Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Infection of epithelioma papulosum cyprini (EPC) cells with spring viremia of carp virus (SVCV) induces autophagy and apoptosis through endoplasmic reticulum stress.

Spring viremia of carp virus (SVCV) is a lethal freshwater pathogen of cyprinid fish that has caused significant economic losses to aquaculture. To reduce the economic losses caused by SVCV, its pathogenic mechanism needs to be studied more thoroughly. Here, we report for the first time that SVCV infection of Epithelioma papulosum cyprini (EPC) cells can induce cellular autophagy and apoptosis through endoplasmic reticulum stress. The presence of autophagic vesicles in infected EPC cells was shown by transmission electron microscopy. Quantitative fluorescence PCR and Western blot results showed that p62 mRNA expression was decreased, and the expression of Beclin1 and LC3 mRNA was increased. The p62 protein was decreased, and the Beclin1 protein and LC3 were increased in the endoplasmic reticulum stress activation state. To further clarify the mode of death of SVCV-infected EPC cells, we examined caspase3, caspase9, BCL-2, and Bax mRNA, which showed that they were all increased. Apoptosis of SVCV-infected cells increased upon activation of endoplasmic reticulum stress. Our results suggest that endoplasmic reticulum stress can regulate SVCV infection-induced autophagy and apoptosis. The results of this study provide theoretical data for the pathogenesis of SVCV and lay the foundation for future drug development and vaccine construction.

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.

Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp.

Aeromonas veronii is an important aquatic zoonotic, which elicits a range of diseases, such as haemorrhagic septicemia. To develop an effective oral vaccine against Aeromonas veronii infection in carp, the Aeromonas veronii adhesion (Aha1) gene was used as a target molecule to attach to intestinal epithelial cells. Two anchored recombinant. Lactic acid bacteria strains (LC-pPG-Aha1 1038 bp and LC-pPG-Aha1-LTB 1383 bp) were constructed by fusing them with the E. coli intolerant enterotoxin B subunit (LTB) gene and using Lactobacillus casei as antigen delivery vector to evaluate immune effects of these in carp. Western blotting and immunofluorescence were used to confirm that protein expression was successful. Additionally, levels of specific IgM in serum and the activities of ACP, AKP, SOD, LYS, C3, C4, and lectin enzymes-were assessed. Cytokines IL-10, IL-1ß, TNF-α, IgZ1, and IgZ2 were measured in the liver, spleen, kidney, intestines, and gills tissue by qRT-PCR, which showed an increasing trend compared with the control group (P < 0.05). A colonization assay showed that the two L. casei recombinants colonized the middle and hind intestines of immunized fish. When immunized carp were experimentally challenged with Aeromonas veronii the relative percentage protection of LC-pPG-Aha1 was 53.57%, and LC-pPG-Aha1-LTB was 60.71%. In conclusion, these results demonstrate that Aha1 is a promising candidate antigen when it is displayed on lactic acid bacteria (Lc-pPG-Aha1 and Lc-pPG-Aha1-LTB) seems promising for a mucosal therapeutic approach. We plan to investigate the molecular mechanism of the L. casei recombinant in regulating the intestinal tissue of carp in future studies.

A novel "prime and pull" strategy mediated by the combination of two dendritic cell-targeting designs induced protective lung tissue-resident memory T cells against H1N1 influenza virus challenge.

Vaccination is still the most promising strategy for combating influenza virus pandemics. However, the highly variable characteristics of influenza virus make it difficult to develop antibody-based universal vaccines, until now. Lung tissue-resident memory T cells (TRM), which actively survey tissues for signs of infection and react rapidly to eliminate infected cells without the need for a systemic immune reaction, have recently drawn increasing attention towards the development of a universal influenza vaccine. We previously designed a sequential immunization strategy based on orally administered Salmonella vectored vaccine candidates. To further improve our vaccine design, in this study, we used two different dendritic cell (DC)-targeting strategies, including a single chain variable fragment (scFv) targeting the surface marker DC-CD11c and DC targeting peptide 3 (DCpep3). Oral immunization with Salmonella harboring plasmid pYL230 (S230), which displayed scFv-CD11c on the bacterial surface, induced dramatic production of spleen effector memory T cells (TEM). On the other hand, intranasal boost immunization using purified DCpep3-decorated 3M2e-ferritin nanoparticles in mice orally immunized twice with S230 (S230inDC) significantly stimulated the differentiation of lung CD11b+ DCs, increased intracellular IL-17 production in lung CD4+ T cells and elevated chemokine production in lung sections, such as CXCL13 and CXCL15, as determined by RNAseq and qRT‒PCR assays, resulting in significantly increased percentages of lung TRMs, which could provide efficient protection against influenza virus challenge. The dual DC targeting strategy, together with the sequential immunization approach described in this study, provides us with a novel "prime and pull" strategy for addressing the production of protective TRM cells in vaccine design.

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.

A phage cocktail in controlling phage resistance development in multidrug resistant Aeromonas hydrophila with great therapeutic potential.

Aeromonas hydrophila (A. hydrophila) is an opportunistic pathogen of fish-human-livestock, which poses a threat to the development of aquaculture. Lytic phage has long been considered as an effective bactericidal agent. However, the rapid development of phage resistance seriously hinders the continuous application of lytic phages. In our study, a new bacteriophage vB_ AhaP_PZL-Ah8 was isolated from sewage and its characteristics and genome were investigated. Phage vB_ AhaP_PZL-Ah8 has been classified as the member of the Podoviridae family, which exhibited the latent period was about 30 min. As revealed from the genomic sequence analysis, vB_ AhaP_PZL-Ah8 covered a double-stranded genome of 40,855 bp (exhibiting 51.89% G + C content), with encoding 52 predicted open reading frames (ORFs). The results suggested that the combination of vB_ AhaP_PZL-Ah8 and another A. hydrophila phage vB_ AhaP_PZL-Ah1 could improve the therapeutic efficacy both in vitro and in vivo. The resistance mutation frequency of A. hydrophila cells infected with the mixture phage (vB_ AhaP_PZL-Ah8+ vB_ AhaP_PZL-Ah1) was significantly lower than cells treated with single phage (P <0.01). Phage therapy in vivo showed that the survival rate in the mixture phage treatment group was significantly higher than that in single phage treatment group.

Oral vaccination with recombinant Lactobacillus casei expressing Aha1 fused with CTB as an adjuvant against Aeromonas veronii in common carp (Cyprinus carpio).

Aeromonas veronii (A. veronii) is a pathogenic that can infect human, animal and aquatic organisms, in which poses a huge threat to the health of many aquatic organisms such as Cyprinus carpio. In this study, Lactobacillus casei (L. casei) strain CC16 was used as antigen deliver carrier and fused with cholera toxin B subunit (CTB) as an adjuvant to construct the recombinant L. casei pPG-Aha1/Lc CC16(surface-displayed) and pPG-Aha1-CTB/Lc CC16(surface-displayed) expressing Aha1 protein of A. veronii, respectively. And the immune responses in Cyprinus carpio by oral route was explored. Our results demonstrated that the recombinant strains could stimulate high serum specific antibody immunoglobulin M (IgM) and induce a stronger acid phosphatase (ACP), alkaline phosphatase (AKP), C3, C4, lysozyme (LZM), Lectin and superoxide dismutase (SOD) activity in Cyprinus carpio compared with control groups. Meanwhile, the expression of Interleukin-10 (IL-10), Interleukin-1ß (IL-1ß), Tumor Necrosis Factor-α (TNF-α), immunoglobulin Z1 (IgZ1) and immunoglobulin Z2 (IgZ2) in the tissues were significantly upregulated compared with Lc-pPG or PBS groups, indicating that humoral and cell immune response were triggered. Additionally, recombinant L. casei could survive and colonize in fish intestine. Significantly, recombinant L. casei provides immune protection against A. veronii infection, which Cyprinus carpio received pPG-Aha1-CTB/Lc CC16 (64.29%) and pPG-Aha1/Lc CC16 (53.57%) had higher survival rates compared with the controls. Thus, we demonstrated that recombinant pPG-Aha1/Lc CC16 and pPG-Aha1-CTB/Lc CC16 may be the promising strategy for the development of an oral vaccine against A. veronii.

Immune enhancement effects of inactivated vaccine against extracellular products of Aeromonas caviae AC-CY on crucian carp.

Aeromonas caviae is a zoonotic pathogen that can cause disease in aquatic organisms and mammals, including humans, and it is widespread in nature, especially in freshwater environments. Previous research has reported that extracellular products (ECPs) secreted by pathogens during growth are effective protective antigens that can induce the host immune response and protect the host from pathogens. However, little is known about how ECPs enhance immunity. Here, we prepared extracellular products by the cellophane plate method, determined the total protein concentration, and analysed the protein composition of the extracellular products by SDS-PAGE. Subsequently, their enzyme activity and pathogenicity were evaluated separately. Crucian carp were randomly divided into four groups to receive formalin-inactivated A. caviae vaccine (FKC), ECPs mixed with the same amount of Freund's complete adjuvant, the same amount of ECPs mixed with an equal volume of A. caviae inactivated vaccine (FKC + ECPs), sterile PBS alone via intraperitoneal injection. On Days 7, 14, 21, and 28 after immunization, the expression levels of IgM, SOD, and CAT and the lysozyme (LYS) activity in the serum were detected by ELISA, and the relative expression levels of the TNF-α, IFN-γ, IL-1ß, and IL-10 genes in the liver, kidney, spleen, intestine, and gills were measured by qPCR. The extracellular products generated five clearly visible protein bands and exhibited lipase, protease, amylase, DNase and lysozyme but no urease or lecithinase activities. In addition, the median lethal doses of A. caviae and ECPs to crucian carp were 411.64 µg/fish and 1.6 × 105 CFU/mL, respectively. Compared with those of the control group, the IgM, SOD, and CAT contents and serum LYS activity were significantly increased in the experimental groups, and the qRT-PCR results showed that the relative expression levels of TNF-α, IFN-γ, IL-1ß, and IL-10 genes in the liver, kidney, spleen, and intestine were significantly increased after injection immunization. In addition, the relative immune protection rates of the three experimental groups were 60%, 65%, and 45%, all of which were significantly higher than those of the control group. Collectively, our findings show that the extracellular products of A. caviae can be used as a vaccine to significantly improve the immune level of crucian carp and have obvious anti-infection ability. This may represent a promising approach to prevent and control infection by A. caviae and provides strong theoretical support for the development of new inactivated vaccines.

Effects of four different adjuvants separately combined with Aeromonas veronii inactivated vaccine on haematoimmunological state, enzymatic activity, inflammatory response and disease resistance in crucian carp.

The purpose of the current study was to explore the immunomodulatory effects of different adjuvants combined with inactivated vaccines under Aeromonas veronii TH0426 infection in crucian carp. This study explored the best conditions for A. veronii as an inactivated vaccine, and included an animal safety test. Furthermore, we expressed the flagellin FlaA of the A. veronii TH0426 strain for use as an adjuvant supplemented in the diet. Crucian carp were fed 12 different experimental diets for 35 days, including the administration of 10 different adjuvants and inactivated vaccine combinations (50% aluminum hydroxide gel and inactivated vaccine combination, and inactivated vaccine with 20%, 30%, or 50% glucan, astragalus polysaccharide or flagellin), inactivated vaccine alone, and PBS control without adjuvant and inactivated vaccine. After the 42 day feeding trials, the fish were challenged with A. veronii TH0426, and the survival rate over 14 days was recorded. In addition, flagellin FlaA can be expressed normally in large amounts. All experimental groups produced higher levels of IgM serum titres than the control group in the different feeding cycles. Moreover, the activity of serum ACP, AKP, SOD, and LZM, and the expression of inflammatory factors were significantly increased in the experimental groups compared with the control group. The results of qRT-PCR analysis showed that the transcription levels of the IL-10, IL-1ß, IFN-γ and TNF-α genes in heart, liver, spleen and kidney tissues were significantly enhanced by adjuvant treatment, indicating that the addition of adjuvants can significantly promote the body's inflammatory response. In addition, the phagocytic activity of leukocytes in each adjuvant treated group was significantly enhanced compared to that in the groups without adjuvant. After the A. veronii challenge, the survival rate of all adjuvant-treated groups was significantly higher than that of the control group, and the 50% flagellin adjuvant group had the highest rate of 78.37%. Overall, our findings strongly indicate that adjuvants not only significantly improve the body's immunity, but also exhibit a strong anti-infection ability. Importantly, this work provides a new perspective for the prevention and control of aquaculture diseases.

Dietary supplementation of probiotics fermented Chinese herbal medicine Sanguisorba officinalis cultures enhanced immune response and disease resistance of crucian carp (Carassius auratus) against Aeromonas hydrophila.

Aeromonas hydrophila, a Gram-negative bacterium, is one of the major pathogens causing bacterial sepsis in aquatic animals due to drug resistance and pathogenicity, which could cause high mortality and serious economic losses to the aquaculture. Sanguisorba officinalis (called DiYu in Chinese, DY) is well known as herbal medicine, which could inhibit the growth of pathogenic bacteria, hemostasis and regulate the immune response. Moreover, the active ingredients in DY could remarkably reduce drug resistance. In this study, we investigated the effects of probiotic fermentation cultures on A. hydrophila through in vitro and in vivo experiments. Three lactic acid bacteria, including Lactobacillus rhamnosus (LGG), Lactobacillus casei (LC) and Lactobacillus plantarum (LP), were selected to ferment the Chinese herbal medicine DY. The assays of antagonism showed that all three fermented cultures could influence the ability of A. hydrophila growth, among which L. rhamnosus fermented DY cultures appeared to be the strongest inhibitory effect. In addition, the biofilm determination revealed that L. rhamnosus fermented DY cultures could significantly inhibit the biofilm formation of A. hydrophila compared to the other groups. Furthermore, protease, lecithinase and urease activities were found in the three fermentation cultures. Three probiotics fermented DY cultures were orally administration with crucian carp to evaluate the growth performance, immunological parameters and pathogen resistance. The results showed that the three fermentation cultures could promote the growth performance of crucian carp, and the immunoglobulins, antioxidant-related enzymes and immune-related genes were significantly enhanced. Besides, the results showed that crucian carp received L. rhamnosus (60.87%), L. casei (56.09%) and L. plantarum (41.46%) fermented DY cultures had higher survival rates compared with the control group after infection with A. hydrophila. Meanwhile, the pathological tissue results revealed that the probiotic fermented cultures could largely improve the tissues damage caused by the pathogenic bacteria. In conclusion, this study proved that the fermentation cultures of three probiotics could effectively inhibit the growth of A. hydrophila, regulate the level of immune response and improve the survival rate against A. hydrophila in crucian carp. The present data suggest that probiotic fermented Sanguisorba officinalis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.

Characterization and genome analysis of two new Aeromonas hydrophila phages, PZL-Ah1and PZL-Ah8.

Aeromonas hydrophila (A. hydrophila) is an opportunistic pathogen of fish, humans, and livestock, and has a severe negative impact on aquaculture development. Phage therapy is considered an alternative strategy for controlling bacterial infections and contamination. In this study, we isolated and characterized the genomes of two A. hydrophila-specific phages, PZL-Ah1 and PZL-Ah8, which, based on transmission electron microscopy, were identified as members of the family Podoviridae. Both of these phages had a relatively narrow host range, with lytic activity against Aeromonas spp. strains. Whole-genome sequence analysis revealed that PZL-Ah1 and PZL-Ah8 have a double-stranded DNA genome of 38,641 bp and 40,855 bp in length, with a GC content of 53.68% and 51.89%, respectively. Forty-four open reading frames (ORFs) were predicted in PZL-Ah1, and 52 were predicted in PZL-Ah8. Twenty-eight (63.6%) ORFs in PZL-Ah1 and 29 (55.8%) ORFs in PZL-Ah8 were predicted to encode functional proteins with homologs in the NCBI database, while the remaining ORFs were classified as encoding hypothetical proteins with unknown functions. A comparison with known phage genes suggested that ORF 02, ORF 29, and ORF 04 of PZL-Ah1 and ORF 2 and ORF 4 of PZL-Ah8 are involved in host cell lysis. This study expands the phage genome database and provides good candidates for phage typing applications.

Essential role of ascO for virulence of Aeromonas veronii and inducing apoptosis.

Aeromonas veronii is a significant pathogen that is capable of infecting humans, animals, and aquatic animals. The type III secretion system (T3SS) is intimately associated with bacterial pathogenicity. The ascO gene is an important core component of T3SS in A. veronii, but its function is still unclear. The ascO gene of A. veronii TH0426 was deleted by using the pRE112 suicide plasmid to study its function. The study results showed that the ability of ∆ascO to adhere and invade EPC cells was significantly reduced by 1.28 times. The toxicity of the mutant strain ΔascO to EPC cells was consistently significantly lower than wild-type strain TH0426 at 1, 2, and 4 h. The LD50 values of ∆ascO against zebrafish and Carassius auratus (C. auratus) were 53 and 15 times that of the wild-type strain. In addition, the bacterial load of the mutant strain ΔascO in blood, heart, liver, and spleen was lower than wild-type strain TH0426. The Hoechst staining showed that the apoptotic degree of EPC cells induced by the mutant strain ΔascO was lower than that of the wild-type strain TH0426. Furthermore, real-time quantitative PCR (RT-qPCR) analysis revealed lower expression levels of pro-apoptotic genes (including cytC, cas3, cas9, TNF-α, and IL-1ß) in C. auratus tissues infected with the mutant strain ΔascO compared to the wild-type strain TH0426. The results of in vivo and in vitro experiments have shown that ascO gene mutation can reduce the adhesion and toxicity of A. veronii to EPC and reduce the level of apoptosis induced by A. veronii. As a result, these insights will help further elucidate the function of the ascO gene and thus contribute to understanding the pathogenesis of A. veronii.

Aeromonas veronii virulence and adhesion attenuation mediated by the gene aodp.

Aeromonas veronii (A. veronii) is an opportunistic pathogen of fish-human-livestock, which poses a threat to the development of aquaculture. Based on our previous studies on proteomics and genomics, we found out that the aodp gene may be related to the virulence of A. veronii TH0426. However, aodp gene encodes a hypothetical protein with an unknown function, and its role in A. veronii TH0426 is not clear. Here, we first constructed a mutant strain (△-aodp) to investigate the functional role of aodp in A. veronii TH0426. Compared with the wild strain A. veronii TH0426, the growth rate of strain △-aodp was slower and was resistant to neomycin and kanamycin, but sensitive to cephalexin. The swimming and swarming ability of △-aodp strain decreased, and the pathogenicity to mice decreased by 15.84-fold. Besides, the activity of caspase-3 in EPCs infected with △-aodp strain was 1.49-fold lower than that of the wild strain. We examined 20 factors closely related to A. veronii virulence, among them 17 genes were down-regulated as a result of aodp deficiency. This study laid a foundation for further studies on the pathogenesis of A. veronii.

Functional analysis of fis in Aeromonas veronii TH0426 reveals a key role in the regulation of virulence.

Aeromonas veronii is a comorbid pathogen that can infect humans, and animals including various aquatic organisms. In recent years, an increasing number of cases of A. veronii infection has been reported, indicating serious risks. This bacterium not only threatens public health and safety but also causes considerable economic loss in the aquaculture industry. Currently, some understanding of the pathogenic mechanism of A. veronii has been obtained. In this study, we first constructed the A. veronii TH0426 fis gene deletion strain Δfis and the complementation strain C-fis through homologous recombination technology. The results showed that the adhesion and invasion ability of the Δfis strain towards Epithelioma papulosum cyprini (EPC) cells and the cytotoxicity were 3.8-fold and 1.38-fold lower, respectively, than those of the wild-type strain. In the zebrafish infection model, the lethality of the deleted strain is 3-fold that of the wild strain. In addition, the bacterial load of the deletion strain Δfis in crucian carp was significantly lower than the wild-type strain, and the load decreased with time. In summary, deletion of the fis gene led to a decrease in the virulence of A. veronii. Our research results showed that the deletion of the fis gene significantly reduces the virulence and adhesion ability of A. veronii TH0426. Therefore, the fis gene plays a vital role in the pathogenesis of A. veronii TH0426. This preliminary study of the function of the fis gene in A. veronii will help researchers further understand the pathogenic mechanism of A. veronii.

Comparative genomics analysis of strains from diverse sources reveals the evolutionary relationship of Aeromonas veronii.

Aeromonas veronii (A. veronii, AV) strains are emerging zoonotic and aquatic pathogens, yet we know very little about their genomics. This study aims to utilize comparative genomics to investigate the intraspecific genetic diversity, differences in virulence factors and evolutionary mechanisms of A. veronii strains from diverse sources and to fundamentally demonstrate their pathogenic mechanisms. We conducted comparative genomics analysis of 39 A. veronii strains from different sources and found that 1993 core genes are shared by these strains and that these shared core genes may be necessary to maintain the basic characteristics of A. veronii. Additionally, phylogenetic relationship analysis based on these shared genes revealed that a distant relationship between the AMC34 strain and the other 38 strains but that, the genetic relationship among the 38 strains is relatively close, indicating that AMC34 may not belong to A. veronii. Furthermore, analysis of shared core genes and average nucleotide identity (ANI) values showed no obvious correlation with the location of A. veronii isolation and genetic relationship. Our research indicates the evolutionary mechanism of A. veronii from different sources and provides new insights for a deeper understanding of its pathogenic mechanism.

Comparative genomic analysis of different virulence strains reveals reasons for the increased virulence of Aeromonas veronii.

Aeromonas veronii is an important zoonotic and aquatic agent. More and more cases have shown that it has caused huge economic losses in the aquaculture industry in addition to threatening human health. But the reasons for the increasing virulence of A. veronii are still unclear. In order to further understand the reasons for the increased virulence of A. veronii, we conducted a comparative analysis of the genomes of A. veronii with different virulence. The analysis revealed that there are multiple virulence factors, such as those related to fimbriae, flagella, toxins, iron ion uptake systems and type II, type III and type VI secretion systems in the virulent strain TH0426 genome. And comparative analysis showed that there were two complete type III secretion systems (API1 and API2), of which the API2 and iron ion transport system were unique to the TH0426 strain. In addition, TH0426 strain also has unique functional gene clusters, which may play important roles in terms of resisting infection, adapting to different environments and genetic evolution. These particular virulence factors and gene clusters may be the important reasons for the increased virulence. These insights will provide a reference for the study of the pathogenesis of A. veronii.

Inactivation of the T6SS inner membrane protein DotU results in severe attenuation and decreased pathogenicity of Aeromonas veronii TH0426.

BACKGROUND: The inner membrane protein DotU of Aeromonas veronii is an important component of the minimal core conserved membrane proteome required for the formation of an envelope-transmembrane complex. This protein functions in a type VI secretion system (T6SS), and the role of this T6SS during the pathogenic process has not been clearly described. RESULTS: A recombinant A. veronii with a partial disruption of the dotU gene (720 bp of the in-frame sequence) (defined as ∆dotU) was constructed by two conjugate exchanges. We found that the mutant ∆dotU allele can be stably inherited for more than 50 generations. Inactivation of the A. veronii dotU gene resulted in no significant changes in growth or resistance to various environmental changes. However, compared with the wild-type strain colony, the mutant ∆dotU colony had a rough surface morphology. In addition, the biofilm formation ability of the mutant ∆dotU was significantly enhanced by 2.1-fold. Conversely, the deletion of the dotU gene resulted in a significant decrease in pathogenicity and infectivity compared to those of the A. veronii wild-type strain. CONCLUSIONS: Our findings indicated that the dotU gene was an essential participant in the pathogenicity and invasiveness of A. veronii TH0426, which provides a novel perspective on the pathogenesis of TH0426 and lays the foundation for discovering potential T6SS effectors.

Nucleoside Diphosphate Kinases (ndk) reveals a key role in adhesion and virulence of Aeromonas veronii.

Aeromous veronii is a severe pathogen that can infect aquatic organisms and mammals also causes irreparable damage to fish aquaculture. Analysis of the results of epidemiological investigations have revealed that its tolerance to drugs and the virulence of A. veronii have increased in recent years. Most of the researches on A. veronii focuse on the strain isolation, identification, and drug susceptibility. However, we do not know so much about the molecular mechanism of the pathogenesis on A. veronii. Here we identified and obtained the highly expressed TH0426 Nucleoside Diphosphate Kinases (NDK) of A. veronii. We first constructed a mutant strain (△-ndk) by generating an in-frame deletion of the ndk gene, to investigate the functional role in A. veronii TH0426. The ability in the adhesion and invasion of EPC cells and biofilm formation significantly reduced of the △-ndk strain. The motility test showed that the ndk gene affected on the swimming ability, while did not affect the swarming motility. Compared with the wild-type strain TH0426, the pathogenicity of △-ndk strain to zebrafish reduced severely. Besides, the ndk gene has affected the apoptosis rate of A. veronii TH0426. These results would help to demonstrate the function of ndk further and realize the pathogenesis on A. veronii.

Construction and immune efficacy of recombinant Lactobacillus casei strains expressing Malt from Aeromonas veronii.

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1ß, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Results also showed that recombinant L. casei could stimulate the mucosa through colonization of the intestine, resulting in increased transcription of IL-10, IL-1ß, TNF-α, and IFN-γ. Immunity and colonization assays also showed that after 34 days of fasting, recombinant L. casei were still present in the intestines of the immunized fish. Common carp that received Lc-MCS-Malt(53.3%) and Lc-pPG-Malt (46.7%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our findings suggested that recombinant L. casei can adequately protect fish and improve immunity, providing a theoretical basis for the future development of an oral Lactobacillus vaccine for use in aquaculture.