Anguillarum Infection Research Articles

Galectins in turbot (Scophthalmus maximus L.): Characterization and expression profiling in mucosal tissues

Galectins, a family of evolutionary conserved β-galactoside-binding proteins, have been characterized in a wide range of species. Many reports have indicated vital roles of galectins in innate immunity, especially in the mucosal tissues against infection. However, the systematic identification of galectin gene family is still lacking in teleost. Here, we characterized the galectin gene family and investigated their expression profiles post bacterial challenge in turbot (Scophthalmus maximus L.). In this study, a total of 13 galectin genes were characterized in turbot, phylogenetic analyses revealed their strong relationships to half smooth tongue sole and puffer fish, and syntenic analyses confirmed the orthology suggested by the phylogenetic analysis. In addition, the copy number of galectin genes is similar across a broad spectrum of species from fish to amphibians, birds, and mammals, ranging from 8 to 16 genes. Furthermore, the galectin genes were widely expressed in all the examined turbot tissues, and most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after Vibrio anguillarum infection in the intestine, gill and skin, suggesting that galectins were involved in the mucosal immune response to V. anguillarum infection in turbot. In addition, subcellular localization analysis showed lgals3a was distributed in the cytoplasm and nucleus. However, the knowledge of galectins are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Characterization and expression analysis of mitochondrial localization molecule: NOD-like receptor X1 (Nlrx1) in mucosal tissues of turbot (Scophthalmus maximus) following bacterial challenge

The NOD-like receptor X1 (NLRX1) is a member of highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family (known as NLR), that localizes to the mitochondrial outer membrane and regulate the innate immunity by interacting with mitochondrial antiviral-signaling protein (MAVS). As one of cytoplasmic PRRs, NLRX1 plays key roles for pathogen recognition, autophagy and regulating of subsequent immune signaling pathways. In this study, we identified the nlrx1 in turbot as well as its expression profiles in mucosal surfaces following bacterial infection. In our results, the full-length nlrx1 transcript consists of an open reading frame (ORF) of 4,886 bp encoding the putative peptide of 966 amino acids. The phylogenetic analysis revealed the SmNlrx1 showed the closest relationship to Cynoglossus semilaevis. In addition, the Nlrx1 mRNA expression could be detected in all the examined tissues, with the most abundant expression level in head kidney, and the lowest expression level in liver. Moreover, Nlrx1 showed similar expression patterns following Vibrio anguillarum and Streptococcus iniae infection, that were both significantly up-regulated following challenge, especially post S. iniae challenge. Finally, fluorescence microscopy unveiled that the SmNlrx1 localized to mitochondria in HEK293T by N-terminal mitochondrial targeting sequence. Characterization of Nlrx1 might have an important implication in bioenergetic adaptation during metabolic stress, oncogenic transformation and innate immunity and will probably contribute to the development of novel intervention strategies for farming turbot.

CD10+ Cells and IgM in Pathogen Response in Lumpfish (Cyclopterus lumpus) Eye Tissues.

Lumpfish (Cyclopterus lumpus), a North Atlantic “cleaner“ fish, is utilized to biocontrol salmon louse (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar) farms. Lumpfish require excellent vision to scan for and eat louse on salmon skin. The lumpfish eye immune response to infectious diseases has not been explored. We examined the ocular response to a natural parasite infection in wild lumpfish and to an experimental bacterial infection in cultured lumpfish. Cysts associated with natural myxozoan infection in the ocular scleral cartilage of wild adult lumpfish harbored cells expressing cluster of differentiation 10 (CD10) and immunoglobulin M (IgM). Experimental Vibrio anguillarum infection, which led to exophthalmos and disorganization of the retinal tissues was associated with disruption of normal CD10 expression, CD10+ cellular infiltration and IgM expression. We further describe the lumpfish CD10 orthologue and characterize the lumpfish scleral skeleton in the context of myxozoan scleral cysts. We propose that lumpfish develop an intraocular response to pathogens, exemplified herein by myxozoan and V. anguillarum infection involving novel CD10+ cells and IgM+ cells to contain and mitigate damage to eye structures. This work is the first demonstration of CD10 and IgM expressing cells in a novel ocular immune system component in response to disease in a teleost.

Elucidation of the potential mechanism of punicalagin for conferring disease resistance in Yesso scallops against Vibrio anguillarum infection

Although punicalagin (PNG) has been demonstrated to exhibit a variety of bioactivities, its disease resistance role in bivalve shellfish remains poorly understood. This study was designed to investigate the effect of PNG on immunoprotective activity in Yesso scallops (Patinopecten yessoensis) infected with Vibrio anguillarum. Surprisingly, PNG exhibited both prophylaxis and curative potential against this pathogen, as indicated by the significant increase in scallop survival rates. Additional in vitro and in vivo experiments were conducted to assess the possible mechanism by which PNG protected against pathogen infection. The results showed that PNG could cause significant damage to the membrane, suggesting that it displayed potent antimicrobial activity against V. anguillarum. At subinhibitory concentrations (SICs), PNG significantly reduced the bacterial swimming ability and inhibited the expression of some essential genes involved in virulence. Bacterial challenge experiments showed that PNG could facilitate the clearance of bacteria from the scallops. Interestingly, hemolymph immune-related enzyme activities significantly increased after PNG treatment. In addition, pre-treatment with PNG suppressed the expression of bacterially-induced immune response genes, indicating that PNG could be applied as an antibacterial drug and immunostimulant to protect pathogen-infected scallops in aquaculture.

GHRH-SST-GH-IGF axis regulates crosstalk between growth and immunity in rainbow trout (Oncorhynchus mykiss) infected with Vibrio anguillarum

An energy trade-off is existed between immunological competence and growth. The axis of growth hormone releasing hormone, somatostatin, growth hormone, insulin-like growth factor (GHRH-SST-GH-IGF axis) regulates growth performances and immune competences in rainbow trout (Oncorhynchus mykiss). The salmonid-specific whole genome duplication event is known to result in duplicated copies of several key genes in GHRH-SST-GH-IGF axis. In this study, we evaluated the physiological functions of GHRH-SST-GH-IGF axis in regulating crosstalk between growth and immunity. Based on principal components analysis (PCA), we observed the overall expression profiles of GHRH-SST-GH-IGF axis were significantly altered by Vibrio anguillarum infection. Trout challenged with Vibrio anguillarum showed down-regulated igf1s subtypes and up-regulated igfbp1a1. The brain sst genes (sst1a, sst1b, sst3b and sst5) and igfpbs genes (igfbp4s and igfbp5b2) were significantly affected by V. anguillarum infection, while the igfbp4s, igfbp5s, igfbp6s and igf2bps genes showed significant changes in peripheral immune tissues in response to V. anguillarum infection. Gene enrichment analyses showed functional and signaling pathways associated with apoptosis (such as p53, HIF-1 or FoxO signaling) were activated. We further proposed a possible model that describes the IGF and IGFBPs-regulated interaction between cell growth and programmed death. Our study provided new insights into the physiological functions and potentially regulatory mechanisms of the GHRH-SST-GH-IGF axis, indicating the pleiotropic effects of GHRH-SST-GH-IGF axis in regulating crosstalk between growth and immunity in trout.

Coadministration of citric acid allows oxytetracycline dose reduction in yellowtail Seriola quinqueradiata infected with Vibrio anguillarum.

Oxytetracycline (OTC) has been commonly used as an effective antibiotic against various fish bacterial diseases, including vibriosis. In this study, the absorption-enhancing effect of citric acid on oral OTC pharmacokinetics and treatment of artificial Vibrio anguillarum infection was evaluated in juvenile yellowtail Seriola quinqueradiata followed by serum OTC concentration analysis. When 25 mg kg-1 body weight (BW) OTC was administered in combination with 1250 mg kg-1 BW citric acid, the serum OTC concentration reached almost the same concentration as that of the group treated with 50 mg kg-1 BW OTC. This coadministration successfully suppressed mortality due to vibriosis similar to the group treated with 50 mg kg-1 BW OTC. Conversely, poor efficacy was observed when only 25 mg kg-1 BW OTC was administered. These results suggest that coadministration of citric acid can be beneficial in reducing the dose of OTC needed for effective treatment, and thus contributes to the goal of reduced use of this antibiotic in aquaculture.

Micro-Transcriptome Analysis Reveals Immune-Related MicroRNA Regulatory Networks of Paralichthys olivaceus Induced by Vibrio anguillarum Infection.

MicroRNAs (miRNAs) are non-coding regulatory RNAs that play a vital part in the host immune response to pathogen infection. Japanese flounder (Paralichthys olivaceus) is an important aquaculture fish species that has suffered from bacterial diseases, including that caused by Vibrio anguillarum infection. In a previous study, we examined the messenger RNA (mRNA) expression profiles of flounder during V. anguillarum infection and identified 26 hub genes in the flounder immune response. In this study, we performed the micro-transcriptome analysis of flounder spleen in response to V. anguillarum infection at 3 different time points. Approximately 277 million reads were obtained, from which 1218 miRNAs were identified, including 740 known miRNAs and 478 novel miRNAs. Among the miRNAs, 206 were differentially expressed miRNAs (DEmiRs), and 104 of the 206 DEmiRs are novel miRNAs identified for the first time. Most of the DEmiRs were strongly time-dependent. A total of 1355 putative target genes of the DEmiRs (named DETGs) were identified based on integrated analysis of miRNA-mRNA expressions. The DETGs were enriched in multiple functional categories associated with immunity. Thirteen key DEmiRs and 66 immune DETGs formed an intricate regulatory network constituting 106 pairs of miRNAs and DETGs that span five immune pathways. Furthermore, seven of the previously identified hub genes were found to be targeted by 73 DEmiRs, and together they formed interlinking regulatory networks. These results indicate that V. anguillarum infection induces complicated miRNA response with extensive influences on immune gene expression in Japanese flounder.

Dietary inclusion effect of yacon, ginger, and blueberry on growth, body composition, and disease resistance of juvenile black rockfish (Sebastes schlegeli) against Vibrio anguillarum

BackgroundTo minimize the use of antibiotics and to obtain a more sustainable fish culture and aquaculture industry, development of alternative natural source of immunostimulant to replace antibiotic in aquafeed is highly needed.ObjectiveDietary inclusion effect of yacon (YC), ginger (GG), and blueberry (BB) on growth, body composition, and disease resistance of black rockfish against Vibrio anguillarum was compared to ethoxyquin (EQ).MethodsFour hundred eighty juvenile (an initial weight of 4.2 g) fish were randomly distributed into 12 of 50 L flow-through tanks (forty fish per tank). Four experimental diets were prepared; the control (Con) diet with 0.01% EQ inclusion, and YC, GG, and BB diets at 1% each additive inclusion. Each additive was included into the experimental diets at the expense of wheat flour. Each diet was assigned to triplicate tanks of fish and hand-fed to satiation twice daily for 8 weeks. At the end of 8-week feeding trial, 20 fish from each tank fish were artificially infected by intraperitoneal injection with 0.1 mL of culture suspension of pathogenic V. anguillarum containing 3.3 × 106 cfu/mL respectively. Fish were monitored for the following 8 days after V. anguillarum infection and dead fish were removed every 6 h for the first 4 days and 12 h for the rest of the study.ResultsWeight gain, specific growth rate (SGR), and feed efficiency ratio (FER) of fish fed the YC diet was higher than those of fish fed all other diets. However, feed consumption, protein efficiency ratio, and protein retention was not affected by dietary additive. Moisture, crude protein, and crude lipid content of the whole body of fish were affected by dietary additive. Analysis of the Kaplan-Meier survival curves showed that survival of fish fed the YC, BB, and GG diets was higher than the Con diet.ConclusionOral administration of YC can improve not only weight gain, SGR, and FER of black rockfish, but lower mortality of rockfish at occurrence of V. anguillarum.

Characterization and Pathological Analysis of a Virulent Edwardsiella anguillarum Strain Isolated From Nile Tilapia (Oreochromis niloticus) in Korea.

Edwardsiella species are one of the top causative pathogens of mortality in various fisheries worldwide. Their role in zoonotic infections and increase in antibiotic-resistance has raised concerns and interests in many research fields. Similar to the studies investigating human clinical cases, there has been an increase in research examining the potential pathogenic role of the bacterium in aquaculture. Within the Edwardsiella family, Edwardsiella anguillarum was lastest group to be differentiated from the Edwardsiella tarda group, and many studies focusing on the virulence of this species have since ensued. In Korea, only E. tarda infections have been reported in aquaculture industries, and there have been no reports on economic losses incurred owing to E. anguillarum infection. There has been a recent report investigating the pathogenicity and pathological changes caused by E. anguillarum infection in a tilapia farm located in the Costa Rica. To the best of our knowledge, as ours is the first report of E. anguillarum infection in a Nile tilapia (Oreochromis niloticus) farm located in an Asian country, the pathogenicity of the bacterial strain was histopathologically compared to that of the past studies. As tilapia is one of the most globally consumed fish species, particularly throughout Asia, Europe, and America, an epidemiological study regarding the disease distribution is necessary for the control and prevention of the disease. Here, we report the first mass mortality case caused by E. anguillarum infection in a Nile tilapia farm located in Korea; the bacterial strain responsible was isolated, characterized, and pathologically analyzed.

Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels (Angullia angullia)

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, an expressed recombinant Outer membrane proteinⅡ (rOmpⅡ) from A. hydrophila was intraperitoneally injected into European eels (Angullia angullia). All examined eels were equally divided into three groups. One group was injected with PBS only (PBS group), one group was injected with 1:1 mixture of PBS and Freund's incomplete adjuvant (PBS + F, adjuvant group), and the third group was injected with 1:1 mixture of 1 mg mL−1 rOmpⅡ and Freund's incomplete adjuvant (rOmpⅡ+F, OmpⅡ group). The immunogenicity of OmpⅡ was studied by detecting the expression of 4 immune-related genes, stimulation index (SI) of the whole blood cell, serum antibody titer, lysozyme and Superoxide Dismutase (SOD) activity, and relative percent of survival (RPS) rate. The results showed that gene expression of MHC-Ⅱ, LysC, SOD and IgM in the OmpⅡ group significantly increased in liver, spleen, kidney and intestine. At 28 days post the immunization (dpi), the SI of whole blood cells in the OmpⅡ group increased significantly; at 14, 21, 28 and 42 dpi, the serum antibody titers against A. hydrophila and E. anguillarum in the OmpⅡ group were significantly higher than that of the PBS and the adjuvant group; the SOD in the OmpⅡ group was found increased significantly in liver, kidney, mucus and serum. On the 28 dpi, eels were challenged by A. hydrophila, E. anguillarum and V. vulnificus for cross protection study. The results showed that the RPS of the OmpⅡ group were 83.33%, 55.56% and 33.33% respectively. These results showed that the expressed OmpⅡ from A. hydrophila significantly improve the immune function of Europena eels and their resistance to the infection of A. hydrophila and E. anguillarum simultaneously.

Immunization of a novel bivalent outer membrane protein simultaneously resisting Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus infection in European eels (Angullia angullia)

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, European eels (Anguilla anguilla) were immunized by the bivalent expression products of the outer membrane protein (Omp) gene from A. hydrophila (OmpⅡ) and E. anguillarum (OmpA), and the effects of the bivalent protein (rOmpⅡ-A) on the immune function of the European eel were detected. Three hundred eels were divided average into three groups of PBS, adjuvant and rOmp. Eels of three goups were injected intraperitoneal with 0.2 mL of PBS (0.01 mol/L, pH7.4), PBS + F (PBS mixed equal volume of freund's uncomplete adjuvant) or rOmpⅡ-A (1 mg mL−1 rOmpⅡ-A mixed equal volume of freund's uncomplete adjuvant). Four immune-related genes expression, proliferation of whole blood cells, serum and skin mucus antibody titer, superoxide dismutase (SOD) activity and the relative percent of survival (RPS) were studied at different days (or hours) post the immunization. The results showed that the igm, lysC, mhc2 and sod gene in the liver, spleen, kidney and intestine tract were significant increased in the Omp group; On the 28 day post the immunization (dpi), blood cell proliferation was increased in the Omp group, and on the 14, 21, 28 and 42 dpi, antibody titers in serum and mucus of the Omp group were significantly higher than that of the PBS and adjuvant group, regardless of coating with bacteria or Omp antigen. The SOD activity of Omp group increased significantly in liver, kidney, skin mucus and serum from 14 to 42 dpi, especially in serum. Eels chanllenged by A. hydrophila, E. anguillarum and V. vulnificus in the bivalent Omp group showed the RPS were 83.33%, 55.56% and 44.44%, respectively. The results of this study showed that immunization of the bivalent Omp could effectively improve the immune function of European eels, and produced effectively protection to A. hydrophila and E. anguillarum infection. Simultaneously, the bivalent Omp also produced distinct cross-protection to the eels challenged by V. vulnificus.

Gene network analysis reveals a core set of genes involved in the immune response of Japanese flounder (Paralichthys olivaceus) against Vibrio anguillarum infection

Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine fish cultured in north Asia. Vibrio anguillarum is a severe bacterial pathogen to Japanese flounder and many other aquaculture species. In order to understand the immune response of flounder during bacterial infection, we systematically examined the transcriptome profiles of flounder spleen at three time points after V. anguillarum challenge. More than one billion high quality reads were obtained, approximately 80.70% of which were successfully mapped to the reference genome of flounder. A total of 6060, 4688 and 4235 differentially expressed genes (DEGs) were captured at 6, 12 and 24-h post-infection, respectively. The DEGs exhibited dynamic changes in expression and were assigned into four different profiles based on expression trend. GO and KEGG analysis showed that the DEGs were enriched in various immune-related terms, including response to stimulation, immune system and pathways of cytokine-cytokine receptor interaction, Jak-STAT signaling and Toll-like receptor signaling. Furthermore, a network of highly interactive DEGs involved in 11 immune-related pathways was detected by utilizing the weighted co-expressing network analysis (WGCNA). Accordingly, 26 hub genes were discovered that constituted an elaborate immune regulatory network and functioned mainly in pathogen recognition, antigen processing, and molecular signaling. The results of this study provided the first systematical transcriptome profile of flounder in association with V. anguillarum infection and can serve as a valuable resource of target genes for future studies on the molecular mechanisms underlying the immune defense of flounder against bacterial infection.

Identification, expression profile and analysis of the antimicrobial activity of collectin 11 (CL-11, CL-K1), a novel complement-associated pattern recognition molecule, in half-smooth tongue sole (Cynoglossus semilaevis)

Collectin 11 (CL-11, also known as collectin kidney-1, CL-K1), a new member of the vertebrate C-type lectin superfamily, plays an important role in innate immunity as a pattern recognition molecule of the lectin complement pathway. However, little is known about CL-11 in teleosts. In the present study, a CL-11 homolog was identified and characterized from half-smooth tongue sole (Cynoglossus semilaevis) (designated as CsCL-11). The full-length cDNA of CsCL-11 is 1220 bp long and includes a 5′untranslated region (5′-UTR) of 180 bp, a 3′-UTR of 218 bp and an open reading frame (ORF) of 819 bp encoding 273 amino acids. Multiple sequence alignment revealed that the deduced CsCL-11 protein has the typical modular architecture (EPN and WTD) conserved throughout vertebrates, suggesting a conserved function of CsCL-11. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed CsCL-11 to be ubiquitously distributed in tissues and highly expressed in the ovary and liver. A pattern of significant upregulation of CsCL-11 expression was observed in the blood, spleen, head kidney and gill at 6 h, 12 h and 24 h after infection with Vibrio anguillarum, and western blotting showed that natural CsCL-11 protein levels in the blood were significantly increased after V. anguillarum infection. Moreover, by binding to various bacteria, recombinant CsCL-11 (rCsCL-11) expressed in HEK-293 T cells displayed strong antibacterial activity. Taken together, these results suggest that CsCL-11 is a unique C-type lectin that is likely involved in host defense against bacterial infection. To our knowledge, this is the first study on CL-11 in marine fish.

Effect of Curcumin on Iron Toxicity and Bacterial Infection in Catfish (Clarias gariepinus).

Iron is an essential element that involved in many vital physiological functions in fish, while excess iron concentration causes many toxic effects. Curcumin is a natural popular spice that used as a dietary supplementation and has iron chelating properties. This study was conducted to evaluate the effect of curcumin on iron toxicity in catfish (Clarias gariepinus). Also this study assess the antibacterial effect of curcumin against Vibrio anguillarum infection. Clarias gariepinus were orally exposed to low and high doses of curcumin (40, 80 mg kg-1 fish) for 3 weeks. Fish were then exposed to 25 mg L-1 of ferric chloride as a source of iron toxicity for another 3 weeks. Some hematological parameters (Total and differential white blood cells count, total red blood cells count, hemoglobin concentration and hematocrit %) and biochemical parameters (Serum ferritin, transferrin, ALT, AST, protein and albumin) were assessed before and after exposure to iron. Iron residues in gills, spleen, liver, kidney, abdominal fats, gonads and muscles were also determined. Moreover the determination of fish survivability after bacterial challenge with Vibrio anguillarum was recorded. Iron caused decrease in total white blood cells count (WBCs), increase in ferritin level and elevation in liver function enzymes (ALT and AST). However, the pretreatment of fish with curcumin significantly increased WBCs, lymphocyte percentage, ferritin level and protein and albumin concentrations with significantly decreased transferrin, ALT and AST levels. Also there were significant decreases in iron concentration in serum, kidney, gonads and muscle in both low and high curcumin pretreated groups compared to Fe group. Results indicated a modulatory effect of curcumin against iron toxicity in catfish, also curcumin had an immune-stimulant effect against Vibrio anguillarum infection.

Beta-adrenergic receptor stimulation influences the function of monocytes/macrophages in ayu (Plecoglossus altivelis)

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor superfamily that can be categorized into αARs and βARs. The specific function of ARs in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of ARs from ayu (Plecoglossus altivelis; PaαAR and PaβAR). Sequence comparisons showed that PaαAR was most closely related to the αAR of the Japanese flounder and Nile tilapia, while PaβAR was most closely related to the βAR of Atlantic salmon. The AR transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaαAR and PaβAR proteins were upregulated in MO/MФ after infection, and PaβAR knockdown resulted in a pro-inflammatory status in ayu MO/MФ upon V. anguillarum infection and lowered the phagocytic activity of MO/MФ. Our results indicate that PaβAR plays the role of an anti-inflammatory mediator in the immune response of ayu against bacterial infection.

The host defense peptide β-defensin confers protection against Vibrio anguillarum in ayu, Plecoglossus altivelis

β-defensin is a cationic host defense peptide actively participating in host innate immune response against pathogens. In teleost fish, β-defensin exhibits a diversity in genotypes and functions. Herein, a β-defensin homolog (PaBD) was identified from ayu, Plecoglossus altivelis, showing multiple tissues’ upregulation against Vibrio anguillarum challenge. In vivo experiments revealed that intraperitoneal injection of chemically synthesized mature PaBD (mPaBD) increased the survival rate of V. anguillarum-infected ayu, accompanied by reduced bacterial load and decreased tissue mRNA levels of tumor necrosis factor α (PaTNF-α) and interleukin 1β (PaIL-1β). However, in vitro, mPaBD showed weak bactericidal activity against V. anguillarum. Interestingly, mPaBD enhanced phagocytosis, intracellular bacterial killing, and respiratory burst of ayu monocytes/macrophages (MO/MΦ). Moreover, it inhibited mRNA levels of PaIL-1β and PaTNF-α in MO/MФ upon V. anguillarum infection. In conclusion, PaBD protects ayu against V. anguillarum challenge not only through its direct antibacterial ability, but also through its immunomodulation in MO/MΦ.

Immune response and protective effect against Vibrio anguillarum induced by DNA vaccine encoding Hsp33 protein

Heat shock proteins (HSPs) are considered as potent vaccine candidates against a wide range of bacterial diseases. In the present study, a recombinant DNA plasmid based on the expression of Hsp33 gene was constructed and intramuscularly administrated to flounder to elucidate whether it induces immune response and prevents the infection of Vibrio anguillarum in flounder model. Meanwhile, the expression of pHsp33 was analyzed in vitro and in vivo. The results revealed that pHsp33 was successfully expressed both in transfected hirame natural embryo cell lines and injected flounder muscle, suggesting the functionality of pHsp33 to express Hsp33 protein. Fish, when intramuscularly injected with pHsp33 vaccine, exhibited the production of specific antibodies, upregulation of immune related genes expression in the head kidney and increase of sIgM+, CD4-1+ and CD4-2+ lymphocytes in peripheral blood, spleen and head kidney, which indicated the activation of humoral and cellular immune responses. Moreover, pHsp33 upregulated the expression of immune related genes at the inoculation site, indicating the activation of local immune response. In addition, pHsp33 vaccinated flounder provided a relative percent survival of 42.86% and inhibited the pathological lesion in liver following V. anguillarum challenge. In general, the results revealed that pHsp33 could elicit local and system immune responses and confer protection for flounder, suggesting pHsp33 could serve as a DNA vaccine candidate for the control of V. anguillarum infection.

Effects of Temperature on Haemocyte and Granulocyte Counts and Expressions of Immunity-Related Genes in Haemocytes of Scallop Chlamys farreri After Vibrio anguillarum Infection

Bivalve live in aquatic environment and the water temperature can affect their immunity directly. In this research, the scallop Chlamys farreri was injected with 104 or 107 CFUmL-1Vibrio anguillarum and cultured at 11°C, 17°C, 23°C, and 28°C, respectively. For the control scallop, only phosphate-buffered saline (PBS) was injected. Then total haemocytes and granulocytes were measured by ELISA using monoclonal antibodies. In the meantime, expressions of six immunity-related genes, including lipopoly-saccharide and β-1, 3-glucan binding protein (CfLGBP), C-type lectin (CfLec-2), Toll-like receptor (CfTLR), Lysozyme (CfLYZ), superoxide dismutase (SOD), and phenoloxidase (CfPO) in haemocytes were measured using quantitative real-time PCR. The results showed that total haemocytes counts in 104 CFU mL-1 injection groups showed no differences compared to the control group at all temperatures. However, they varied significantly in 107 CFU mL-1 injection groups at 3 h at 11°C, 6–12 h at 17°C, 3–48 h at 23°C, and 12–48 h at 28°C. Granulocytes counts in 104 CFU mL-1 injection groups showed no variance compared to the control group at all temperatures, except for 12 h at 23°C, and 24–36 h at 28°C. They were significantly decreased in 107 CFU mL-1 injection groups during 6–48 h at 11°C, 12–48 h at 17°C, 3–48 h at 23°C, and 3–72 h at 28°C. The expression levels of six immunity-related genes in haemocytes of 107 CFU mL-1 injection groups were significantly higher than those of control group and 104 CFU mL-1 injection groups at all temperatures. The results indicated that infected with high concentration of vibrios, haemocyte counts, granulocyte counts and the expressions of immunity-related genes in scallop C. farreri were significantly affected by environmental temperature.

Generation and functional evaluation of a DNA vaccine co-expressing Vibrio anguillarum VAA protein and flounder interleukin-2

In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and demonstrated to confer moderated protection against V. anguillarum challenge. Here, a bicistronic DNA vaccine (pVAA-IRES-IL2), co-expressing the VAA gene of V. anguillarum and Interleukin-2 (IL2) gene of flounder, was constructed to increase the protective efficacy of VAA DNA vaccine. The potential of pVAA-IRES-IL2 to express both VAA and IL2 in transfected HINAE cell lines was confirmed by immunofluorescence assay. Further, the variation of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of VAA-specific antibodies in flounder, which was intramuscularly immunized with three DNA plasmids (pIRES, pVAA-IRES, pVAA-IRES-IL2), were investigated, respectively. The bacterial burden and relative percentage survival (RPS) of flounder exposed to V. anguillarum infection were both analyzed to evaluate the efficacy of bicistronic DNA plasmid. Our results revealed that the percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and antibodies specific to VAA were remarkably increased in pVAA-IRES or pVAA-IRES-IL2 immunized fish. Moreover, the co-expression of IL2 enhanced the immune response in response to VAA DNA vaccination, as shown by the higher percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of specific antibody. Importantly, the RPS in pVAA-IRES-IL2 and pVAA-IRES groups reached 64.1% and 51.3%, respectively, when compared with the 97.5% cumulative mortality in pIRES group. Furthermore, the number of V. anguillarum in liver, spleen and kidney of pVAA-IRES or pVAA-IRES-IL2 immunized flounder after V. anguillarum challenge was significantly reduced, as compared to that in pIRES group. These suggest that the bicistronic DNA vaccine can be an effective immunization strategy in inducing immune response against V. anguillarum infection and IL2 has the potential as the adjuvant for VAA DNA vaccine.

The interleukin-6 regulates the function of monocytes/macrophages (MO/MФ) via the interleukin-6 receptor β in ayu (Plecoglossus altivelis)

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor β (PaIL-6Rβ) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rβ knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rβ knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.