Genome-wide DNA N6-methyladenosine in Aeromonas veronii and Helicobacter pylori.

BACKGROUND: DNA N6-methyladenosine (6mA), as an important epigenetic modification, widely exists in bacterial genomes and participates in the regulation of toxicity, antibiotic resistance, and antioxidant. With the continuous development of sequencing technology, more 6mA sites have been identified in bacterial genomes, but few studies have focused on the distribution characteristics of 6mA at the whole-genome level and its association with gene expression and function. RESULTS: This study conducted an in-depth analysis of the 6mA in the genomes of two pathogenic bacteria, Aeromonas veronii and Helicobacter pylori. The results showed that the 6mA was widely distributed in both strains. In A. veronii, 6mA sites were enriched at 3' end of protein-coding genes, exhibiting a certain inhibitory effect on gene expression. Genes with low 6mA density were associated with cell motility. While in H. pylori, 6mA sites were enriched at 5' end of protein-coding genes, potentially enhancing gene expression. Genes with low 6mA density were closely related to defense mechanism. CONCLUSIONS: This study elucidated the distribution characteristics of 6mA in A. veronii and H. pylori, highlighting the effects of 6mA on gene expression and function. These findings provide valuable insights into the epigenetic regulation and functional characteristics of A. veronii and H. pylori.

Integration analysis of miRNA-mRNA uncovers the molecular immune mechanism of macrophage response to Aeromonas veronii infection.

Macrophages are innate immunity cells which play pivotal roles in infectious immunity. Aeromonas veronii is a zoonotic agent capable of causing sepsis and poses a serious threat to public health. However, few studies have focused on miRNA-mRNA integration analysis to address the immune mechanisms of macrophage response to A. veronii infection. Herein, we characterized the immunophysiological, biochemical, and transcriptome changes of macrophage under A. veronii infection. We found that macrophages infected with A. veronii released large amounts of cytokines and triggered NLRP3-dependent pyroptosis. Subsequently, 603 differentially expressed miRNAs (DEMIs) and 3693 differentially expressed mRNAs (DEMs) were identified by RNA-seq analysis under A. veronii infection. Moreover, integrated analysis of miRNA-mRNA yielded 66 miRNA-target gene pairs composed of 41 DEMIs and 27 DEMs. We next identified the Toll-like receptor, NOD-like receptor, TNF and NF-κB pathways as necessary for macrophage to respond to A. veronii infection. miR-847 and miR-627 were involved in macrophage response to A. veronii infection by negatively regulating Pannexin-1 and thioredoxin interacting protein (TXNIP). Our findings elucidate the molecular mechanism of macrophage response to A. veronii infection at the miRNA level, providing many candidate miRNAs and mRNAs therapeutic targets for the prevention and treatment of A. veornii infectious diseases.

Identification and characterization of a novel type II toxin-antitoxin system in Aeromonas veronii.

The bacterial type II toxin-antitoxin (TA) system is a rich genetic element that participates in various physiological processes. Aeromonas veronii is the main bacterial pathogen threatening the freshwater aquaculture industry. However, the distribution of type II TA system in A. veronii was seldom documented and its roles in the life activities of A. veronii were still unexplored. In this study, a novel type II TA system AvtA-AvtT was predicted in a fish pathogen Aeromonas veronii biovar sobria with multi-drug resistance using TADB 2.0. Through an Escherichia coli host killing and rescue assay, we demonstrated that AvtA and AvtT worked as a genuine TA system, and the predicted toxin AvtT actually functioned as an antitoxin while the predicted antitoxin AvtA actually functioned as a toxin. The binding ability of AvtA with AvtT proteins were confirmed by dot blotting analysis and co-immunoprecipitation assay. Furthermore, we found that the toxin and antitoxin labelled with fluorescent proteins were co-localized. In addition, it was found that the transcription of AvtAT bicistronic operon was repressed by the AvtAT protein complex. Deletion of avtA gene and avtT gene had no obvious effect on the drug susceptibility. This study provides first characterization of type II TA system AvtA-AvtT in aquatic pathogen A. veronii.

Evaluate the potential use of TonB-dependent receptor protein as a subunit vaccine against Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus).

Aeromonas veronii is an emerging bacterial pathogen that causes serious systemic infections in cultured Nile tilapia (Oreochromis niloticus), leading to massive deaths. Therefore, there is an urgent need to identify effective vaccine candidates to control the spread of this emerging disease. TonB-dependent receptor (Tdr) of A. veronii, which plays a role in the virulence factor of the organism, could be useful in terms of protective antigens for vaccine development. This study aims to evaluate the potential use of Tdr protein as a novel subunit vaccine against A. veronii infection in Nile tilapia. The Tdr gene from A. veronii was cloned into the pET28b expression vector, and the recombinant protein was subsequently produced in Escherichia coli strain BL21 (DE3). Tdr was expressed as an insoluble protein and purified by affinity chromatography. Antigenicity test indicated that this protein was recognized by serum from A. veronii infected fish. When Nile tilapia were immunized with the Tdr protein, specific antibody levels increased significantly (p-value <0.05) at 7 days post-immunization (dpi), and peaked at 21 dpi compared to antibody levels at 0 dpi. Furthermore, bacterial agglutination activity was observed in the fish serum immunized with the Tdr protein, indicating that specific antibodies in the serum can detect Tdr on the bacterial cell surface. These results suggest that Tdr protein has potential as a vaccine candidate. However, challenging tests with A.veronii in Nile tilapia needs to be investigated to thoroughly evaluate its protective efficacy for future applications.

Characteristics of CD4-1 gene and its immune responses against Aeromonas veronii infection by activating NF-κB signaling in Qihe crucian carp Carassius auratus.

CD4-1 found in bony fish contains four extracellular immunoglobulin (Ig)-like domains similar to that of mammalian CD4, which is crucial for the activation of CD4+ helper T-cell. However, there is limited knowledge regarding the molecular markers, immune functions and regulation mechanism of CD4-1 in teleosts due to their vast diversity. In this study, we cloned and characterized two isoforms of Qihe crucian carp CD4-1, designated as CaCD4-1.1 and CaCD4-1.2. We further explored their expression responses upon stimulation with Aeromonas veronii, and the regulation of their immune responses against A. veronii by NF-κB. The ORF of CaCD4-1.1 and CaCD4-1.2 cDNA encoded 477 and 466 amino acids, respectively. Both proteins contained seven conserved cysteine residues in the extracellular domain, and a CCC motif in their cytoplasm, respectively. However, CaCD4-1.1 exhibited a relatively limited similarity with CaCD4-1.2 in the ectodomain. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the mRNA expression of CaCD4-1.1 and CaCD4-1.2 exhibited differential constitutive expression across all examined tissues. Furthermore, the expression level of CD4-1.2 was higher than that of CD4-1.1 in the gills, head kidney, and spleen of Qihe crucian carp subjected to A. veronii challenge, while it was lower in the trunk kidney. Inhibition of NF-κB activity resulted in a decrease in the expression levels of CD4-1.1 and CD4-1.2 mRNA in the gill, while inducing an increase in expression levels in the spleen, in accordance with the observed ultrastructural changes in both organs. Interestingly, the impact of NF-κB on the mRNA expression level of CD4-1.1 appears to be stronger than that of CD4-1.2. Our results suggest that CaCD4-1.1 and CaCD4-1.2 could be expressed on T cells and antigen-sampling cells that exhibit similar characteristics to mammalian M cells, respectively, and differentially regulated by NF-κB in adaptive immune responses against bacterial infection. This research contributes to a better understanding of the crucial role of CD4-1 in the immune response of Qihe crucian carp and provide novel insights for the prevention and treatment of fish diseases in aquaculture.

Rapid discrimination methods for clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using the N-terminal sequence of the flaA gene and investigation of antimicrobial resistance.

Although the genus Aeromonas inhabits the natural environment, it has also been isolated from hospital patient specimens as a causative agent of Aeromonas infections. However, it is not known whether clinical strains live in the natural environment, and if these strains have acquired antimicrobial resistance. In this study, we performed the typing of flagellin A gene (flaA) of clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using Polymerase Chain Reaction (PCR) assay with newly designed primers. Detection rates of the clinical and environmental flaA types of A. hydrophila were 66.7% and 88.2%, and the corresponding rates for A. veronii biovar sobria were 66.7% and 90.9%. The PCR assays could significantly discriminate between clinical and environmental strains of both species in approximately 4 h. Also, among the 63 clinical Aeromonas strains used, only one extended-spectrum ß-lactamase-producing bacteria, no plasmid-mediated quinolone resistance bacteria, and only four multidrug-resistant bacteria were detected. Therefore, the PCR assays could be useful for the rapid diagnosis of these Aeromonas infections and the monitoring of clinical strain invasion into water-related facilities and environments. Also, the frequency of drug-resistant Aeromonas in clinical isolates from Okinawa Prefecture, Japan, appeared to be low.

Inhibition mechanism of crude lipopeptide from Bacillus subtilis against Aeromonas veronii growth, biofilm formation, and spoilage of channel catfish flesh.

Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.

The Hanks-like serine/threonine protein kinase YihE is crucial for Aeromonas veronii virulence and adhesion.

Aeromonas veronii is an important pathogen found in various aquatic environments and products, posing a threat to public health. The Hanks-like serine/threonine protein kinase is closely linked to the pathogenesis of pathogenic bacteria, but the exact role of YihE in A. veronii remains still unknown. To study the specific function of the YihE kinase, we constructed a knockout mutant of the yihE gene in A. veronii. The deletion of the yihE gene resulted in changes to the metabolism of L-arginine-AMC and acetic acid, as well as enhanced resistance to ampicillin and kanamycin in A. veronii. Additionally, the ΔyihE strain demonstrated a 1.4-fold increase in biofilm formation ability and a 1.8-fold decrease in adhesion and invasion to EPCs when compared to the wild-type strain. A significant decrease in cytotoxicity was observed at 2 and 3 h post-infection with EPCs compared to the wild-type strain. Additionally, the deletion of the yihE gene was associated with a significant decrease in motility of the strain. Furthermore, the deletion of the yihE gene resulted in a 1.44-fold increase in the LD50 of A. veronii in zebrafish. These findings offer valuable insights into the pathogenic mechanisms of A. veronii.

Expression profiles of four Nile Tilapia innate immune genes during early stages of Aeromonas veronii infection.

OBJECTIVE: During Egypt's hot summer season, Aeromonas veronii infection causes catastrophic mortality on Nile Tilapia Oreochromis niloticus farms. Egypt is ranked first in aquaculture production in Africa, sixth in aquaculture production worldwide, and third in global tilapia production. This study aimed to investigate, at the molecular level, the early innate immune responses of Nile Tilapia to experimental A. veronii infection. METHODS: The relative gene expression, co-expression clustering, and correlation of four selected immune genes were studied by quantitative real-time polymerase chain reaction in four organs (spleen, liver, gills, and intestine) for up to 72 h after a waterborne A. veronii challenge. The four genes studied were nucleotide-binding oligomerization domain 1 (NOD1), lipopolysaccharide-binding protein (LBP), natural killer-lysin (NKL), and interleukin-1 beta (IL-1ß). RESULT: The four genes showed significant transcriptional upregulation in response to infection. At 72 h postchallenge, the highest NOD1 and IL-1ß expression levels were recorded in the spleen, whereas the highest LBP and NKL expression levels were found in the gills. Pairwise distances of the data points and the hierarchical relationship showed that NOD1 clustered with IL-1ß, whereas LBP clustered with NKL; both genes within each cluster showed a significant positive expression correlation. Tissue clustering indicated that the responses of only the gill and intestine exhibited a significant positive correlation. CONCLUSION: The results suggest that NOD1, LBP, NKL, and IL-1ß genes play pivotal roles in the early innate immune response of Nile Tilapia to A. veronii infection, and the postinfection expression profile trends of these genes imply tissue-/organ-specific responses and synchronized co-regulation.

Pathogenicity and inactivated vaccine treatment of Aeromonas veronii JW-4 on crucian carp.

Aeromonas veronii is a common bacterium found in a variety of aquatic environments, capable of causing a diverse array of diseases in both aquatic animals and humans. Therefore, evaluating the pathogenicity of A. veronii and implementing measures to control its spread are essential. In this study, a strain JW-4, identified as A. veronii, was isolated from diseased Scaphesthes macrolepis, a grade Ⅱ protected animal in China. To investigate the pathogenicity of the strain, fish were fed with serial levels JW-4 supplemented diet or basal diet (control group 1, CG1) for 28 days (d). Results showed that JW-4 stimulated an immune response, evidenced by an increase in immune-related enzyme activities (GOT and GPT) of serum and liver and an upregulation of genes expression levels (TNF-α and IFN-γ) of liver and spleen, and these effects gradually decreased over time. Histopathological examination revealed that JW-4 could alter the tissue structure of immune organs, such as liver and kidney. These changes were accompanied by vacuolar degeneration, nuclear dissolution, and an increased lymphocyte count. To assess protective effects of a vaccine against this strain, fish were injected with an inactivated vaccine (immunization group, IG) or 0.85% sterile saline (control group 2, CG2) for 28-day observation period, then challenged with JW-4 on the 28th day. The inactivated vaccine enhanced total and specific IgM to A. veronii levels of the fish, resulting in a relative percentage survival of 75% in IG. These findings provide a foundation for identifying pathogenic bacteria and developing more effective prophylactic strategies in aquaculture.

OmpR (TCS response regulator) of Aeromonas veronii plays a major role in drug resistance, stress resistance and virulence by regulating biofilm formation.

Aeromonas veronii (A. veronii), a highly pathogenic bacteria with a wide range of hosts, widely exists in the environment of humans, animals and aquatic animals, and can cause a variety of diseases. In this study, the receptor regulator ompR in the envZ/ompR of two-component system was selected to construct a mutant strain (Δ ompR) and a complement strain (C-ompR) to explore the regulatory effect of ompR on the biological characteristics and virulence of TH0426. The results showed that the ability of biofilm formation and osmotic stress of TH0426 were significantly reduced (P < 0.001), the resistance to ceftriaxone and neomycin were slightly down-regulate when the ompR gene was deleted. At the same time, animal pathogenicity experiments showed that the virulence of TH0426 was significantly down-regulated (P < 0.001). These results indicated that ompR gene regulates the biofilm formation of TH0426, and regulates some biological characteristics of TH0426, including drug sensitivity, resistance to osmotic stress, and also affects its virulence.

Detection of changes in biological characteristics of Aeromonas veronii TH0426 after deletion of lsrB gene by homologous recombination.

Aeromonas veronii is a widespread pathogenic microorganism that can infect humans, animals, and a variety of aquatilia, at the same time, can cause diseases, mainly sepsis and ulcer syndrome. In this research, we first deleted the gene of lsrB's nucleotide sequences by homologous recombination. The results showed that the median lethal dose (LD50) of the mutant strain (ΔlsrB) for zebrafish was 1.28-times higher than that of the TH0426 strain. The toxicity of TH0426 to epithelioma papulosum cyprini (EPC) cells was 1.15-times and 1.64-times higher than that of ΔlsrB, 1 and 2 h after infection. The production ability of the biofilm of ΔlsrB decreased by 1.38-times, and the adhesion ability of ΔlsrB to EPC cells greatly decreased by 1.96-times than the TH0426. The result of motility detection pointed out that the swimming ability of ΔlsrB was down by 1.67-times. The results indicated that almost all of them lost their flagella after deleting the lsrB gene. In general, the virulence of TH0426 was reduced after deleting the lsrB gene. The final results point out that the lsrB gene of TH0426 is related to motility, biofilm formation, adhesion, and virulence.

Construction and efficacy of Aeromonas veronii mutant Δhcp as a live attenuated vaccine for the largemouth bass (Micropterus salmoides).

Aeromonas veronii is a human and animal co-pathogenic bacterium that could have a significant negative impact on both human health and aquaculture. In this study, a mutant strain of A. veronii with deletion of the hemolysin co-regulated protein (hcp) gene was constructed (Δhcp-AV). Compared with the wild strain, Δhcp-AV showed significantly reduced growth capacity and biofilm formation ability. Motility tests showed that the hcp gene had no significant effect on the swimming and swarming ability. In addition, the pathogenicity was also reduced. To evaluate the efficacy of Δhcp-AV as a live attenuated vaccine for prevention of Aeromonas veronii infection, we compared the immune response of largemouth bass (Micropterus salmoides) after immunization with 500 µL of 1.47 × 105 CFU/mL of Δhcp-AV and 4 × 108 CFU/mL of inactivated A. veronii. Obvious increases of serum immune related enzyme activity were observed in immunization groups. Expression levels of immune-related genes in Δhcp-AV group were up-regulated, and higher than those in inactivated A. veronii group. After challenging with live A. veronii, the relative percent survival (RPS) was 100% in Δhcp- AV group, whereas the RPS was 76.67% in inactivated A. veronii group. Our data suggest that the live attenuated vaccine Δhcp- AV could elicit a stronger immune response and provide a higher RPS than inactivated A. veronii. These data suggest that hcp gene is an important virulence factor of A. veronii, and the live attenuated vaccine Δhcp-AV is safe and effective for prevention A. veronii infection in M. salmoides farming.

Characterization of CD3γ/δ gene and its immune response in Qihe crucian carp Carassius auratus after challenged by Aeromonas veronii and Poly(I:C).

CD3γ/δ found in non-mammalian vertebrates is a CD3 homolog with structural characteristics similar to both mammalian CD3γ and CD3δ, and plays important roles in T cell recognization and immune response in fish. In this study, the full-length of CD3γ/δ from Qihe crucian carp (named CaCD3γ/δ) was cloned and characterized, then the expression response profiles and potential immune functions was explored after Aeromonas veronii and Poly(I:C) challenge. The results showed that the full-length of CaCD3γ/δ was 819 bp including a 5'-UTR of 141 bp, a 3'-UTR of 168 bp, and an ORF of 510 bp encoding a putative 169-aa protein with an estimated MW of 18.71 kD and a theoretical pI of 8.77. The protein sequence of CaCD3γ/δ contained a Leu-Leu and a CXXXC motif in the extracellular domain, and an ITAM and a Leu-Ile motif in the cytoplasm, and a residue of Asn in the transmembrane. CaCD3γ/δ was constitutively expressed in the spleen, liver, gill, and blood of Qihe crucian carp. After the carp were challenged with Poly(I:C) and Aeromonas veronii, the mRNA expression levels of CaCD3γ/δ were significantly changed in the spleen, head kidney, intestine and gill, according to the results of qPCR. However, compared with A. veronii, Poly(I:C) challenge can rapidly induce the CaCD3γ/δ expression levels in head kidney, intestine and spleen, which suggested CaCD3γ/δ may be differentially modulated by different pathogens. Moreover, the results of immunohistochemical analysis showed that the CaCD3γ/δ+ secreted cells in the spleen and gills of Qihe crucian were increased after challenged with Poly(I:C), as well as the spleen challenged with A. veronii, but at different levels. Combined with the fact that vascular congestion, necrosis of parenchymal cells, and inflammatory cells including lymphocytes infiltration were also observed in the gill and spleen of Qihe crucian carp treated with A. veronii and Poly(I:C) revealed by pathological analysis, it was predicted that CaCD3γ/δ+ T lymphocytes may participated in the immune response against pathogens. This study will contribute to understand the important role of CaCD3γ/δ+ T lymphocytes in the immune response of Qihe crucian carp, and provide new insights for the prevention and treatment of the diseases of Qihe crucian carp.

Identification and characterisation of emerging fish pathogens Aeromonas veronii and Aeromonas hydrophila isolated from naturally infected Channa punctata.

Naturally infected Channa punctata exhibiting bacterial septicemic syndrome including ulcerations along with mortality records were collected from a fish farm in Assam during winter season (early November 2020 to early January 2021). The moribund fishes were subjected for bacterial isolation followed by identification of the bacteria. Two dominant emerging bacterial pathogens were identified as Aeromonas veronii (isolate ZooGURD-01) and Aeromonas hydrophila (isolate ZooGURD-05) by standard biochemical characterization and 16S rRNA and rpo B gene amplification. Re-infection experiments of both the bacterial isolates in healthy disease-free C. punctata showed similar symptoms to that of natural infection thus confirming their virulence. The LD50 calculated during challenge test for both the isolates ZooGURD-01 and ZooGURD-05 found to be pathogenic at 2.6 × 104 and 1.6 × 104 CFU/fish respectively. Further PCR amplification of specific virulent genes (aerolysin, hemolysin and enterotoxin) confirmed pathogenicity for both isolates. Histopathological examinations of liver and kidney in re-infection experiments showed prominent changes supporting bacterial septicaemia. Antibiotic sensitivity pattern showed that the isolates ZooGURD-01 and ZooGURD-05 were sensitive to 22 and 19 out of 25 antimicrobials respectively. The present study was the first report on the mortality of farmed C. punctata associated with natural infection caused by A. veronii and A. hydrophila with no record of pathogenicity of A. veronii in C. punctata.

Contribution of flagellar cap gene in virulence and pathogenicity of Aeromonas veronii.

Aeromonas veronii is an important zoonotic and aquatic pathogen that causes a number of illnesses in both humans and animals. It is related to gastroenteritis, skin and soft tissue infections and bacteremia in humans, as well as causing significant economic losses in aquaculture owing to fish sepsis. Here, we constructed the flagellar cap gene (fliD) mutant strain of A. veronii by suicide plasmid-mediated homologous recombination system and analysed its characteristics. It was found that the deletion of fliD had no effect on growth and biochemical properties and could be inherited stably. However, the motility of A. veronii ΔfliD was significantly reduced, the flagellum was defective and the biofilm formation was attenuated compared with that of A. veronii wild-type strain. In vivo experiments revealed that the colonization capacity of ΔfliD was significantly lower than that of the wild-type strain in the period of first 24 h, and the median lethal dose (LD50 ) was 56 times higher than that of the wild-type strain. The Cyprinus carpio infected with the wild-type strain indicated faster death speed and more severe clinical signs compared to ΔfliD strain. These results suggest that fliD is closely related to the virulence of A. veronii and plays an important role in pathogenicity, providing the foundation for pathogenic mechanism studies of A. veronii.

Critical roles of VipB protein on virulence and oxidative stress tolerance in Aeromonas veronii.

Aeromonas veronii is a zoonotic pathogen capable of causing sepsis and ulceration in freshwater fish. Recently, reports of numerous cases indicate a marked increase in pathogenicity. Nonetheless, little is known about the pathogenesis of A. veronii infections. In this study, an in-frame mutant of the A. veronii vipB gene was generated to investigate its biological function. Deletion of the vipB gene resulted in a significant 204.71-fold decrease in the LD50 of A. veronii against zebrafish and a 2-fold and 4-fold reduction in the toxicity to EPC cells at 1 h and 2 h of infection, respectively. The virulence-related genes of the mutant ΔvipB all showed significantly reduced expression levels compared to the wild strain. In addition, the motility of the mutant ΔvipB decreased significantly, the adhesion ability to EPC cells was 3.25-fold lower than that of the parental strain, and the oxidative stress tolerance was 2.31-fold lower than that of TH0426 strain. In contrast, the biofilm formation amount of ΔvipB strain increased by 1.65-fold at both 12 h and 24 h. Our findings suggest that the vipB gene is associated with flagella stability, virulence, and oxidative stress tolerance and plays critical roles in the pathogenesis of A. veronii infections.

Concurrent infections of Streptococcus iniae and Aeromonas veronii in farmed Giant snakehead (Channa micropeltes).

The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram-positive cocci and cream-coloured, round and convex colonies of rod-shaped gram-negative bacteria. Additional biochemical and species-specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole-trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole-trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies.

Transcriptomic insights into the immune response of the intestine to Aeromonas veronii infection in northern snakehead (Channa argus).

Intestinal inflammation is a protective response that is implicated in bacterial enteritis triggered by gastrointestinal infection. The immune mechanisms elicited in teleost against the infection of Aeromonas veronii are largely unknown. In this study, we performed a de novo northern snakehead (Channa argus) transcriptome assembly using Illumina sequencing platform. On this basis we performed a comparative transcriptomic analysis of northern snakehead intestine from A. veronii-challenge and phosphate buffer solution (PBS)-challenge fish, and 2076 genes were up-regulated and 1598 genes were down-regulated in the intestines infected with A. veronii. The Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes (DEGs) were enriched to 27, 21 and 20 GO terms in biological process, cellular component, and molecular function, respectively. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 420 DEGs were involved in 194 pathways. Moreover, 33 DEGs were selected for quantitative real-time PCR analysis to validate the RNA-seq data. The results reflected the consistency of the expression levels between qRT-PCR and RNA-seq data. In addition, a time-course analysis of the mRNA expression of 33 immune-related genes further indicated that the intestinal inflammation to A. veronii infection simultaneously regulated gene expression alterations. The present study provides transcriptome data of the teleost intestine, allowing us to understand the mechanisms of intestinal inflammation triggered by bacterial pathogens. DATA AVAILABILITY STATEMENT: All data supporting the findings of this study are available within the article and Supplementary files. The RNA-seq raw sequence data are available in NCBI short read archive (SRA) database under accession number PRJNA615958.

Dual RNA-seq of spleens extracted from channel catfish infected with Aeromonas veronii reveals novel insights into host-pathogen interactions.

Interactions between host and pathogen are involving various dynamic changes in transcript expression and critical for understanding host immunity against infections and its associated pathogenesis. Herein, we established a model of channel catfish infected with Aeromonas veronii. The infected fish had prominent body surface bleeding, and the spleen showed hyperemia and swelling. Then, the spleen of channel catfish infected with A. veronii was analyzed by dual RNA sequencing (RNA-seq), and the transcriptome data were compared with uninfected channel catfish spleen or bacteria cultured in vitro. The transcript expression profile of pathogen-host interaction between A. veronii and channel catfish was successfully studied. During infection, the host was enriched for multiple immune-related signaling pathways, such as the Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, and T cell receptor signaling pathway; and significantly upregulated for many innate immune-related genes, including IL-8. At the same time, we found that A. veronii mainly harmed the host spleen through hemolysin. Our current findings are of great significance in clarifying the pathogenesis of channel catfish induced by A. veronii and provide gene targets for developing preventive measures.