Piscicida Infection Research Articles

Dietary supplementation of propolis enhanced the innate immune response against Edwardsiella piscicida challenge in turbot (Scophthalmus maximus)

Propolis is non-hazardous resinous substance mixture containing bioactive ingredients such as polyphenols, flavonoids and organic acid. It has been widely used as food supplement and immune adjuvant due to its benefits in anti-microbial and immunomodulation. Edwardsiella piscicida is a kind of threatening pathogen which could cause high mortality in turbot. However, whether propolis could enhance the innate immune response against E. piscicida infection in turbot remains unknown. In this study, we found dietary propolis addition could improve the expression of anti-oxidative stress related enzymes, e.g., SOD, CAT and GPT, and relieved the histopathological changes of juvenile turbot after E. piscicida infection. Moreover, propolis addition increased the expression of cytokines such as il-1β, il-6 and tnf-α in different organs of juvenile turbot. Importantly, rescued survival and decreased bacterial loads were observed in propolis feeding group. Taken together, these findings suggest that the important roles of propolis in protecting juvenile turbot from E. piscicida infection, indicating propolis might be applied as a promising immunopotentiator candidate in aquaculture.

Enhancement of Edwardsiella piscicida infection, biofilm formation, and motility caused by N-acetylneuraminate lyase.

Sialic acid and its catabolism are involved in bacterial pathogenicity. N-acetylneuraminate lyase (NAL), which catalyzes the reversible aldol cleavage of sialic acid to form N-acetyl-D-mannosamine in the first step of sialic acid degradation, has been recently investigated to elucidate whether NAL enhances bacterial virulence; however, the role of NAL in bacterial pathogenicity remains unclear. In the present study, we demonstrated that the existence of two enzymes in Edwardsiella piscicida, referred to as dihydrodipicolinate synthase (DHDPS) and NAL, induced the cleavage/condensation activity toward sialic acids such as N-acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid. NAL enhanced cellular infection in vitro and suppressed the survival rate in zebrafish larvae in bath-infection in vivo, whereas DHDPS did not. Furthermore, NAL strongly activated the expression of E. piscicida phenotypes such as biofilm formation and motility, whereas DHDPS did not. Besides, the gene expression level of nanK, nanE, and glmU were up-regulated in the NAL-overexpressing strain, along with an increase in the total amount of N-acetylglucosamine.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Characterization of IL-17/IL-17R gene family in Sebastes schlegelii and their expression profiles under Aeromonas salmonicida and Edwardsiella piscicida infections

Interleukin 17 (IL-17) is a hallmark inflammatory cytokine that can induce a variety of cells to produce immune-related factors, and plays a critical role in host defend itself against invasion by pathogens, which signals through a distinct receptor subclass- IL-17R. In our study, seven IL-17 and eight IL-17R family members were identified in black rockfish. Subsequently, phylogenetic relationships, gene structures and syntenic analysis were performed to confirm the annotation and homology of these genes during evolution. Except for eight positive selection sites were detected in IL-17RA1, IL-17RC1 and IL-17RD, most of gene sites were underwent purification selection. The mRNA levels of IL-17 and IL-17R genes were higher in immune-related tissues. Additionally, the expressions of IL-17RA1 and IL-17RE1 in spleen were dramatically induced by A. salmonicida treatment, as well as IL-17RA1, IL-17RB, IL-17RC1 and IL-17RE1 in liver, which indicated their important roles in antimicrobial infections. Furthermore, the expression patterns of IL-17 and IL-17R genes in mucosal tissues in response to E. piscicida infection were investigated, of which some members were dramatically induced, particularly for IL-17A/F1 and IL-17A/F3 in skin, IL-17A/F2 in gill, IL-17RB in intestine, IL-17RA1 and IL-17RC1 in skin and gill, suggests their potential essential roles in innate immune response. In summary, this study provides a systemic overview of the IL-17 and IL-17R gene families in black rockfish, which is of great importance to further study the functional characteristics of these genes.

Alteration of hemoglobin ß gene expression in mucosal tissues of Japanese flounder, Paralichthys olivaceus, in response to heat stress, Edwardsiella piscicida infection, and immunostimulants administration

Hemoglobin beta (Hbß) is a heme-binding protein capable of oxygen delivery. The oligopeptides derived from Hbβ in fish mucus are active against a variety of gram-negative bacteria and protozoa. To gain information on the physiological and immunological roles of Hbβ in the mucosal tissues of fish, we analyzed changes in Hbß gene expression levels in the epidermis, gills, and intestine of Japanese flounder, Paralichthys olivaceus, in response to heat stress, Edwardsiella piscicida infection, and trial feeding of immunostimulants, high-concentration ascorbic acid (AsA) or lactoferrin (LF). The results of quantitative real-time PCR showed that expression of the Hbß gene in the gills decreased markedly when exposed to heat stress, whereas that in the epidermis exhibited an increase 3h after infection with E. piscicida. Seven days after starting to feed either immunostimulant, epidermal Hbß gene expression in all AsA or LF dose groups was significantly higher than in the control group. The results of in situ hybridization showed that the abundance and intensity of the stained cells in the epidermis and in the gills were consistent with the expression levels of Hbß gene obtained from the infection and immunosuppressant experiments and the heat stress experiment, respectively. Our results suggest that mucosal Hbβ gene expression is closely related to physiological and immunological status and could be a useful indicator for monitoring condition of fish health.

Edwardsiella piscicida, a pathogenic bacterium newly detected in spotted sea bass Lateolabrax maculatus in China

In this study, Edwardsiella piscicida strain 18BJ136 was isolated from diseased spotted sea bass Lateolabrax maculatus, in China, and its pathogenicity in this newly recognized host was investigated. Physiological and biochemical tests showed that 18BJ136 did not produce H2S but utilized d-maltose, and was positive for succinate alkalinization, coumarate, and the ellman reaction. Its growth characteristic showed that it proliferated in a wide range of salinities, but was sensitive to acidity and alkalinity. A PCR analysis showed that 18BJ136 carried at least nine virulence genes: gadB, katB, citC, usp13, esrB, sodB, luxS, fimA, and mukF. A spot test showed that the strain had a variety of extracellular enzyme activities, including hemolysin and protease. Artificial infection experiments indicated that 18BJ136 was strongly virulent in the spotted sea bass, with a median lethal dose (LD50) of 1.23 × 103 cfu/g. Similar to the naturally infected fish, the main clinical symptoms of the artificially infected fish were festering scores of skin, scale shedding, and the swelling of multiple internal organs. The 18BJ136 caused the following histopathological lesions in the spotted sea bass: different degrees of inflammation in each tissue; multiple necrotic foci in the liver, spleen, and head kidney; and severe hemorrhage in the spleen and head kidney. The expression of IL-8, TNF-α, TLR-23, and HSP70 was significantly upregulated after infection (P < 0.05), indicating that the pathogen activated the host’s innate immune response. These results extended our understanding of E. piscicida infection in the spotted sea bass.

The Zebrafish Antiapoptotic Protein BIRC2 Promotes Edwardsiella piscicida Infection by Inhibiting Caspases and Accumulating p53 in a p53 Transcription-Dependent and -Independent Manner.

IAPs (inhibitors of apoptosis) are endogenous caspase inhibitors with multiple biological activities. In the present study, we show functional characteristics of antiapoptotic protein BIRC2 (cIAP1) in response to Edwardsiella piscicida infection. Overexpression of BIRC2 in zebrafish larvae promoted the proliferation of E. piscicida, leading to a decreased larvae survival. The expression levels of caspases including casp3, casp8, and casp9 were significantly inhibited by BIRC2 overexpression in the case of E. piscicida infection. Treatment of zebrafish larvae microinjected with BIRC2 with the caspase activator PAC-1 completely blocked the negative regulation of BIRC2 on the E. piscicida infection, with the reduced inhibition on the casp3 and without inhibition on casp8 and casp9. In contrast to the regulation of BIRC2 on the caspases, BIRC2 overexpression significantly induced the expression of p53, especially at 24 hpi. In addition to the cytoplasmic p53 expression, BIRC2 overexpression also induced the expression of the nuclear p53 protein. Further analysis demonstrated that BIRC2 could interact and colocalize with p53 in the cytoplasm. The numbers of E. piscicida in larvae overexpressed with BIRC2 and treated with pifithrin-μ (an inhibitor of mitochondrial p53) or pifithrin-α (an inhibitor of p53 transactivation) were lower than those of larvae without pifithrin-μ or pifithrin-α treatment. Critically, the p53 inactivators pifithrin-μ and pifithrin-α had no significant effect on larval survival, but completely rescued larval survival for zebrafish microinjected with BIRC2 in the case of E. piscicida infection. Collectively, the present study suggest that piscine BIRC2 is a negative regulator for antibacterial immune response in response to the E. piscicida infection via inhibiting caspases, and accumulating p53 in a p53 transcription-dependent and -independent manner.

Rapid on-the-spot detection of Edwardsiella piscicida using recombinase polymerase amplification with lateral flow

Edwardsiella piscicida is one of the important pathogens in aquaculture, which takes huge losses to the economic benefits of turbot industry. Thus, a simple, rapid and accurate diagnostic assay of E. piscicida becomes an urgent need. In the present study, a detection method of E. piscicida based on recombinase polymerase amplification combined with lateral flow strip (RPA-LF) was established. The RPA-LF assay targeted E. piscicida evpP gene and could detect as low as 10 fg DNA of E. piscicida, which was approximately 1000 times more sensitive than conventional PCR assay. Meanwhile, the RPA-LF assay showed no cross-reactivity with DNA from other major bacterial pathogens in aquaculture. Besides, to improve the convenience of RPA-LF assay, a rapid on-the-spot sample preparation using nucleic acid extraction by cellulose dipsticks was established. Results indicated that bacteria could be detected as low as 1 copy and completed within 30 min. Moreover, this modified RPA-LF assay could be used to monitor E. piscicida infection in turbot industry.

Pyroptosis Mediates Neutrophil Extracellular Trap Formation during Bacterial Infection in Zebrafish

The formation of neutrophil extracellular trap (NET) is a critical host defense when neutrophils migrate to infection sites. Pyroptosis is a newly identified programmed cell death, which is tightly regulated by inflammasome activation. However, the mechanism of pyroptotic signaling participating in NET production remains to be elucidated. In this study, the zebrafish larvae otic vesicle microinjection model was used to infect larvae with hemolysin-overexpressing Edwardsiella piscicida (EthA+), and a rapid migration of neutrophils to infection sites was observed. Intriguingly, EthA+ infection effectively induced significant neutrophil membrane rupture in vivo, which was dependent on caspase-B (caspy2) and gasdermin Eb (GSDMEb) but not caspase-A or gasdermin Ea. Specifically, the EthA+ E. piscicida infection induced pyroptosis along with NETosis in vitro, and depletion of either caspy2 or GSDMEb impaired NET formation in vivo. Consequently, inhibition of the caspy2-GSDMEb axis-gated NETosis impaired bacterial clearance in vivo. Altogether, these data provide evidence that teleost fish innate immune cells, including neutrophils, express features of pyroptosis that are critical for NETosis in teleost innate immunity.

Two CD9 tetraspanin family members of Japanese flounder (Paralichthys olivaceus): characterization and comparative analysis of the anti-infectious immune function

CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.

Effects of a short pulse administration ofUlva rigidaon innate immune response and intestinal microbiota inSparus auratajuveniles

Given the potential of algae as a new aquafeed ingredient, this study evaluated the effect of the tank-cultivated macroalgae Ulva rigida C. Agardh (Chlorophyta) on innate immune response, intestinal microbiota and their resistance against Photobacterium damselae subsp. piscicida in juvenile gilthead seabream (Sparus aurata L.) after a short pulse of administration. An algae-free diet was used as control, and an experimental diet was formulated containing 25% of U. rigida (UL-25). After 2, 7, 15 and 30 days biological samples were taken. No statistical significance (p > 0.05) was detected in respiratory burst activity. Diet UL-25 significantly enhanced alternative complement pathway activity respect to control diet from the beginning of the experiment (p < 0.05). When fish were challenged against P. damselae subsp. piscicida, UL-25-fed fish were more resistant against the pathogen than the fish fed control diet, obtaining a relative survival percentage of 54.55%. The ability of UL-25 diet to modify the intestinal microbiota was also confirmed. In conclusion, the use of a short pulse of dietary administration of 25% U. rigida in S. aurata diet modulates the immune response, intestinal microbiota and reduced the mortality rate during P. damselae subsp. piscicida infection.

Edwardsiella piscicida HigB: A type II toxin that is essential to oxidative resistance, biofilm formation, serum survival, intracellular propagation, and host infection

Edwardsiella piscicida (formerly included in E. tarda) is the leading pathogen threatening worldwide fresh and seawater aquaculture industries and has been considered as a model organism for studying intracellular and systemic infections. Toxin-antitoxin (TA) systems are omnipresent in bacteria and participate widely in stress responses, biofilm formation and pathogenicity. However, the roles of TA systems are completely unknown in aquatic pathogenic bacteria. In this study, we identified and characterized a type II TA system HigBA in E. piscicida, where HigB is the toxin and HigA is the antitoxin. higB and higA are expressed in a bicistronic operon. To examine the biological role of HigBA, two markerless higB and higBA in-frame mutant strains, TX01ΔhigB and TX01ΔhigBA, were constructed. Compared to the wild strain TX01, TX01ΔhigB exhibited markedly reduced resistance against oxidative stress, but TX01ΔhigBA remained unchanged; the biofilm growth of TX01ΔhigB was decreased, but that of TX01ΔhigBA was increased. The deletion of higB enhanced bacterial motility but impaired bacterial resistance against host serum killing. In vitro infection experiment showed that the inactivation of higB dramatically abated bacterial capability of adhesion, invasion, and propagation in host cells. Consistently, in vivo experiment suggested that the higB mutation had an obvious negative effect on bacteria dissemination of fish tissues and general virulence. Introduction of a trans-expressed higB restored the lost virulence of TX01ΔhigB. However, the virulence of TX01ΔhigBA was compared to that of TX01. These findings indicate that HigB is essential for responding adverse circumstance and pathogenicity of E. piscicida. Based on the attenuated virulence of TX01ΔhigB, we further investigated its potential as a live vaccine. Vaccination experiment showed that TX01ΔhigB induced effective protection against lethal E. piscicida infection. In addition, we found that antitoxin HigA negatively autoregulated the expression of higBA by binding directly with own promoter, and toxin HigB promoted the regulatory function of HigA. This study provides new insights into the biological activity of type II TA system HigBA in aquatic pathogenic bacteria and contributes to understand the pathogenesis of E. piscicida.

The function of zebrafish gpbar1 in antiviral response and lipid metabolism

G protein-coupled bile acids receptor 1 (GPBAR1 or TGR5) has been widely studied as a metabolic regulator involved in bile acids synthesis, glucose metabolism and energy homeostasis. Several recent studies have shown that mammalian GPBAR1 is also involved in antiviral innate immune responses. However, the functions of piscine GPBAR1 in antibacterial or antiviral immune responses and lipid metabolism remain unclear. In the present study, we report the functional characterization of zebrafish gpbar1. Similar to mammalian GPBAR1, zebrafish gpbar1 contains similar domain composition, shows a dose-dependent activation by bile acids including INT777, LCA, DCA, CDCA and CA, and can be induced by viral infection. Compared with corresponding control groups, a significant antiviral activity against spring viremia of carp virus (SVCV) infection was observed in ZF4 cells overexpressing zebrafish gpbar1 with INT777 treatment, but not in ZF4 cells overexpressing zebrafish gpbar1 without INT777 treatment. The activation of zebrafish gpbar1 had no significant antibacterial effect against Edwardsiella piscicida infection in ZF4 cells in vitro. Transcriptome analysis revealed that zebrafish gpbar1 activation played a crucial role in activating RLR signaling pathway and inducing the production of ISGs, but not for bile acid biosynthesis and transportation. The co-occurrence analysis for antiviral-related and bile acids metabolism-related DEGs suggested a strong interaction among 2 bile acid receptors (gpbar1 and nr1h4), slco2b1 and the antiviral DEGs. The lipidomic analysis showed that zebrafish gpbar1 activation in ZF4 cells resulted a change of glycerophospholipids, but none of bile acids nor their derivatives, which were different from mammalian GPBAR1. All together, these results firstly demonstrate the conserved antiviral role of gpbar1 and its function in regulating glycerophospholipids metabolism in teleost.

Effect on health status and pathogen resistance of gilthead seabream (Sparus aurata) fed with diets supplemented with Gracilaria gracilis

Aquaculture intensification turns the reared populations prone to disease outbreaks, leading the industry to use antibiotics and chemotherapeutics. As the industry is searching for solutions, the development of sustainable aquafeeds with immunostimulant properties is emerging. This work aimed to assess the health benefits of Gracilaria gracilis as a supplement in Sparus aurata feed as well as its protective effect in a bacterial challenge. G. gracilis supplementation improved growth performance and influenced the haematological profile, namely in the constitution of the leukocytic population and erythrocytes size. Algae powder supplementation did not compromise the hepatic antioxidant systems and intestinal health and morphology were not affected by the algal supplementations. Algal extract improved lysozyme plasmatic concentration and a 5% algae powder inclusion was able to successfully protect the fish against a Photobacterium damselae subsp. piscicida infection, granting a 95% survival. In general, a 5% G. gracilis powder diet supplementation was able to improve growth, health, and bacterial resistance in gilthead seabream.

NLRC3-like 1 inhibits NOD1-RIPK2 pathway via targeting RIPK2

Both NLRC3 and NOD1 belong to regulatory NLR subfamily based on their best-characterized function. In mammals, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs. In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing. However, the functions of many NLRC3-like genes, especially the fish-specific NLRC3-like genes, remain unclear. In the present study, we report the functional characterization of a novel category of NLRC3-like proteins (named as NLRC3-like 1) from the zebrafish, which consists of a fish-specific FISNA, a conserved NACHT and five C-terminal LRRs domains. The expression of zebrafish NLRC3-like 1 was inducible in response to Edwardsiella piscicida infection. During bacterial infection, the in vitro and in vivo studies revealed that zebrafish NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, together with the decreased survival rate of zebrafish larvae infected with E. piscicida. The attenuated response by zebrafish NLRC3-like 1 in response to bacterial infection were characterized by the impaired expression of antibacterial genes, proinflammatory cytokines and Nox genes. Furthermore, zebrafish NLRC3-like 1 interacted with the adaptor protein RIPK2 of NODs signaling via the FISNA (Fish-specific NACHT associated domain) and NACHT domains. However, the interaction between zebrafish NLRC3-like 1 and RIPK2 inhibited the assembly of the NOD1-RIPK2 complex. Importantly, zebrafish NLRC3-like 1 inhibited NOD1-mediated antibacterial activity, NF-κB and MAPK pathways and proinflammatory cytokine production. All together, these results firstly demonstrate that zebrafish NLRC3-like 1 inhibits NOD1-RIPK2 antibacterial pathway via targeting the adaptor protein RIPK2.

Phenotype, Virulence and Immunogenicity of Edwardsiella piscicida Cyclic AMP Receptor Protein (Crp) Mutants in Catfish Host.

Edwardsiella piscicida, a facultative aerobic pathogen belonging to the Enterobacteriaceae family, is the etiological agent of edwardsiellosis that causes significant economic loses in the aquaculture industry. cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. Here we characterize the crp gene and report the effect of a crp deletion in E. piscicida. The crp-deficient mutant lost the capacity to utilize maltose, and showed significantly reduced motility due to the lack of flagella synthesis. We further constructed a ΔPcrp mutant to support that the phenotype above was caused by the crp deletion. Evidence obtained in fish serum killing assay and competitive infection assay strongly indicated that the inactivation of crp impaired the ability of E. piscicida to evade host immune clearance. More importantly, the virulence of the crp mutant was attenuated in both zebrafish and channel catfish, with reductions in mortality rates. In the end, we found that crp mutant could confer immune protection against E. piscicida infection to zebrafish and channel catfish, indicating its potential as a live attenuated vaccine.

Inclusion of dietary Ulva ohnoi 5% modulates Solea senegalensis immune response during Photobacterium damselae subsp. piscicida infection

Macroalgae represent valuable sources of functional ingredients for fish diets, and the influence of supplemented aquafeeds on growth performance has been studied for some fish and seaweed species. In the present work, the potential immunomodulation exerted by U. ohnoi (5%) as dietary ingredient was investigated in Senegalese sole. After feeding with the experimental diets for 90 d, fish immune response before and after challenge with Photobacterium damselae subsp. piscicida (Phdp) was assessed. In absence of infection, systemic immune response was not modified by 5% U. ohnoi dietary inclusion for 90 d. Thus, no differences in liver and head kidney immune gene transcription or serum lysozyme, peroxidase, antiprotease and complement activities were observed based on the diet received by Senegalese sole specimens. Regarding mucosal immune parameters, no changes in gene transcription were detected in the skin and gills, whilst only tnf, cd4 and cd8 were significantly up-regulated in the intestine of fish fed with U. ohnoi, compared to the values obtained with control diet. On the contrary, when S. senegalensis specimens were challenged with Phdp, modulation of the immune response consisting in increased transcription of genes encoding complement (c1q4, c3, c9), lysozyme g (lysg), tumor necrosis factor alpha (tnfα) as well as those involved in the antioxidant response (gpx, sodmn) and iron metabolism (ferrm, hamp-1) was observed in the liver of fish fed with U. ohnoi. In parallel, decreased inflammatory cytokine and complement encoding gene transcription was displayed by the spleen of fish receiving the algal diet. Though mortality rates due to Phdp challenge were not affected by the diet received, lower pathogen loads were detected in the liver of soles receiving U. ohnoi diet. Further research to investigate the effects of higher inclusion levels of this seaweed in fish diets, feeding during short periods as wells as to assess the response against other pathogens needs to be carried out.

An Edwardsiella piscicida esaS mutant reveals contribution to virulence and vaccine potential

Edwardsiella piscicida is a Gram-negative pathogen that causes disease in diverse aquatic organisms. The disease leads to extensive losses in commercial aquaculture species, including farmed U.S. catfish. The type III secretion system (T3SS) often contributes to virulence of Gram-negative bacteria. The E. piscicida esaS gene encodes a predicted T3SS export apparatus protein. In the current study, an E. piscicida esaS mutant was constructed and characterized to increase our understanding of the role of T3SS in E. piscicida virulence. Deletion of esaS did not significantly affect biofilm formation and hemolytic activity of E. piscicida, but it had significant effects on expression of hemolysis and T3SS effector genes during biofilm growth. EpΔesaS showed significantly (P < 0.05) reduced virulence in catfish compared to the parent strain. No mortalities occurred in fish infected with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU/fish compared to 26% mortality in fish infected with wild-type E. piscicida at 7.5 × 105 CFU/fish. Bioluminescence imaging indicated that EpΔesaS invades catfish and colonizes for a short period in the organs. Furthermore, catfish immunized with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU provided 47% and 87% relative percent survival, respectively. These findings demonstrated that esaS plays a role in E. piscicida virulence, and the deletion mutant has vaccine potential for protection against wild-type E. piscicida infection.

Potential immune regulatory role of miR-146a upon Aeromonas hydrophila and Edwardsiella piscicida infections in zebrafish.

This study was aimed to understand the expression of miR-146a in zebrafish (Danio rerio) and its role in regulating immune responses during Aeromonas hydrophila and Edwardsiella piscicida infections. The miR-146a expression was observed from the 1-h post fertilization (hpf) stage and gradually increased up to the early larval stage of zebrafish. The ubiquitous expression of miR-146a was detected in all tested tissues, with the highest level in gills. The expression of miR-146a was significantly increased in larvae when exposed to E. piscicida infection at 24 and 48h post exposure (hpe). Intraperitoneally (i.p.) injected A. hydrophila and E. piscicida into adult zebrafish showed significant upregulation of miR-146a in gills. Furthermore, immune-related genes, toll-like receptor,tlr-4, transducing signaling pathway molecules, traf-6 and myd88 (bacteria-infected larvae and adults), transcription factor relA and mcp-1b (bacteria-infected adults), pro-inflammatory, il-6 (A. hydrophila-exposed larvae) and mmp-9 (bacteria-exposed larvae) were significantly repressed. In contrast, il-1β, tnf-α, cxcl-18b, and ccl-34a.4 were induced in both bacteria-challenged larvae and adults. Based on the results, it is suggested that endogenous miR-146a could act as an infection inducible miRNA in zebrafish upon A. hydrophila and E. piscicida infections; also, it could potentially regulate the immune responses in zebrafish.

Trxlp, a thioredoxin-like effector from Edwardsiella piscicida inhibits cellular redox signaling and nuclear translocation of NF-κB

Redox signaling and homeostasis are essential for cell survival and the immune response. Peroxiredoxin (Prx) modulates the level of H2O2 as a redox signal through H2O2 decomposition. The redox activity of thioredoxin (Trx) is required as a reducing equivalent to regenerate Prx. Edwardsiella piscicida is an opportunistic Gram-negative enteric pathogen that secretes a novel Trx-like effector protein, ETAE_2186 (Trxlp). Trxlp has unique structural properties compared with other Trx proteins. In enzymatic and binding assays, we confirmed Trxlp to be redox-inactive due to the low reactivity and flexibility of the resolving cysteine residue, C35, at the active site motif “31WCXXC35”. We identified key residues near the active site that are critical for reactivity and flexibility of C35 by site-directed mutagenesis analysis. NMR titration experiment demonstrated prolong inhibitory interaction of Trxlp with Prx1 resulting in the repression of Prx1–mediated H2O2 decomposition leading to increased ROS accumulation in infected host cells. Increased ROS in turn prevented nuclear translocation of NF-κB and inhibition of NF-κB target genes, leading to bacterial survival and enhanced replication inside host cells. Targeting Trxlp-mediated virulence promises to attenuate E. piscicida infection.