Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Prevalence, genetic diversity, and antimicrobial susceptibility of Vibrio spp. infected gilthead sea breams from coastal farms at Damietta, Egypt.

BACKGROUND: Vibriosis is one of the most serious bacterial diseases and causes high morbidity and mortality among cultured sea breams. This study was undertaken to track the surveillance of Vibrio infection and its correlation to environmental factors. A total of 115 gilthead sea breams were collected seasonally from a private earthen pond fish farm in the Shatta area of Damietta, Egypt from September 2022 to July 2023. Physicochemical parameters of water were analyzed, and heavy metal levels were measured. The fish samples were subjected to clinical, bacteriological, Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting, and hematoxylin and Eosin histopathological staining. RESULTS: The results revealed significant variations in the water quality parameters over different seasons, in addition to an increase in heavy metals. Naturally infected fish showed external signs and postmortem lesions that were relevant to bacterial infection. Two dominant Vibrio subspecies of bacteria were identified: V. alginolyticus (205 isolates) and V. fluvialis (87 isolates). PCR confirmed the presence of V. alginolyticus using the species-specific primer collagenase at 737 bp. The highest prevalence of V. alginolyticus was detected during the summer season (57.72%), and the lowest prevalence was observed in autumn (39.75%). The correlation analysis revealed a positive relationship between V. alginolyticus and water temperature (r = 0.69). On the other hand, V. fluvialis showed a high prevalence during the autumn season (25.30%) and the lowest prevalence during the summer season (10.56%), where it was negatively correlated with water temperatures (r =-0.03). ERIC fingerprinting showed genetic variation within the Vibrio isolates. Antimicrobial susceptibility testing revealed sensitivity to ciprofloxacin and doxycycline, and resistance to amoxicillin and erythromycin. The multiple antibiotic resistance (MAR) index values for V. alginolyticus and V. fluvialis ranged from 0.3 to 0.7, with a multi-drug resistance pattern to at least three antibiotics. Histopathological alterations in the affected tissues revealed marked hemorrhage, vascular congestion, and hemosiderosis infiltration. CONCLUSION: This study provides insights into the potential propagation of waterborne diseases and antibiotic resistance in the environment. Ensuring that the environment does not serve as a reservoir for virulent and contagious Vibrio species is a critical concern for regional aquaculture industries. Therefore, we recommend implementing environmental context-specific monitoring and surveillance tools for microbial resistance.

Epidemiological and Genomic analysis of Vibrio parahaemolyticus isolated from imported travelers at the port of Shanghai, China (2017-2019).

BACKGROUND: Vibrio parahaemolyticus is the predominant etiological agent of seafood-associated foodborne illnesses on a global scale. It is essential to elucidate the mechanisms by which this pathogen disseminates. Given the existing research predominantly concentrates on localized outbreaks, there is a pressing necessity for a comprehensive investigation to capture strains of V. parahaemolyticus cross borders. RESULTS: This study examined the frequency and genetic attributes of imported V. parahaemolyticus strains among travelers entering Shanghai Port, China, between 2017 and 2019.Through the collection of 21 strains from diverse countries and regions, Southeast Asia was pinpointed as a significant source for the emergence of V. parahaemolyticus. Phylogenetic analysis revealed clear delineation between strains originating from human and environmental sources, emphasizing that underlying genome data of foodborne pathogens is essential for environmental monitoring, food safety and early diagnosis of diseases. Furthermore, our study identified the presence of virulence genes (tdh and tlh) and approximately 120 antibiotic resistance-related genes in the majority of isolates, highlighting their crucial involvement in the pathogenesis of V. parahaemolyticus. CONCLUSIONS: This research enhanced our comprehension of the worldwide transmission of V. parahaemolyticus and its antimicrobial resistance patterns. The findings have important implications for public health interventions and antimicrobial stewardship strategies, underscoring the necessity for epidemiological surveillance of pathogen at international travel hubs.

Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

Introduction: The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination. Methods: Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment. Results: Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%. Discussion: These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii).

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

Glycosaminoglycan lyase: A new competition between bacteria and the pacific white shrimp Litopenaeus vannamei.

Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.

Tracking Vibrio: population dynamics and ecology of Vibrio parahaemolyticus and V. vulnificus in an Alabama estuary.

Vibrio is a genus of halophilic, gram-negative bacteria found in estuaries around the globe. Integral parts of coastal cultures often involve contact with vectors of pathogenic Vibrio spp. (e.g., consuming raw shellfish). High rates of mortality from certain Vibrio spp. infections demonstrate the need for an improved understanding of Vibrio spp. dynamics in estuarine regions. Our study assessed meteorological, hydrographic, and biological correlates of Vibrio parahaemolyticus and V. vulnificus at 10 sites in the Eastern Mississippi Sound System (EMSS) from April to October 2019. During the sampling period, median abundances of V. parahaemolyticus and V. vulnificus were 2.31 log MPN/L and 2.90 log MPN/L, respectively. Vibrio spp. dynamics were largely driven by site-based variation, with sites closest to freshwater inputs having the highest abundances. The E-W wind scalar, which affects Ekman transport, was a novel Vibrio spp. correlate observed. A potential salinity effect on bacterial-particle associations was identified, where V. vulnificus was associated with larger particles in conditions outside of their optimal salinity. Additionally, V. vulnificus abundances were correlated to those of harmful algal species that did not dominate community chlorophyll. Correlates from this study may be used to inform the next iteration of regionally predictive Vibrio models and may lend additional insight to Vibrio spp. ecology in similar systems. IMPORTANCE: Vibrio spp. are bacteria found in estuaries worldwide; some species can cause illness and infections in humans. Relationships between Vibrio spp. abundance, salinity, and temperature are well documented, but correlations to other environmental parameters are less understood. This study identifies unique correlates (e.g., E-W wind scalar and harmful algal species) that could potentially inform the next iteration of predictive Vibrio models for the EMSS region. Additionally, these correlates may allow existing environmental monitoring efforts to be leveraged in providing data inputs for future Vibrio risk models. An observed correlation between salinity and V. vulnificus/particle-size associations suggests that predicted environmental changes may affect the abundance of Vibrio spp. in certain reservoirs, which may alter which vectors present the greatest vibrio risk.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas.

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

Vibrio infection in freshwater fish in Poland.

Vibrio species are common inhabitants of aquatic environments and have been described in connection with fish and human diseases. Six Vibrio species were isolated from diseased freshwater and ornamental fish in Poland. The strains were identified based on morphological and biochemical characteristics and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as V. albensis (n=3) from Gymnocephalus cernua, Sander lucioperca, Paracheirodon innesi, and Xiphophorus hellerii; V. mimicus (n=1) from Xiphophorus maculatus; and V. vulnificus (n=1) from Nematobrycon palmeri. This is the first time that Vibrio species have been isolated and described from ornamental fish in Poland. The isolates were resistant to ampicillin (83.3%), gentamicin (16.6%), ciprofloxacin (16.6%), sulfamethoxazole-trimethoprim (16.6%), and chloramphenicol (16.6%). The multiple antibiotic resistance (MAR) index was 0.00-0.08 for V. albensis, 0.17 for V. mimicus, and 0.33 for V. vulnificus. Our study confirmed the presence of potentially pathogenic Vibrio species in freshwater and ornamental fish. Therefore, further monitoring of the presence of Vibrio species, mainly in ornamental fish, is necessary.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

[Clinical characteristics of 11 patients with Vibrio vulnificus infection and the establishment of a rapid diagnosis procedure for this disease].

Objective: To analyze the clinical characteristics of patients with Vibrio vulnificus infection, share diagnosis and treatment experience, and establish a rapid diagnosis procedure for this disease. Methods: This study was a retrospective case series study. From January 2009 to November 2022, 11 patients with Vibrio vulnificus infection who met the inclusion criteria were admitted to the Department of Burns and Wound Repair of Guangdong Provincial People's Hospital Affiliated to Southern Medical University. The gender, age, time of onset of illness, time of admission, time of diagnosis, route of infection, underlying diseases, affected limbs, clinical manifestations and signs on admission, white blood cell count, hemoglobin, platelet count, C-reactive protein (CRP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, procalcitonin, albumin, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and blood sodium levels on admission, culture results and metagenomic next-generation sequencing (mNGS) results of pathogenic bacteria and the Vibrio vulnificus drug susceptibility test results during hospitalization, treatment methods, length of hospital stay, and outcomes of all patients were recorded. Comparative analysis was conducted on the admission time and diagnosis time of patients with and without a history of exposure to seawater/marine products, as well as the fatality ratio and amputation of limbs/digits ratio of patients with and without early adequate antibiotic treatment. For the survived patients with hand involvement, the hand function was assessed using Brunnstrom staging at the last follow-up. Based on patients' clinical characteristics and treatment conditions, a rapid diagnosis procedure for Vibrio vulnificus infection was established. Results: There were 7 males and 4 females among the patients, aged (56±17) years. Most of the patients developed symptoms in summer and autumn. The admission time was 3.00 (1.00, 4.00) d after the onset of illness, and the diagnosis time was 4.00 (2.00, 8.00) d after the onset of illness. There were 7 and 4 patients with and without a history of contact with seawater/marine products, respectively, and the admission time of these two types of patients was similar (P>0.05). The diagnosis time of patients with a history of contact with seawater/marine products was 2.00 (2.00, 5.00) d after the onset of illness, which was significantly shorter than 9.00 (4.25, 13.00) d after the onset of illness for patients without a history of contact with seawater/marine products (Z=-2.01, P<0.05). Totally 10 patients had underlying diseases. The affected limbs were right-hand in 8 cases, left-hand in 1 case, and lower limb in 2 cases. On admission, a total of 9 patients had fever; 11 patients had pain at the infected site, and redness and swelling of the affected limb, and 9 patients each had ecchymosis/necrosis and blisters/blood blisters; 6 patients suffered from shock, and 2 patients developed multiple organ dysfunction syndrome. On admission, there were 8 patients with abnormal white blood cell count, hemoglobin, and albumin levels, 10 patients with abnormal CRP, procalcitonin, and NT-proBNP levels, 5 patients with abnormal creatinine and blood sodium levels, and fewer patients with abnormal platelet count, ALT, and AST levels. During hospitalization, 4 of the 11 wound tissue/exudation samples had positive pathogenic bacterial culture results, and the result reporting time was 5.00 (5.00, 5.00) d; 4 of the 9 blood specimens had positive pathogenic bacterial culture results, and the result reporting time was 3.50 (1.25, 5.00) d; the mNGS results of 7 wound tissue/exudation or blood samples were all positive, and the result reporting time was 1.00 (1.00, 2.00) d. The three strains of Vibrio vulnificus detected were sensitive to 10 commonly used clinical antibiotics, including ciprofloxacin, levofloxacin, and amikacin, etc. A total of 10 patients received surgical treatment, 4 of whom had amputation of limbs/digits; all patients received anti-infection treatment. The length of hospital stay of 11 patients was (26±11) d, of whom 9 patients were cured and 2 patients died. Compared with that of the 6 patients who did not receive early adequate antibiotic treatment, the 5 patients who received early adequate antibiotic treatment had no significant changes in the fatality ratio or amputation of limbs/digits ratio (P>0.05). In 3 months to 2 years after surgery, the hand function of 8 patients was assessed, with results showing 4 cases of disabled hands, 2 cases of incompletely disabled hands, and 2 cases of recovered hands. When a patient had clinical symptoms of limb redness and swelling and a history of contact with seawater/marine products or a pre-examination triage RiCH score of Vibrio vulnificus sepsis ≥1, the etiological testing should be initiated immediately to quickly diagnose Vibrio vulnificus infection. Conclusions: Vibrio vulnificus infection occurs most frequently in summer and autumn, with clinical manifestations and laboratory test results showing obvious infection characteristics, and may be accompanied by damage to multiple organ functions. Both the fatality and disability ratios are high and have a great impact on the function of the affected limbs. Early diagnosis is difficult and treatment is easily delayed, but mNGS could facilitate rapid detection. For patients with red and swollen limbs accompanied by a history of contact with seawater/marine products or with a pre-examination triage RiCH score of Vibrio vulnificus sepsis ≥1, the etiological testing should be initiated immediately to quickly diagnose Vibrio vulnificus infection.

Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.

Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.

In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei.

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2500 ppm and 5000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

YTHDC1/CRM1 Facilitates m6A-Modified circRNA388 Nuclear Export to Induce Coelomocyte Autophagy via the miR-2008/ULK Axis in Apostichopus japonicus.

N 6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic RNA, was able to mediate circular RNA (circRNA) function in many immune processes. Nevertheless, the functional role of m6A-modified circRNAs in innate immunity of invertebrates remained unclear. In this study, we identified m6A-modified circRNA388 from cultured sea cucumber (Apostichopus japonicus) coelomocytes, which was mainly detected in cytoplasm after Vibrio splendidus infection. A knockdown assay indicated that cytoplasm circRNA388 promoted coelomocyte autophagy and decreased the number of intracellular V. splendidus. Mechanistically, the circRNA388 in the cytoplasm directly sponged miR-2008 to block its interaction with Unc-51-like kinase 1 from A. japonicus (AjULK) and further promoted autophagy to resist V. splendidus infection. More importantly, we found that m6A modification was vital to circRNA388 nuclear export with YTH domain-containing protein 1 from A. japonicus (AjYTHDC1) as the reader. AjYTHDC1 facilitated the nuclear export of m6A-modified circRNA388 via interaction with exportin-1 (chromosomal maintenance 1) from A. japonicus (AjCRM1). Knockdown of AjCRM1 could significantly decrease the content of cytoplasm circRNA388. Overall, our results provide the first evidence that nuclear export of m6A-modified circRNA388 is dependent on the novel AjCRM1 to our knowledge, which was further promoted coelomocyte autophagy by miR-2008/AjULK axis to clear intracellular V. splendidus.

Immuno-protective response of Asian seabass (Lates calcarifer) to inactivated vaccines against Streptococcus iniae and Vibrio harveyi.

BACKGROUND: In this study, the protective immunity and immunogenicity of the monovalent and bivalent Streptococcus iniae and Vibrio harveyi vaccine were evaluated in Asian seabass. To analyze immune responses, 1200 Asian seabass with an average weight of 132.6 ± 25.4 g were divided into eight treatments in triplicates (50 fish per tank) as follows: S. iniae immunized by injection (SI), V. harveyi immunized by injection (VI), bivalent S. iniae and V. harveyi (SVI) immunized by injection, S. iniae immunized by immersion (SIM), V. harveyi (VIM) immunized by immersion, bivalent S. iniae and V. harvei (SVIM) immunized by immersion, phosphate-buffered saline (PBS) by injection (PBSI) and control group without vaccine administration (CTRL). Blood and serum samples were taken at the end of the 30th and 60th days. Then the vaccinated groups were challenged with two bacteria (S. iniae) and (V. harveyi) separately and mortality was recorded for 14 days. RESULTS: This study reveals that there is no significant difference in the hematological parameters on the 30th and 60th days of the experiment in the vaccine-immunized groups compared to the CTRL group (P > 0.05). Meanwhile, there was no significant difference in the amount of serum albumin level, respiratory burst activity, and serum bactericidal activity in the vaccine-immunized groups compared to the CTRL group on the 30th and 60th days of the experiment (P > 0.05). Total protein on the 60th day (in the VI and SVI groups), globulin on the 30th day (in the VI and SVI groups) and the 60th day (in the VI group) compared to the CTRL and PBSI groups had a significant increase (P < 0.05). Complement activity (in the VI and SVI groups) and lysozyme (in the SI and SVI groups) increased significantly compared to the control group (P < 0.05). Serum antibody titer against S. iniae had a significant increase in the SI, VI, SVI and SVIM groups compared to the CTRL and PBSI groups (P < 0.05). Serum antibody titer against V. harveyi had a significant increase in the groups immunized with the vaccine compared to the CTRL and PBSI groups (P < 0.05). A significant increase in the relative percentage survival (RPS) following challenge with S. iniae in the SVI (86.6%), SI (83.3%,) and VI (73.3%) groups were observed compared to the CTRL (43.3%) and PBSI (40%) groups (P < 0.05). Also, a significant increase in the RPS after challenge with V. harveyi in the SVI group, VI 86.6%, SVI 83.3%, VIM 80% and SVIM 76.6% were observed compared to the CTRL (46.6%) and PBSI (50%) groups (P < 0.05). CONCLUSION: Overall, the results demonstrated that the bivalent vaccine of S. iniae and V. harveywas able to produce significant immunogenicity and RPS in Asian seabass.

Morinda citrifolia fruit extract enhances the resistance of Penaeus vannamei to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1-26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection.

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Construction and analysis of the immune effect of two different vaccine types based on Vibrio harveyi VgrG.

Vibrio harveyi poses a significant threat to fish and invertebrates in mariculture, resulting in substantial financial repercussions for the aquaculture sector. Valine-glycine repeat protein G (VgrG) is essential for the type VI secretion system's (T6SS) assembly and secretion. VgrG from V. harveyi QT520 was cloned and analyzed in this study. The localization of VgrG was determined by Western blot, which revealed that it was located in the cytoplasm, secreted extracellularly, and attached to the membrane. The effectiveness of two vaccinations against V. harveyi infection-a subunit vaccine (rVgrG) and a DNA vaccine (pCNVgrG) prepared with VgrG was evaluated. The findings indicated that both vaccines provided a degree of protection against V. harveyi challenge. At 4 weeks post-vaccination (p.v.), the rVgrG and pCNVgrG exhibited relative percent survival rates (RPS) of 71.43% and 76.19%, respectively. At 8 weeks p.v., the RPS for rVgrG and pCNVgrG were 68.21% and 72.71%, respectively. While both rVgrG and pCNVgrG elicited serum antibody production, the subunit vaccinated fish demonstrated significantly higher levels of serum anti-VgrG specific antibodies than the DNA vaccine group. The result of qRT-PCR demonstrated that the expression of major histocompatibility complex (MHC) class Iα, tumor necrosis factor-alpha (TNF-α), interferon γ (IFNγ), and cluster of differentiation 4 (CD4) were up-regulated by both rVgrG and pCNVgrG. Fish vaccinated with rVgrG and pCNVgrG exhibited increased activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme. These findings suggest that VgrG from V. harveyi holds potential for application in vaccination.