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.

Efficacy of a recombinant Lactobacillus plantarum Lp-pPG-Malt as an oral vaccine candidate against Aeromonas hydrophila infection in crucian carp.

Aeromonas hydrophila (A. hydrophila), a gram-negative bacterium, causes serious diseases with various clinical symptoms in farm raised fish. Thus, different ways to prevent and control A. hydrophila infection need to be explored, including a vaccine. In this study, we evaluated the protective efficacy of an oral vaccine prepared from the A. hydrophila TPS maltoporin (Malt) with Lactobacillus plantarum (L. plantarum) against A. hydrophila infection in crucian carp (Carassius auratus). For the in vivo experiment, the oral vaccine was administered to crucian carp by feeding them fish diets containing Lp-pPG-Malt, Lp-pPG and PBS for 28 days. The enzyme-linked immunosorbent assay (ELISA), leukocyte phagocytosis assay and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to measure the protective efficacy of the Lp-pPG-Malt. ELISA and leukocyte phagocytosis assay confirmed that Lp-pPG-Malt significantly enhanced the IgM level and nonspecific immune response of crucian carp compared with the control groups (Lp-pPG and PBS). The RT-qPCR results showed that the Lp-pPG-Malt increased the relative expression of immune-related genes (IL-10, IL-1ß, TNF-α, IFN-γ) of crucian carp in various tissues (liver, spleen, head kidney and hind intestine). Moreover, Lp-pPG-Malt significantly increased the relative percent survival of fish after intraperitoneal injection with A. hydrophila (55%) compared with the Lp-pPG and PBS groups (0%). These findings suggest that Lp-pPG-Malt can serve as an oral vaccine candidate for A. hydrophila infection and that Malt can be used as an effective antigen in crucian carp farming.

Lc-pPG-612-OmpU-CTB: A promising oral vaccine for protecting Carassius auratus against Vibrio mimicus infection.

Vibrio mimicus (V. mimicus) is known to cause severe bacterial diseases with high mortality rates in fish, resulting in significant economic losses in the global aquaculture industry. Therefore, the objective of this study was to develop a safe and effective vaccine for protecting Carassius auratus (C. auratus) against V. mimicus infection. Recombinant Lactobacillus casei (L. casei) strains, Lc-pPG-612-OmpU and Lc-pPG-612-OmpU-CTB (surface-displayed), were constructed using a L. casei strain (ATCC 393) as an antigen delivery carrier and the cholera toxin B subunit (CTB) as an adjuvant. The two recombinant strains of L. casei were administered to C. auratus via oral immunization, and the protective efficacy of the oral vaccines was assessed. The results demonstrated that oral immunization with the two strains significantly increased the levels of nonspecific immune indicators in C. auratus, including alkaline phosphatase (AKP), lysozyme (LYS), acid phosphatase (ACP), complement 3 (C3), complement 4 (C4), lectin, and superoxide dismutase (SOD). Moreover, the experiment groups exhibited significant increases in specific immunoglobulin M (IgM) antibodies against OmpU, as well as the transcription of immune-related genes (ie., IL-1ß, TNF-α, IL-10, and TGF-ß), when compared to the control groups. Following infection of C. auratus with V. mimicus, the mortality rate of the recombinant L. casei-treated fish was observed to be lower compared to the control group. This finding suggests that recombinant L. casei demonstrates effective protection against V. mimicus infection in C. auratus. Furthermore, the addition of the immune adjuvant CTB was found to induce a more robust adaptive and innate immune response in C. auratus, resulting in reduced mortality after infection with V. mimicus.

Oral vaccination with recombinant Lactobacillus casei with surface displayed OmpK fused to CTB as an adjuvant against Vibrio mimicus infection in Carassius auratus.

Vibrio mimicus (V. mimicus) is a pathogenic bacterium that causes diseases in humans and various aquatic animals. A particularly efficient way to provide protection against V. mimicus is through vaccination. However, there are few commercial vaccines against V. mimics, especially oral vaccines. In our study, two surface-display recombinant Lactobacillus casei (L. casei) Lc-pPG-OmpK and Lc-pPG-OmpK-CTB were constructed using L. casei ATCC393 as an antigen delivery vector, outer membrane protein K (OmpK) of V. mimicus as an antigen, and cholera toxin B subunit (CTB) as a molecular adjuvant; furthermore, the immunological effects of recombinant L.casei in Carassius auratus (C. auratus) were assessed. The results indicated that oral recombinant L.casei Lc-pPG-OmpK and Lc-pPG-OmpK-CTB stimulated higher levels of serum-specific immunoglobulin M (IgM) and increased the activity of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LYS), lectin, C3, and C4 in C. auratus, compared with control groups (Lc-pPG group and PBS group). Furthermore, the expression of interleukin-1ß (IL-1ß), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and transforming growth factor-ß (TGF-ß) in the liver, spleen, head kidney, hind intestine and gills of C. auratus was significantly increased, compared with that in the controls. These results demonstrated that the two recombinant L. casei strains could effectively trigger humoral and cellular immunity in C. auratus. In addition, two recombinant L.casei strains were able to survive and colonize the intestine of C. auratus. Importantly, after being challenged with V. mimicus, C. auratus fed Lc-pPG-OmpK and Lc-pPG-OmpK-CTB exhibited greater survival rates than the controls (52.08% and 58.33%, respectively). The data showed that recombinant L. casei could elicit a protective immunological response in C. auratus. The effect of the Lc-pPG-OmpK-CTB group was better than that of the Lc-pPG-OmpK group, and Lc-pPG-OmpK-CTB was found to be an effective candidate for oral vaccination.

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.

Dietary supplementation with Weissella cibaria C-10 and Bacillus amyloliquefaciens T-5 enhance immunity against Aeromonas veronii infection in crucian carp (Carassiu auratus).

With the aim to discover novel lactic acid bacteria and Bacillus strains from fish as potential probiotics to replace antibiotics in aquaculture, the present study was conducted to isolate lactic acid bacteria and Bacillus from intestinal tract of healthy crucian carp (Carassiu auratus) and largemouth bass (Micropterus salmoides) and evaluate their resistance against Aeromonas veronii. Based on the evaluation of antibacterial activity and tolerance test, one strain of lactic acid bacteria (Weissella cibaria C-10) and one strain of Bacillus (Bacillus amyloliquefaciens T-5) with strong environmental stability were screened out. The safety evaluation showed that these two strains were non-toxic to crucian carp and were sensitive to most antibiotics. In vivo study, the crucian carps were fed a basal diet supplemented with W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5), respectively, for 5 weeks. Then, various immune parameters were measured at 35 days of post-feeding. Results showed both probiotics could improve the activities of related immune enzymes, immune factors and non-specific immune antibodies in blood and organs (gill, gut, kidney, liver, and spleen) of crucian carp in varying degrees. Moreover, after 7 days of challenge experiment, the survival rates after challenged with A. veronii of W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5) supplemented groups to the crucian carps were 20%, 33% and 22%, respectively. Overall, W. cibaria C-10 and B. amyloliquefaciens T-5 could be considered to be developed into microecological preparations for the alternatives of antibiotics in aquaculture.

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.

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.

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.

Bacillus subtilis BSH has a protective effect on Salmonella infection by regulating the intestinal flora structure in chickens.

Salmonellosis is a worldwide zoonotic disease that poses a serious threat to the reproduction of livestock and poultry and the health of young animals. Probiotics including Bacillus species, have received increasing attention as a substitute for antibiotics. In this study, chicks infected with Salmonella were fed feed supplemented with the BSH to observe the pathological changes in the liver, detect the number of viable bacteria in the liver and spleen, and record the death of the chicks. The results showed that BSH could reduce the pathological changes in the liver and the invasion of Salmonella into the liver and spleen of chicks. In addition, the survival rate of chicks in the BSH experimental group was 60%, while that in the infected control group was 26%, indicating that BSH had a protective effect on chicks infected with Salmonella. Finally, the fecal microflora of 9-day-old chicks was analyzed by 16S rRNA high-throughput sequencing. The results showed that Salmonella infection could cause intestinal flora changes, while BSH could alleviate this change. In addition, BSH also promoted the proliferation of Lactobacillus salivarius in the cecum of chick. This study emphasized that BSH has anti- Salmonella infection effects in chickens and can be used as a candidate microecological preparation strain.

Antitumour metastasis and the antiangiogenic and antitumour effects of a Eimeria stiedae soluble protein.

Some protozoa (Plasmodium falciparum, Toxoplasma gondii, etc) are used to treat cancer because they can improve tumour-induced immunosuppression. This study aims to evaluate the antitumour effect of Eimeria stiedae oocyst soluble protein (ESSP). ESSP was extracted, and mice were injected with 5 × 105 CT26 cells in the right axilla, and then, 50 µg of ESSP was intraperitoneally injected for 5 continuous days. The effect of ESSP on tumour immunity was detected by flow cytometry 25 days after the CT26 inoculation. The results showed that ESSP can inhibit the growth of CT26 subcutaneous tumours; significantly increase the expression of MHC I, MHC II, CD80 and CD86 on the surface of splenic dendritic cells; and enhance the level of IL-12 secretion. ESSP induced an increase in the number of NK cells in the mouse spleen, and the levels of IFN-γ and CD107 were upregulated in the NK cells and CD8+ T cells. The number of metastatic nodules in the lung tumours in the mice was significantly reduced, and the number of tubes, area of the loops and total length of the tubes were significantly reduced. ESSP enhances the antitumour immune response and inhibits tumour growth, metastasis and angiogenesis.

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.

Dendritic Cells Targeting Lactobacillus plantarum Strain NC8 with a Surface-Displayed Single-Chain Variable Fragment of CD11c Induce an Antigen-Specific Protective Cellular Immune Response.

Influenza A virus (H1N1) is an acute, highly contagious respiratory virus. The use of lactic acid bacteria (LAB) to deliver mucosal vaccines against influenza virus infection is a research hot spot. In this study, two recombinant Lactobacillus plantarum strains expressing hemagglutinin (HA) alone or coexpressing aCD11c-HA to target HA protein to dendritic cells (DCs) by fusion to an anti-CD11c single-chain antibody (aCD11c) were constructed. The activation of bone marrow dendritic cells (BMDCs) by recombinant strains and the interaction of activated BMDCs and sorted CD4+ or CD8+ T cells were evaluated through flow cytometry in vitro, and cellular supernatants were assessed by using an enzyme-linked immunosorbent assay kit. The results demonstrated that, compared to the HA strain, the aCD11c-HA strain significantly increased the activation of BMDCs and increased the production of CD4+ gamma interferon-positive (IFN-γ+) T cells, CD8+ IFN-γ+ T cells, and IFN-γ in the cell culture supernatant in vitro Consistent with these results, the aCD11c-HA strain clearly increased the activation and maturation of DCs, the HA-specific responses of CD4+ IFN-γ+ T cells, CD8+ IFN-γ+ T cells, and CD8+ CD107a+ T cells, and the proliferation of T cells in the spleen, finally increasing the levels of specific antibodies and neutralizing antibodies in mice. In addition, the protection of immunized mice was observed after viral infection, as evidenced by improved weight loss, survival, and lung pathology. The adoptive transfer of CD8+ T cells from the aCD11c-HA mice to NOD/Lt-SCID mice resulted in a certain level of protection after influenza virus infection, highlighting the efficacy of the aCD11c targeting strategy.

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.

Cadaverine reverse transporter (CadB protein) contributes to the virulence of Aeromonas veronii TH0426.

Aeromonas veronii is one of the main pathogens causing sepsis and ulcer syndrome in freshwater fish. Analysis of the results of epidemiological investigations in recent years has revealed that the virulence of A. veronii and its tolerance to drugs have been increasing year by year. Currently, most of the research on A. veronii focuses on its isolation, identification, and drug susceptibility, whereas research on its virulence factors and pathogenesis mechanisms is relatively rare. In this study, we identified and obtained the highly expressed TH0426 cadaverine reverse transporter (CadB) of A. veronii. We used efficient suicide plasmid-mediated homologous recombination to delete the cadB gene in TH0426 and constructed a cadB deletion strain. The LD50 of ΔcadB was 93.2 times higher than that of TH0426 in zebrafish, the toxicity of ΔcadB was 9.5 times less than that of TH0426 in EPC cells, and the biofilm formation ability of ΔcadB was 5.6-fold greater than that of TH0426. In addition, motility detection results indicated that ΔcadB had lost its swimming ability. The results of flagellar staining and TEM demonstrated that ΔcadB shed the flagella. In summary, the virulence and adhesion of A. veronii TH0426 were significantly decreased by the deletion of cadB, which might provide a theoretical basis for research into A. veronii virulence factors.

Effects of recombinant Lactobacillus casei on growth performance, immune response and disease resistance in crucian carp, Carassius auratus.

In the present study, we constructed two recombinant Lactobacillus casei (L. casei) Lc-pPG-1-AcrV (surface-displayed) and Lc-pPG-2-AcrV (secretory) constitutively expressing AcrV protein of Aeromonas veronii (A. veronii). Expression of recombinant AcrV protein was verified by western blot and immunofluorescence technique. Compared with PBS group, the final weight (FW), weight gain (WG) and specific growth rate (SGR) of fish fed Lc-pPG-1-AcrV, Lc-pPG-2-AcrV and Lc-pPG diets after 56 days observed significantly increase (p < 0.05), while the feed conversion ratio (FCR) showed a significantly decrease (p < 0.05). The recombinant L. casei strains were orally administrated to crucian carp, and significant increased (p < 0.05) the immunoglobulin M (IgM), elevated the acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM) and superoxide dismutase (SOD) activity in serum. Moreover, leukocytes phagocytosis percentage and index of the recombinant L. casei were both enhanced. The results demonstrated that the recombinant L. casei could elicit systemic immune responses and increase the serum immunological index. The Interleukin-10 (IL-10), Interleukin-1ß (IL-1ß), interferon-γ (IFN-γ) and Tumor Necrosis Factor-α (TNF-α) levels in liver, spleen, kidney and intestine have up regulated significantly in tissues (p < 0.05), suggesting that the recombinant L. casei has the ability to induce expression of cytokines and enhance the innate immune response. Higher survival rates were exhibited that crucian carp immunized with Lc-pPG-1-AcrV (67.5%) and Lc-pPG-2-AcrV (52.5%) after challenge with A. veronii. In conclusion, these two recombinant L. casei vaccine were effective in improving crucian carp growth, immunity response and disease resistance. The recombinant L. casei strains may be a promising candidate for the development of an oral vaccine against A. veronii.

An effective live attenuated vaccine against Aeromonas veronii infection in the loach (Misgurnus anguillicaudatus).

Aeromonas veronii is a major pathogenic bacterium in humans and animals. When it causes outbreaks, there are enormous economic losses to the aquaculture industry. An effective live attenuated vaccine strain, ΔhisJ, was obtained in our previous studies by gene knockout in Aeromonas veronii TH0426 using the suicide vector pRE112. Here, we evaluated whether the live attenuated vaccine ΔhisJ was suitable for prevention of Aeromonas veronii infection by injection and immersion in loaches. Compared with that of the TH0426 wild-type strain, the virulence of the live vaccine was significantly weakened. Vaccine safety assessment results also indicated that 1 × 107 CFU/mL live vaccine was safe and did not induce clinical symptoms or obvious pathological changes. Additionally, after challenging loaches with Aeromonas veronii TH0426, the relative percent survival of the IN3 injection group was 65.66%, and that of the IM group was 50.78%. Our data show that the live attenuated vaccine ΔhisJ can improve the immune protection rate of loaches. Furthermore, increased enzyme activity parameters (SOD, LZM, ACP, and AKP) in the skin mucus, increased enzyme activity parameters (SOD, LZM, ACP, AKP, and GPx) in the serum, increased specific IgM antibodies and cytokine IL-1ß contents in the serum, and increased cytokine (IL-15, pIgR, IL-1ß, and TNF-α) expression in the liver and spleen were observed. These data are the first to indicate that the live attenuated vaccine ΔhisJ is suitable for the development of a safe and effective vaccine against Aeromonas veronii infection in loach aquaculture.