gradually disappearing. The burdens of clpV-RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV-RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems.

Variations in the lipid peroxidation and antioxidant biomarkers in some tissues of anadromous cyprinid fish during migration

Alburnus tarichi is an endemic fish species inhabiting the Lake Van basin of Turkey. This anadromous cyprinid fish migrates for spawning to the freshwater inlets pouring into Lake Van, which has highly alkaline and brackish water. During the migration, the fish moves to a new habitat from a different habitat and encounters many challenges. The objective of the present study is to investigate whether antioxidant responses occur in the tissues of A. tarichi during its anadromous migration. To this end, fish were sampled at three different points and in two different periods from the migration route, including in Lake Van at prespawning, at the entrance of a freshwater stream (Karasu) at spawning and in the upstream of the freshwater stream at spawning. Malondialdehyde (MDA) content and antioxidant defenses including activities of the enzymes superoxide dismutase, glutathione peroxidase, glutathione-S-transferase and catalase and the level of glutathione were assayed in liver, gill, white muscle, trunk kidney and anterior intestine tissues. Our results showed increased MDA levels in liver, white muscle and anterior intestine and tissue-specific antioxidant responses in the freshwater environment. This study reports that alterations occurred in the antioxidant defense system indicators in the tissues of anadromous cyprinid fish during migration and that the antioxidant defenses might reflect an important role in spawning migration that ultimately leads to accomplishment of reproductive activity.