Vibrio Harveyi Infection Research Articles

Understanding the effects of salinity and Vibrio harveyi on the gut microbiota profiles of Litopenaeus vannamei

Increasing evidence have revealed a positive correlation between gut microbiota and shrimp health, in which a healthy shrimp gut consists of a complex and stable microbial community. Given that both abiotic and biotic factors constantly regulate shrimp gut microbiota, any changes affecting the levels of these factors could cause modification to the gut microbiota assemblage. The goal of this study was to explore the effects of salinity levels and pathogenic Vibrio harveyi infection on the diversity, structure, composition, interspecies interaction, and functional pathways of Litopenaeus vannamei gut microbiota. Juvenile shrimp were cultured at 5 ppt, 20 ppt, and 30 ppt for two months prior to Vibrio harveyi infection. After pathogenic V. harveyi challenge test, genomic DNA was isolated from the shrimp gut, and subjected to the 16S rRNA metagenomic sequencing analysis. We observed that gut microbiota diversity of shrimp cultured at 5 ppt and 30 ppt were lower than those cultured at 20 ppt after exposure to V. harveyi infection, suggesting that shrimp cultured at the two former salinity levels were more susceptible to V. harveyi infection. Network analysis also showed that shrimp cultured at 20 ppt exhibit a more stable bacterial network with complex interspecies interaction, even after induced by V. harveyi. Moreover, the presence of a high number of beneficial bacteria such as Pseudoruegeria, Rhodovulum, Ruegeria, Shimia and Lactobacillus in shrimp cultured at 20 ppt might have played a role in inhibiting the growth of V. harveyi and other potentially pathogenic bacteria. Besides, bacterial functional pathway prediction has also shown that metabolic pathways such as phenylalanine metabolism, glycine, serine and threonine metabolism, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, carbon metabolism and biofilm formation process were significantly higher in shrimp cultured at 20 ppt. Collectively, our results suggested that 20 ppt is an optimal salinity that suppresses the growth of V. harveyi and potential pathogenic bacteria in the shrimp gut, which could possibly minimize the risk of pathogenic infection for sustainable production of healthy shrimp.

Similarities and differences between vibriosis in European sea bass caused by Vibrio anguillarum and Vibrio harveyi

Vibrioses caused by different members of the genus Vibrio are common diseases in aquaculture characterised by systemic infections, high mortalities, and economic losses of fish and shellfish. Farming of European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) has rapidly grown over the last decade in Croatia but its economic efficacy is significantly jeopardized by losses due to Vibrio anguillarum and Vibrio harveyi infection. Therefore, we studied and compared the most prominent similarities and differences in the environmental conditions, clinical signs and conventional and molecular diagnostic methods of vibriosis caused by both bacterial pathogens. Outbreaks in sea bass and sea bream caused by V. anguillarum mostly occur during the spring and autumn, following a fast increase or decrease in the sea temperature, whereas infections with V. harveyi occur during the summer months at a temperature above 20o C. They have a similar clinical appearance in the acute form but in subacute forms they differ. V. anguillarum infection is characterized by massive haemorrhages in the abdominal wall, while during infection with V. harveyi keratitis, corneal opacity and uncoordinated swimming behaviour are noticed. Necropsy disclosed haemorrhages in the liver, intestine and stomach during infection with V. anguillarum, and sero-catarrhal enteritis with distension of the intestine and ascites in the case of V. harveyi. Although molecular tools enable correct identification, in this study we defined the main differences capable of distinguishing V. anguillarum from V. harveyi based on traditional bacteriological identification such as sensitivity to O/129 (10 µg) and novobiocin, and the enzymatic activity of β-galactosidase, arginine dihydrolase and amygdalin fermentation.

Zootechnical performance, immune response, and resistance to hypoxia stress and Vibrio harveyi infection in Pacific white shrimp (Litopeneaus vannamei) fed different fishmeal diets with and without addition of sodium butyrate

The dietary effect of a chemically protected sodium butyrate, Butirex®C4 (BU), on growth performance, digestive enzymes activity, immunity, gut microflora, and resistance against hypoxia stress and infectious disease were studied in Pacific white shrimp fed different levels of fishmeal (FM) diets in earthen outdoor ponds. Nine isonitrogenous and isolipidic experimental diets were formulated to contain different levels of FM (45 %, 30 %, and 15 %) and enriched with the incremental levels of BU (0, 0.3%, and 0.6%). After 60 days, the shrimp fed diets containing 45% and 30 % FM along with 0.3 % and 0.6 % BU, respectively, showed higher levels of final weight and weight gain, while, the group fed diet with 15 % FM free from BU showed the highest level of feed conversion ratio. The levels of protease and trypsin activity significantly decreased in the group fed 15 % FM without BU, while the administration of BU improved the digestive enzymes activity in low FM diets. The total hemocyte count, phenoloxidase, and lysozyme were negatively affected by decreasing levels of FM in the diets, but BU-supplemented diets improved these parameters. The dietary administration of BU increased lactic acid bacteria and decreased total Vibrio sp. counts in the intestine of shrimp fed different levels of FM. Although the diets with low levels of FM had no significant effect on the hypoxia stress tolerance, the cumulative mortality was decreased in the low FM groups fed with different BU levels. The mortality of shrimp against Vibrio harveyi infection was notably decreased in BU-added groups compared to the groups that did not receive BU in their diets. Overall, dietary BU administration at 0.6 % mitigates the adverse impacts of the diets with the lowest FM (15 %) on overall performance and immunity in Pacific white shrimp.

Exosomal miRNAs in the plasma of Cynoglossus semilaevis infected with Vibrio harveyi: Pleiotropic regulators and potential biomarkers involved in inflammatory and immune responses.

Exosomes are a class of extracellular vesicles with diameters ranging from ~50 to 150 nm. Incorporating diverse biological substances and being present extensively in biofluids, exosomes are involved in intracellular communication in various physiological and pathological processes and emerging as promising biomarkers for the prognosis and diagnosis of many diseases. Accumulating evidence shows that exosomes also play important roles in the inflammatory and immune responses to bacterial infection. However, the study of exosomes in teleost fish remains scarce. In the present study, we focused on the exosomal microRNAs (miRNAs) in the plasma of Chinese tongue sole (Cynoglossus semilaevis) in response to Vibrio harveyi infection. After bacterial challenge, the plasma was sampled at both the early (6 and 16h) and late stages. (48, 72, and 96h) of infection, followed by exosome isolation and exosomal miRNA sequencing. Results showed that the expression profile of 85 exosomal miRNAs was significantly different among the control, early-, and late-infection groups. The predictive genes targeted by exosomal miRNAs were extensively involved in various inflammatory and immune processes by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, suggesting that a series of processes were regulated by exosomal miRNAs in the plasma, including the pathogen invasion and recognition and the activation and regulation of signaling pathways related to cytokine production. Moreover, the spleen was found to be a preference for exosome enrichment and the underlying mechanism of interleukin-6 production regulated by ZC3H12A, ARID5A, and exosomal ssa-miR-146a-5p were probably present in Chinese tongue sole. Additionally, the enhanced levels of ssa-miR-146a-5p and nbr-miR-731 in plasma exosomes and the spleen of the infection groups were identified, indicating their application as biomarkers in favor of the prognosis and diagnosis of V. harveyi infection in Chinese tongue sole. Therefore, the collective results in the present study indicated the pleiotropic roles of exosomal miRNAs in the regulation of inflammatory and immune responses and their potential utilization as biomarkers in teleost fish.

Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus)

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys251, His397, and Asn419). It shares high homology (69.47%–90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn2+ and Ca2+ but inhibited by Fe2+ and Cu2+. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1β), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

The efficiency of indigenous isolates Bacillus sp. RCS1 and Bacillus cereus RCS3 on growth performance, blood biochemical indices and resistance against Vibrio harveyi in cobia fish (Rachycentron canadum) juveniles

In this study, cobia (Rachycentron canadum) juveniles were utilised as an aquatic model to explore growth performance and immune indices after 70 days of feeding with supplementation of viable isolated microbes’ concentrations of 1 × 1010 and 1 × 1012 CFU/mL indigenous isolates Bacillus sp. RCS1 and Bacillus cereus strain RCS3, respectively. Regarding growth performance, fish provided with the indigenous isolates exhibited a significant (P < 0.05) enhancement. The hematological indices such as red blood cells, white blood cells, corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin, and Mean corpuscular haemoglobin were all increased (P < 0.05) in the juvenile cobia fed with indigenous isolates Bacillus sp. RCS1 and B. cereus RCS3 as a nutritional supplement. This study also significantly differed in serum analysis (albumin, globulin, lysozyme, total protein) between all the treatments with the Bacillus sp. RCS1 1 × 1010 proven to be the best. In the present study, the expression of myostatin and insulin-like growth factor 2 showed no significant change (P > 0.05) among the treatment and control groups tested; nonetheless, their mRNA expression exhibited variations in all trial groups. The present study showed significant differences (P < 0.05) among groups in the case of insulin-like growth factor 1, peroxisome proliferator-activated receptors. The improved antioxidant enzyme activities of catalase (CAT) and superoxide dismutase (SOD) digestive enzyme activities (amylase, trypsin, pepsin, lipase, and cellulose) may be linked to the enhanced resistance of fish against Vibrio harveyi infection. The enhanced activities of digestive enzymes in indigenous isolates treatments could be linked to the improved digestion and enhanced food absorption that ultimately helps enhance feed utilisation and growth performance. The results showed that indigenous isolates addition to a commercial diet, had the prospective to enhance cobia juveniles' growth and immune response regardless of their dosage.

The Effect of Adding White Tumeric (Curcuma zedoaria) Extract on the Immune System of Vaname Shrimp (Litopenaeus vannamei) Tested Against Vibrio harveyi Bacteria

The entry of vaname shrimp into Indonesia was to replace tiger prawns (Penaeus monodon) which at that time experienced a decline in quality. Although vaname shrimp is famous for having various advantages, there are still obstacles encountered during the cultivation period, one of which is the attack of vibriosis caused by the bacterium Vibrio harveyi. This study aims to determine the effect of adding white turmeric extract (Curcuma zedoaria) on the immune system of white shrimp (Litopenaeus vannamei) which was challenged with Vibrio harveyi bacteria. This research was conducted for 60 days from March to May 2021 using experimental research methods. The research container used a container container (size 40x30x28 cm) and a stocking density of 20 fish/container with a volume of 20 L. The research design used was a completely randomized design consisting of 5 treatments with 3 replications where P1: artificial feed + Vibrio harveyi infection, P2: artificial feed + 0.1 mL NaCl injection, P3: artificial feed + 0,5% white turmeric extract + V.harveyi infection, P4: Artificial Feed + 1% white turmeric extract + V.harveyi infection, P5: Artificial feed + 2% white turmeric extract + V.harveyi infection. The results showed that administration of white turmeric extract (C.zedoaria) with a dose of 1% gave the best results and had a significant effect on the survival rate of 68.3%, Total Haemocyte Count of 21.63x10⁶ cells/mL, and Differential Haemocyte Count (hyaline cells). by 58.7%, semi-granulocytes by 18.33%, granulocytes by 23%) which play a role in the immune system of white vaname shrimp. In addition, the value of phagocytic activity was 68.8% and was able to suppress the growth of bacteria in the intestine at the lowest at 57.7×108 CFU/mL and suppress the number of vibrios to the lowest number at 26×106 CFU/mL

Genome‐wide characterization of caspase genes in Japanese pufferfish, Takifugu rubripes, and expression profiles in response to Vibrio harveyi infection

AbstractCaspases, also known as cysteine‐dependent aspartate‐directed proteases, play an important role in regulating apoptosis during development and inflammation. In this study, 10 caspase genes were characterized by searching the Japanese pufferfish genome using various bioinformatics methods, such as multiple sequence alignment and phylogenetic analysis. Additionally, we characterized the caspase genes and examined the expression profiles of every member in both healthy tissues and tissues infected with Vibrio harveyi in the liver and intestine. Our results revealed that 10 caspase genes were detected in all examined tissues, and the expression levels varied among different tissues and genes. Vibrio harveyi infection induced significant changes in the expression of 10 caspases in a time‐dependent manner, suggesting that they might play key roles in the defense process of Japanese pufferfish. Our findings offer strong support for future studies aiming to provide an initial assessment of caspase genes in teleosts.

Effectiveness of Meniran Leaf Extract (Phyllanthus niruri L.) as Immunostimulant in Vannamei Shrimp (Litopenaeus vannamei) Against Vibriosis Disease

Vibrio harveyi infection was an obstacle in shrimp culture. Meanwhile, the use of antibiotics in disease control was no longer effective, so the use of immunostimulants such as meniran leaves was the safest alternative. The purpose of this study was to determine the effect of adding different doses of meniran leaf extract (Phyllanthus niruri L.) to feed on the immune system of vannamei shrimp (Litopenaeus vannamei) which was challenged with Vibrio harveyi. This research was an experimental study using completely randomized design (CRD) consist of 5 treatments and 3 replications, namely, P1 (positive control): commercial feed + V. harveyi infection; P2 (negative control): commercial feed + 0.9% NaCl; P3: commercial feed + 0.5% Meniran leaf extract + V. harveyi infection; P4: commercial feed + 1% Meniran leaf extract + V. harveyi infection; and P5: commercial feed + 2% Meniran leaf extract + V. harveyi infection. The parameters observed were survival and immune response. Immune response analysis was carried out by counting hemocytes, phagocytic activation and total bacteria present in the shrimp intestines at the end of the study. The results of this study indicate that the addition of meniran leaf extract affects the immune system and the viability of vannamei shrimp. In conclusion, the addition of meniran extract to vannamei shrimp feed with a dose of 1% can improve the immune system and viability of vannamei shrimp against vibriosis.

Conventional Soybean Meal as Fishmeal Alternative in Diets of Japanese Seabass (Lateolabrax japonicus): Effects of Functional Additives on Growth, Immunity, Antioxidant Capacity and Disease Resistance.

Aiming to optimize soymeal-based diets for Japanese seabass (Lateolabrax japonicas), a 105-day feeding trial was conducted to evaluate the effects of functional additives, including antioxidants (ethoxyquin, thymol and carvacrol) and chelated trace elements (Cu, Mn and Zn), on the growth, immunity, antioxidant capacity and disease resistance of fish fed diets with conventional soybean meal replacing 50% of fishmeal. Three isonitrogenous (45%) and isolipidic (11%) diets were formulated: (1) standard reference diet (FM, 42% fishmeal); (2) soymeal-based diet (SBM, 21% fishmeal and 30% conventional soybean meal); (3) SBM diet supplemented 0.0665% functional additives (FAS). Each experimental diet was randomly fed to quadruplicate groups of forty feed-trained Japanese seabass (initial average body weight = 125.6 ± 0.6 g) stocked in a saltwater floating cage. Upon the conclusion of the feeding trial, lower feed intake was observed in fish fed SBM compared to those fed FM and FAS. Fish fed FM showed the highest growth performance, estimated as the weight gain rate. Notably, FAS supported faster growth of fish than those fed SBM, indicating the optimal growth performance of dietary functional additives. The feed conversion rate showed the opposite trend among dietary treatments, with the highest value in fish fed SBM. Regarding immunity, fish fed soymeal-based diets suppressed the serum alternative complement pathway activities compared to FM, whereas the respiratory burst activity in macrophages of head kidneys showed a similar picture, but no statistical differences were observed. Further, fish fed soymeal-based diets had lower serum Cu-Zn SOD, CAT and GPx activities as well as liver vitamin E levels and scavenging rates of hydroxyl radical but higher liver MDA contents compared to the FM-fed group. Fish fed FAS had higher serum Cu-Zn SOD and GPx activities and liver vitamin E levels than those fed SBM, suggesting the enhancement of antioxidant capacity of dietary functional additives. For the disease resistance against Vibrio harveyi infection, fish fed SBM had the highest cumulative mortality, followed by the FAS and FM groups. Additionally, the biomarkers related to the immune and antioxidant capacities had a positive correlation with the relative abundance of Paracoccus and Pseudomonas, while liver MDA levels had a negative correlation with the relative abundance of Pseudomonas and Psychrobacter. Collectively, soymeal replacing 50% of fishmeal suppressed the growth, immunity, antioxidant capacity and disease resistance of Japanese seabass, while dietary supplementation of antioxidants and chelated trace elements could mitigate soymeal-induced adverse effects on growth and disease resistance through the improvement in antioxidant capacity and regulation of gut microbiota.

Transcriptome Analysis Indicates Immune Responses against Vibrio harveyi in Chinese Tongue Sole (Cynoglossus semilaevis).

Simple SummaryLimited understanding of molecular mechanisms of immune response constrains marine fish farming. Analyzing the dynamic gene expression profile of fish in response to pathogen infection is gaining interest. We analyzed the expression changes of the Chinese tongue sole kidney after Vibrio harveyi infection with a series of transcriptome data. Notably, we observed rapid up-regulation of IL-17, TNF and TLR signaling pathways, indicating treatment measures should be taken in the early stage after infection. We also found a close connection between the immune system and neuroendocrine system, which may be the new strategy to improve immune function. Our research provides insights into disease prevention and treatment in fish farming.Pathogenic infection of fishes is an important constraining factor affecting marine aquaculture. Insufficient understanding of the molecular mechanisms has affected the diagnosis and corresponding treatment. Here, we reported the dynamic changes of gene expression patterns in the Chinese tongue sole kidney at 16 h, 48 h, 72 h and 96 h after Vibrio harveyi infection. In total, 366, 214, 115 and 238 differentially expressed genes were obtained from the 16 h−vs. −C, 48 h−vs. −C, 72 h−vs. −C and 96 h−vs. −C group comparisons, respectively. KEGG enrichment analysis revealed rapid up-regulation of several immune-related pathways, including IL-17, TNF and TLR signaling pathway. More importantly, time-series analyses of transcriptome showed that immune genes were specifically up-regulated in a short period of time and then decreased. The expression levels of chemokines increased after infection and reached a peak at 16 h. Specifically, Jak-STAT signaling pathway played a crucial role in the regulation during Vibrio harveyi infection. In the later stages of infection, genes in the neuroendocrine pathway, such as glucocorticoid-related genes, were activated in the kidney, indicating a close connection between the immune system and neuroendocrine system. Our dynamic transcriptome analyses provided profound insight into the gene expression profile and investigation of immunogenetic mechanisms of Chinese tongue sole.

Dietary astaxanthin modulated the performance, gastrointestinal histology, and antioxidant and immune responses and enhanced the resistance of Litopenaeus vannamei against Vibrio harveyi infection

Astaxanthin (AX) is one of the commonly used feed supplements to enhance the growth performance and provide antioxidant and immune functions of several aquatic animals. In the current study, juveniles of white-leg shrimp (Litopenaeus vannamei) with mean initial weight of 0.340 ± 0.041 g were fed with diets supplemented with 0 (control), 25, 50, 100, and 200 mg/kg feed for 8 weeks. At the end of the feeding trial, shrimps were exposed to Vibrio harveyi, and their mortality rates were observed for additional 10 days. The growth indices in the AX-fed groups were significantly (P < 0.05) higher than what were observed in shrimps in the control group. Dietary AX stimulated the final weight, weight gain, and specific growth rate and optimum growth levels were achieved at 100–200 mg AX/kg feed. Furthermore, the AX-enriched diets significantly enhanced feed intake more than the control diet, and the amount of AX had no effects on feed conversion ratios. In comparison to the control group, the AX-fed animals had significantly (P < 0.05) higher villi length, villi width, and absorption area and their optimum values were observed at 100–200 mg AX/kg feed treatments. Moreover, the intestinal morphometry especially villi and its crypt, both internal and external tunica muscularis, and submucosal tissues did not show any inflammatory and/or degenerative changes in AX-fed shrimp. Furthermore, the dietary AX at escalating levels linearly and quadratically enhanced (P < 0.05) the activities of serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and increased levels of total antioxidant capacity and reduced glutathione. In addition, malondialdehyde levels decreased significantly in AX-fed animals, and the highest levels were observed in the control group (without AX). The expression levels of cMn-SOD, CAT, and GPx genes were significantly upregulated in the hepatopancreas of L. vannamei fed with AX-enriched diets (especially in the 200-mg/kg feed treatment) as compared with the control diet. The immunity indices of the AX treatments (hematocyte count, total protein, lysozyme, phagocytic activity, and phenoloxidase) of L. vannamei were linearly (P < 0.05) and quadratically (P < 0.05) increased. This study revealed the antibacterial activity of AX against V. harveyi abundance. After the bacterial challenge, feeding L. vannamei with dietary AX significantly increased (P < 0.05) the relative percentage of survival, especially in the treatment of 200-mg/kg diet (82.7%). The findings of the current study demonstrate that dietary AX (100–200 mg/kg feed) was effective in enhancing the growth, antioxidant status, immune response, and increasing the resistance of L. vannamei against V. harveyi infection.

Improved growth performance, digestive ability, antioxidant capacity, immunity and Vibrio harveyi resistance in coral trout (Plectropomus leopardus) with dietary vitamin C

An 8-week growth trial was conducted to assess the effects of dietary vitamin C (VC) on the growth performance, digestive enzyme activity, antioxidant ability, immunity, and resistance to Vibrio harveyi infection of coral trout (Plectropomus leopardus, initial weight 17.42 ± 0.06 g). Four kinds of iso-nitrogenous and iso-lipidic experimental feeds were formulated with covering VC levels of 0 (C0), 50 (C1), 100 (C2), and 200 (C3) mg/kg, respectively. Supplementation of an appropriate amount of VC in diet (a) increased specific growth rate (SGR) and weight gain rate (WGR); (b) increased amylase, lipase, and trypsin activities in the liver; (c) enhanced glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and reduced malonic dialdehyde (MDA) level in serum and liver; (d) increased lysozyme (LZ) and acid phosphatase (ACP) activities in serum and liver, as well as complement (C3 and C4) and immunoglobulin M (IgM) contents in the liver; (e) upregulated the relative expression of copper-zinc superoxide dismutase (SOD-1), manganese superoxide dismutase (SOD-2), CAT, GSH-Px1a, ACP6, AKP, LZ-c, IgM, C3, and C4-b in the liver; (f) improved the survival rate of V. harveyi challenge. In summary, an appropriate amount of VC could improve growth, digestive ability, antioxidant capacity, immunity, and the ability to resist the challenge of V. harveyi in coral trout. In order to ensure that coral trout has good growth performance, digestive and antioxidant capacity, it is recommended that the levels of VC in the feed is between 255.25 and 273.84 mg/kg. The immunity of coral trout is stronger when the VC level in the diet is between 251.95 and 314.03 mg/kg.

Designing a novel mRNA vaccine against Vibrio harveyi infection in fish: an immunoinformatics approach.

Vibrio harveyi belongs to the family Vibrionaceae of class Gammaproteobacteria. Around 12 Vibrio species can cause gastroenteritis (gastrointestinal illness) in humans. A large number of bacterial particles can be found in the infected cells, which may cause death. Despite these devastating complications, there is still no cure or vaccine for the bacteria. As a result, we used an immunoinformatics approach to develop a multi-epitope vaccine against the most pathogenic hemolysin gene of V. harveyi. The immunodominant T- and B-cell epitopes were identified using the hemolysin protein. We developed a vaccine employing three possible epitopes: cytotoxic T-lymphocytes, helper T-lymphocytes, and linear B-lymphocyte epitopes, after thorough testing. The vaccine was developed to be antigenic, immunogenic, and non-allergenic, as well as have a better solubility. Molecular dynamics simulation revealed significant structural stiffness and binding stability. In addition, the immunological simulation generated by computers revealed that the vaccination might elicit immune reactions Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher codon adaptation index value, which was then included in the cloning vector pET2+ (a). Altogether, our experiment implies that the proposed peptide vaccine might be a good option for vibriosis prophylaxis.

Mechanism of enrofloxacin-induced multidrug resistance in the pathogenic Vibrio harveyi from diseased abalones

Vibrio harveyi infection had caused severe economic losses in aquaculture. A pathogenic V. harveyi strain had been successfully induced to be a multiple-resistant strain by enrofloxacin (EFX), then the mechanism of multidrug resistance was analyzed. It suggested that the minimum inhibitory concentration of EFX increased by 32-folds. Results of the Kirby-Bauer test showed that the inhibitory zone diameter was 25.3 mm for the sensitive strain (labeled as HL-S) and 8.5 mm for the resistant strain (labeled as HL-R). After 20 serial passages, even when the stress of EFX was removed, the resistance persisted. After induction of EFX, HL-R resisted to other fluoroquinolones, it even resisted to furazolidone and streptomycin, although it was sensitive to these antibiotics initially. Its sensitivity to rifampicin and doxycycline also decreased obviously. Results showed that 3522 differentially expressed genes were identified. Expression of the multidrugs efflux resistance-nodulation-cell division was significantly upregulated (164.61-folds) in HL-R. Other key genes connected with drug efflux were also upregulated significantly (p<0.05). Notably, recA encoded for recombination protein was upregulated significantly, lexA was downregulated significantly in HL-R. Research results showed that the efflux system and the save our souls system have played crucial roles during the development of multidrug resistance of V. harveyi.

Molecular cloning, expression analysis of the IgT gene and detection of IgT+ B cells in the half-smooth tongue sole (Cynoglossus semilaevis)

IgT is a specific Ig isotype in teleosts, which plays extremely important roles in the mucosal immunity of fish. In the present study, the membrane-bound and secretory IgT of the half-smooth tongue sole (Cynoglossus semilaevis) were identified for the first time. The V-D-J-C structure of two forms of csIgT are translated by the same Cτ gene, and the secretory tail and transmembrane domain were encoded through alternative splicing at the 3′ end of the Cτ4. The CH regions of csIgT had high similarity with that of other flatfish (P. olivaceus and S. maximus). In healthy C. semilaevis, sIgT and mIgT were mainly expressed in mucus related tissues such as skin, intestine and gill. The transcript levels of sIgT and mIgT mRNA showed a significant induction in the immune-related tissues upon Vibrio Harveyi infection. A polyclonal rabbit anti-csIgT was successfully prepared using the csIgT heavy chain recombinant protein. Using this antibody, we detected the native IgT with the molecular mass at 220 kDa in skin total protein under non-reducing SDS-PAGE condition. Immunofluorescence analysis indicated that IgT+ B lymphocytes were intensively located in the skin, gill, intestine, and head kidney of C. semilaevis. These results suggest that IgT may participate in the immune response of C. semilaevis, which will facilitate the investigations of the immunoglobulins of marine fish.

Effects of dietary black soldier fly (Hermetia illucens Linnaeus) on the disease resistance of juvenile grouper (Epinephelus coioides)

An experiment was performed to study the effects of dietary levels of black soldier fly larva meal (BSFLM) on the growth performance, immunity and disease resistance of juvenile grouper (Epinephelus coioides). Four isoproteic and isoenergetic diets were formulated with dietary BSFLM levels of 0 g/kg (T0), 25 g/kg (T2.5), 50 g/kg (T5) and 100 g/kg (T10). Each diet was randomly fed to triplicate groups, each containing 40 fish. The results of the 30-day study indicated that fish growth performance was not affected in the T2.5 and T5 groups compared with the T0 group. In the group with a dietary BSFLM level of 100 g/kg, the feed coefficient was significantly higher than that in the other three groups. The superoxide dismutase, catalase, glutathione peroxidase, lysozyme activity, and malondialdehyde content in the liver, and the interleukin-1 beta (IL-1β), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α) and heat shock protein 70 (HSP70) expression in the gills, head kidney, liver and spleen remained consistent in all groups. In addition, no significant differences in the cumulative mortality or parasite abundance in groupers after Vibrio harveyi and Cryptocaryon irritans infection were observed. These results suggested that BSFLM supplemented diets did not inhibit disease resistance in groupers.

RNA-Sequencing Analysis of the Spleen and Gill of Takifugu rubripes in Response to Vibrio harveyi Infection.

Takifugu rubripes is commonly subjected to the disease-causing bacterium, Vibrio harveyi. However, the mechanism involved in the immune response of T. rubripes to V. harveyi infection is unclear. We conducted a transcriptomic analysis of the spleen and gill from T. rubripes infected with V. harveyi. We obtained 60,981,357 and 60,760,550 clean reads from the control and infected spleens, and 57,407,586 and 57,536,651 clean reads from the control and infected gills, respectively. We also identified 1,560 and 1,213 differentially expressed genes in the spleen and gill, respectively. Gene ontology analysis revealed that the most enriched biological process in both the spleen and gill was “immune response”. The most enriched Kyoto Encyclopedia of Genes and Genomes immune response–related pathways were the NOD-like receptor signaling pathway in the spleen and cytokine–cytokine receptor interaction in the gill. We found 10 candidate immune-related genes in the spleen and gill. These putative immune pathways and candidate genes will provide insight into the immune response mechanisms of T. rubripes against V. harveyi.

Synbiotic feed supplementation improves antioxidant response and innate immunity of juvenile barramundi, Lates calcarifer subjected to bacterial infection

The positive effect of dietary synbiotic supplementation on fish exposed to bacterial infection is poorly researched. The present study addressed the knowledge gap of dietary supplementation of Lactobacillus casei and garlic, Allium sativum, termed as synbiotic on the growth performance, lipid utilization, antioxidant activities, skin mucosal status, cytokine gene expression and intestinal epithelial responses following Vibrio harveyi infection in juvenile barramundi, Lates calcarifer. Juvenile barramundi with an initial weight of 9.16 ± 0.08 g were randomly allocated into 8 tanks of 300 L capacity each at a density of 20 fish per tank. The quadruplicate groups were fed either a non-supplemented control diet or the same control diet supplemented with 1% of garlic powder and 1% of L. casei (109 CFU/mL) for 56 days. Results indicated that although the synbiotic supplemented diet did not produce any significant improvement in growth rate and feed utilization, lipid utilization and glutathione peroxidase activity were improved (P < 0.05). Synbiotic diet also reduced the intraperitoneal adipocyte cells size and increased the number of skin mucin cells. TEM micrographs showed an enhanced microvilli length with a normal intestinal morphological pattern in synbiotic fed fish compared to a leaky intestinal tight junction in the control when fish are subjected to V. harveyi infection. In addition, a significant improvement of the expression of immune responsive genes were found in synbiotic fed fish when compared to control. Overall, the results indicate that dietary synbiotic supplementation has the potential to improve antioxidant response and health welfare of barramundi.

Dietary astaxanthin improves the antioxidant capacity, immunity and disease resistance of coral trout (Plectropomus leopardus)

The effects of astaxanthin on growth performance, digestive enzyme activity, antioxidant capacity, immune ability, resistance to Vibrio harveyi infection of coral trout (Plectropomus leopardus, initial weight 17.44 ± 0.05 g) were studied by 8-week feeding trial. Four iso-nitrogenous and iso-lipidic experimental diets containing astaxanthin 0 (A0), 0.05 (A1), 0.1 (A2) and 0.2 (A3) g/kg were formulated with the addition of Haematococcus pluvialis powder (astaxanthin content accounts for 100 g/kg) of 0, 0.5, 1.0 and 2.0 g/kg, separately. The feeding experiment lasted for 56 days, and it was found that supplementing the diet with astaxanthin-rich H. pluvialis powder had no significant impact on the growth performance about coral trout (P > 0.05). Compared with the A0 group, the activities of amylase, lipase, and trypsin in the liver of the A2 group was dramatically increased (P < 0.05); catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities and total antioxidant capacity (T-AOC) level in serum and liver were dramatically higher in the A2 group before as well as after the challenge (P < 0.05); after the challenge, the acid phosphatase (ACP) and lysozyme (LZ) activities, and complement (C3 and C4) contents in serum and liver were significantly raised for the A2 group (P < 0.05); the liver relative expressions of copper-zinc superoxide dismutase (sod-1), manganese superoxide dismutase (sod-2), cat, acp6, akp, lz-c, immunoglobulin M (igm), c3, and c4-b in the A2 group were significantly up-regulated before and after the challenge (P < 0.05); the rate of survival follow V. harveyi challenge in the group A2 was dramatically higher (P < 0.05). In summary, this study indicated that adding 1.0 g/kg astaxanthin-rich H. pluvialis powder (the content of astaxanthin is 0.091 g/kg) could improve the digestive enzyme activity, antioxidant capacity, immunity, and the ability to resist the challenge of V. harveyi in coral trout.