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Aeromonas veronii is a pathogen which can induce diseases in humans, animals and aquatic organisms, but its pathogenic mechanism and virulence factors are still elusive. In this study, we successfully constructed a mutant strain (ΔascP) by homologous recombination. The results showed that the deletion of the ascP gene significantly down-regulated the expression of associated effector proteins in A. veronii compared to its wild type. The adhesive and invasive abilities of ΔascP to EPC cells were 0.82-fold lower in contrast to the wild strain. The toxicity of ΔascP to cells was decreased by about 2.91-fold (1 h) and 1.74-fold (2 h). Furthermore, the LD

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-1β) 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.

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-1β, 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.

Aeromonas veronii (A. veronii) is an important zoonotic pathogen that causes substantial economic losses in aquaculture. In this study, we aimed to develop a safe and effective immune enhancer to protect Carassius auratus (C. auratus) from A. veronii infections. With recognized safety, lactic acid bacteria are used as antigen delivery vehicles to present antigens. Lipopolysaccharide (LPS), a protective antigen, induces immune responses in animals. Therefore, we created recombinant Lactobacillus plantarum (L. plantarum) with surface-displayed LPS of A. veronii TH0426 and tested its effects on immune responses in C. auratus. The results showed that recombinant L. plantarum Lp-pPG-611.1-LPS, as an immune enhancer, could improve the innate and adaptive immune responses of C. auratus when it was added to the diet of C. auratus. The challenge test showed that the survival rate of C. auratus fed with L. plantarum Lp-pPG-611.1-LPS was higher than that of the control groups, indicating that the recombinant L. plantarum Lp-pPG-611.1-LPS increased the resistance of C. auratus to A. veronii infection. The present results provide a theoretical basis for the development of recombinant L. plantarum Lp-pPG-611.1-LPS as an immune enhancer in aquaculture.

Chicken coccidiosis is a protozoan parasitic disease that leads to considerable economic losses in the poultry industry. In this study, we used invasive Lactobacillus plantarum (L.P) expressing the FnBPA protein as a novel bacterial carrier for DNA delivery into epithelial cells to develop a live oral DNA vaccine. A fusion DNA vaccine co-expressing EtMIC2 and chicken IL-18 (chIL-18) was constructed and then delivered to the host by invasive L.P. Its efficacy against Eimeria tenella challenge was evaluated in chickens by examining the relative weight gain rate; caecal lesion score; OPG; anti-coccidial index (ACI); levels of EtMIC2 antibody, FnBPA, IL-4, IL-18, IFN-γ and SIgA; and proliferation ability and percentages of CD4+ and CD8+ splenocytes. The experimental results showed that chickens immunized with invasive L.P carrying the eukaryotic expression vector pValac-EtMIC2 (pValac-EtMIC2/pSIP409-FnBPA) had markedly improved immune protection against challenge compared with that of chickens immunized with non-invasive L.P (pValac-EtMIC2/pSIP409). However, invasive L.P co-expressing EtMIC2 with the chIL-18 vector exhibited the highest protection efficiency against E. tenella. These results indicate that invasive Lactobacillus-expressing FnBPA improved humoural and cellular immunity and enhanced resistance to E. tenella. The DNA vaccine delivered by invasive Lactobacillus provides a new concept and method for the prevention of E. tenella.

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-1β (IL-1β), 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.

Eukaryotic cells can initiate several distinct self-destruction mechanisms to display essential roles for the homeostasis maintenance, development, and survival of an organism. Pyroptosis, a key response mode in innate immunity, also referred to as caspase-1-dependent proinflammatory programmed necrotic cell death activated by human caspase-1/4/5, or mouse caspase-1/11, plays indispensable roles in response to cytoplasmic insults and immune defense against infectious diseases. These inflammatory caspases are employed by the host to eliminate pathogen infections such as bacteria, viruses, protozoans, and fungi. Gasdermin D requires to be cleaved and activated by these inflammatory caspases to trigger the pyroptosis process. Physiological rupture of cells results in the release of proinflammatory cytokines, the alarmins IL-1β and IL-18, symbolizing the inflammatory potential of pyroptosis. Moreover, long noncoding RNAs play direct or indirect roles in the upstream of the pyroptosis trigger pathway. Here, we review in detail recently acquired insights into the central roles of inflammatory caspases, inflammasomes, and pyroptosis, as well as the crosstalk between pyroptosis and long noncoding RNAs in mediating infection immunity and pathogen clearance.

To evaluate the efficiency of preventing pathogenic avian influenza by vaccination with recombinant Lactobacillus plantarum (L. plantarum) that expresses conserved antigens, the mucosal and systemic immune responses in animals vaccinated with recombinant L. plantarum NC8-409-1 (NC8-pSIP409-pgsA'-HA2) and NC8-409-2 (NC8-pSIP409-pgsA'-3M2e-HA2) were evaluated. Our results showed that recombinant L. plantarum NC8-409-1 and NC8-409-2 could substantially stimulate the specific IgA titer in the intestine and the specific IgG antibody titer in the serum. We also found that recombinant L. plantarum induced increases in the number of B220+ IgA+ cells in Peyer's patches (PPs), in lymphocyte proliferation and in the number of IFN-γ+ producing CD8+ T cells after immunization in mice. Most importantly, the mice that were vaccinated with recombinant L. plantarum NC8-409-2 and NC8-409-1 were to some extent protected against infection challenge with the H9N2 and H1N1 viruses. In particular, NC8-409-2 provided up to 80% protection against the H9N2 virus, and NC8-409-1 provided up to 60% protection. Lung tissue pathology was also reduced. Therefore, recombinant L. plantarum NC8-409-2 and NC8-409-1 may be candidate oral vaccines against bird flu.

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.

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.

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.

Citrobacter freundii refers to a fish pathogen extensively reported to be able to cause injury and high mortality. Phage therapy is considered a process to alternatively control bacterial infections and contaminations. In the present study, the isolation of a virulent bacteriophage IME-JL8 isolated from sewage was presented, and such bacteriophage was characterized to be able to infect Citrobacter freundii specifically. Phage IME-JL8 has been classified as the member of the Siphoviridae family, which exhibits the latent period of 30–40 min. The pH and thermal stability of phage IME-JL8 demonstrated that this bacteriophage achieved a pH range of 4–10 as well as a temperature range of 4, 25, and 37°C. As revealed from the results of whole genomic sequence analysis, IME-JL8 covers a double-stranded genome of 49,838 bp (exhibiting 47.96% G+C content), with 80 putative coding sequences contained. No bacterial virulence- or lysogenesis-related ORF was identified in the IME-JL8 genome, so it could be applicable to phage therapy. As indicated by the in vitro experiments, phage IME-JL8 is capable of effectively removing bacteria (the colony count decreased by 6.8 log units at 20 min), and biofilm can be formed in 24 h. According to the in vivo experiments, administrating IME-JL8 (1 × 107 PFU) was demonstrated to effectively protect the fish exhibiting a double median lethal dose (2 × 109 CFU/carp). Moreover, the phage treatment led to the decline of pro-inflammatory cytokines in carp with lethal infections. IME-JL8 was reported to induce efficient lysis of Citrobacter freundii both in vitro and in vivo, thereby demonstrating its potential as an alternative treatment strategy for infections attributed to Citrobacter freundii.

Aims Genotype VII Newcastle disease (ND) is one of the most epidemic and serious infectious diseases in the poultry industry. A novel vaccine targeting VII Newcastle disease virus (NDV) is still proving elusive. Methods and results In this study, we constructed regulated delayed lysis Salmonella strains expressing either a fusion protein (F) alone under an eukaryotic CMV promoter or together with chicken IL-18 (chIL-18) as a molecular adjuvant under prokaryotic Ptrc promoter, named pYL1 and pYL23 respectively. Oral immunization with recombinant strains induced NDV-specific serum IgG antibodies in both pYL1- and pYL23-immunized chickens. The presence of chIL-18 significantly increased lymphocyte proliferation in immunized chickens, as well as the percentages of CD3+ CD4+ and CD3+ CD8+ T cells in serum, even if a statistically significant difference did not exist. After a virulent challenge, pYL23 immunization provided about 80% protection at day 10 postinfection, compared with 60% of protection offered by pYL1 immunization and 100% protection in the inactivated vaccine group, indicating the enhanced immune response provided by chIL-18, which was also confirmed by histochemical analysis. Conclusions Recombinant lysis Salmonella-vectored DNA vaccine could provide us a novel potential option for controlling NDV infection. Significance and impact of the study This study took use of a regulated delayed lysis Salmonella vector for the design of an orally administrated vaccine against NDV.

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.

Aeromonas veronii is a serious pathogen which can infect mammals and aquatic organisms and causes irreparable damage to fish aquaculture. It has been demonstrated that adhesion to host surface and cells is the initial step in bacterial pathogenesis. Previous study found that bacterial weaken motility probably caused by the absence of flagellar related genes. In this study, we generated the aha deletion and complementary strains and found that two strains can be stably inherited for more than 50 generations. No significant change was found in the growth of mutant △aha. But the ability of biofilm formation, the adhesion and invasion to EPC cells significantly decreased for 3.7-fold and 2.3-fold respectively. Due to aha gene deletion, the stability of A. veronii flagellar was severely declined and the mutant △aha with no mobility. Compared with the wild-type TH0426, the pathogenicity of A. veroniiaha-deleted strain to zebrafish and mice reduced significantly and virulence attenuated severely. Cytotoxicity experiment also proved that mutant △aha showed much weaker virulence at the same time infection. The consequences declared that the stability of flagellar decreased severely with porin missing and lost the motility. Porin regulated by aha gene is essential for the adhesion and virulence of A. veronii. Thence, the mutant △aha of A. veronii provides an important tool for further concentration on the pathogenic mechanism of A. veronii.

Aeromonas veronii is a type of human-livestock-aquatic animal pathogen; it is widely found in nature and causes many deaths among aquatic animals. Extracellular products (ECPs) are secreted by the pathogen during growth and reproduction. These products are considered effective protective antigens that can induce the host to produce an immune response. In this study, the ECPs of A.veronii TH0426 were prepared by ultrafiltration, and then the pathogenicity and enzymatic activity of the ECPs were determined. All the groups were injected intraperitoneally, as follows: group one: ECP protein with an equal volume of Freund's adjuvant; group two: ECPs and formalin-killed cells (FKC) of A.veronii combined with an equal volume of Freund's adjuvant (FKC + ECPs); group three: formalin-killed cells (FKC) of A.veronii combined with an equal volume of Freund's adjuvant (FKC); and, group four: sterile PBS as the control group. The expression levels of IgM, IL-1β, and TNF-α and the lysozyme activity in blood were examined at 7, 14, and 21 days after the immunizations. The results show that the ECPs can produce protease, lipase, amylase and hemolyase, and there was no lecithinase, urease, or gelatinase activity. The results indicate that the ECPs were clearly pathogenic to koi fish, and the LD50 dose was 391.6 μg/fish. Throughout this study, the RPS of the three experimental groups were 75%, 50%, and 70%. This study indicates that the ECPs of A.veronii can effectively enhance the ability of kio fish to resist bacterial invasion.

The highly infectious porcine transmissible gastroenteritis virus (TGEV), which belongs to the coronaviruses (CoVs), causes diarrhea and high mortality rates in piglets, resulting in severe economic losses in the pork industry worldwide. In this study, we used Lactobacillus plantarum (L. plantarum) to anchor the expression of TGEV antigen (S) to dendritic cells (DCs) via dendritic cell-targeting peptides (DCpep). The results show that S antigen could be detected on the surface of L. plantarum by different detection methods. Furthermore, flow cytometry and ELISA techniques were used to measure the cellular, mucosal, and humoral immune responses of the different orally gavaged mouse groups. The obtained results demonstrated the significant effect of the constructed L. plantarum expressing S-DCpep fusion proteins in inducing high expression levels of B7 molecules on DCs, as well as high levels of IgG, secretory IgA, and IFN-γ and IL-4 cytokines compared with the other groups. Accordingly, surface expression of DC-targeted antigens successfully induced cellular, mucosal, and humoral immunity in mice and could be used as a vaccine.

Avian influenza virus (AIV) can infect poultry, mammals, and other hosts and causes enormous economic losses to the global poultry industry. In this study, to develop a novel and potent oral vaccine based on Lactobacillus plantarum (L. plantarum) for controlling the spread of AIV in the poultry industry, we constructed a recombinant L. plantarum strain displaying the 3M2e-HA2 protein of the influenza virus and determined the effect of N/pgsA'-3M2e-HA2 against AIV in chicks. We first confirmed that the 3M2e-HA2 fusion protein was expressed on the surface of L. plantarum via flow cytometry and immunofluorescence experiments. Our experimental results demonstrated that chicks immunized with N/pgsA'-3M2e-HA2 could induce specific humoral, mucosal, and T cell-mediated immune responses, eliciting the host body to protect itself against AIV. Additionally, compared to oral administration, the intranasal immunization of chicks with N/pgsA'-3M2e-HA2 provided a stronger immune response, resulting in a potent protective effect that hindered the loss of body weight, decreasing pulmonary virus titers and reducing lung and throat pathological damages. Thus, our results indicate that our novel approach is an effective method of vaccine design to promote mucosal immunity.

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.

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-1β contents in the serum, and increased cytokine (IL-15, pIgR, IL-1β, 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.

Aeromonas veronii is a virulent fish pathogen that causes extensive economic losses in the aquaculture industry worldwide. In this study, a virulent strain of A. veronii TH0426 was used to establish an in vitro biofilm model. The results show that the biofilm-forming abilities of A. veronii TH0426 were similar in different media, peaking under conditions of 20 &deg, C and pH 6. Further, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to compare the differential expression of A. veronii between the biofilm and planktonic cells. The results show alterations in 277 proteins, with 130 being upregulated and 147 downregulated. Pathway analysis and GO (Gene Ontology) annotations indicated that these proteins are mainly involved in metabolic pathways and the biosynthesis of secondary metabolites and antibiotics. These proteins are the main factors affecting the adaptability of A. veronii to its external environment. MRM (multiple reaction 27 monitoring) and qPCR (qPCR) were used to verify the differential proteins of the selected A. veronii. This is the first report on the biofilm and planktonic cells of A. veronii, thus contributing to studying the infection and pathogenesis of A. veronii.

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.

Interleukin-4 (IL-4), an immunomodulatory cytokine derived from activated T lymphocytes were shown to regulate Th2-type immune responses. It plays an important role in anti-parasitic infections. In this study, a recombinant plasmid was designed using murine IL-4 co-expressed with pgsA anchor system of Lactobacillus plantarum NC8 and was delivered by live Lactobacillus plantarum NC8, which exhibited an enhanced immunogenicity in protection of BALB/c mice from infection with Trichinella spiralis. The results showed that the levels of serum IgG1 and mucosal secretory IgA (sIgA) were both increased significantly in mice orally inoculated with NC8-pSIP409-pgsA-mIL-4, and the Th2 phenotype immune response was up-regulated. A 29.9 % reduction in adult worm burden at 7 days post-infection (dpi) and 83.3 % reduction in muscle larvae burden at 28 dpi were observed in immune-stimulated mice with NC8-pSIP409-pgsA-mIL-4. Moreover, weight loss and pathological changes were also improved in mice of NC8-pSIP409-pgsA-mIL-4 group. Taken together, it suggests that NC8-pSIP409-pgsA-mIL-4 could improve the intestinal mucosal immunity and promoted the elimination of the adult worm in Trichinella-infected mice. This study laid the foundation for the development of a novel vaccines against Trichinellosis.

Additional file 1: Figure S1. Confirmation of the success and genetic stability of mutant strain ∆dotU and complemented strain C-dotU. (a) PCR detection of the mutant strain ∆dotU, M: DL5000 Marker; 1: the mutant strain ∆dotU; 2: wild type TH0426. (b) PCR detection of the complemented strain C-dotU, M: DL2000 Marker; 1: C-dotU; 2: TH0426. (c) Genetic stability of the partial deletion strain ∆dotU, M: DL5000 Marker; 1–10: ∆dotU; 11–12: TH0426; 13: control group. (d) Genetic stability of the partial complemented strain C-dotU, M: DL2000 Marker; 1–10: C-dotU; 11–12: TH0426; 13: control group. Figure S2. Flagellum straining and light microscopy observation of the three strains (the wild-type, ∆dotU and C-dotU). Light microscopy images of parental strain (A), ∆dotU (B) and C-dotU (C). Magnifications, 1000 × (A, B and C).

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. This bacterium has caused serious economic losses in the aquaculture industry worldwide, and it has become an important zoonotic and aquatic agent. However, little is known about the molecular mechanism of pathogenesis of A. veronii. In this study, we first constructed an unmarked mutant strain (&Delta, preA) by generating an in-frame deletion of the preA gene, which encodes a periplasmic binding protein, to investigate its role in A. veronii TH0426. Our results showed that the motility and biofilm formation ability of &Delta, preA were similar to those of the wild-type strain. However, the adhesion and invasion ability in epithelioma papulosum cyprini (EPC) cells were significantly enhanced (2.0-fold). Furthermore, the median lethal dose (LD50) of &Delta, preA was 7.6-fold higher than that of the wild-type strain, which illustrates that the virulence of the mutant was significantly enhanced. This finding is also supported by the cytotoxicity test results, which showed that the toxicity of &Delta, preA to EPC cells was enhanced 1.3-fold relative to the wild type. Conversely, tolerance test results showed that oxidative stress resistance of &Delta, preA decreased 5.9-fold compared to with the wild-type strain. The results suggest that preA may negatively regulate the virulence of A. veronii TH0426 through the regulation of resistance to oxidative stress. These insights will help to further elucidate the function of preA and understand the pathogenesis of A. veronii.

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1&beta, TNF-&alpha, and IFN-&gamma, in the tissue of fish immunized with recombinant L. casei was significantly (p &lt, 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. This bacterium has caused serious economic losses in the aquaculture industry worldwide, and it has become an important zoonotic and aquatic agent. However, little is known about the molecular mechanism of pathogenesis of A. veronii. In this study, we first constructed an unmarked mutant strain (ΔpreA) by generating an in-frame deletion of the preA gene, which encodes a periplasmic binding protein, to investigate its role in A. veronii TH0426. Our results showed that the motility and biofilm formation ability of ΔpreA were similar to those of the wild-type strain. However, the adhesion and invasion ability in epithelioma papulosum cyprini (EPC) cells were significantly enhanced (2.0-fold). Furthermore, the median lethal dose (LD50) of ΔpreA was 7.6-fold higher than that of the wild-type strain, which illustrates that the virulence of the mutant was significantly enhanced. This finding is also supported by the cytotoxicity test results, which showed that the toxicity of ΔpreA to EPC cells was enhanced 1.3-fold relative to the wild type. Conversely, tolerance test results showed that oxidative stress resistance of ΔpreA decreased 5.9-fold compared to with the wild-type strain. The results suggest that preA may negatively regulate the virulence of A. veronii TH0426 through the regulation of resistance to oxidative stress. These insights will help to further elucidate the function of preA and understand the pathogenesis of A. veronii.

Aeromonas veronii is an emerging aquatic pathogen causing hemorrhagic septicemia in humans and animals. Probiotic is an effective strategy for controlling enteric infections through reducing intestinal colonization by pathogens. Here we report that the consumption of Bacillus velezensis regulated the intestinal innate immune response and decreased the degree of intestinal inflammation damage caused by the A. veronii in Crucian carp. In this study, we isolated four strains of B. velezensis, named C-11, S-22, L-17 and S-14 from apparently healthy Crucian carp, which exerted a broad-spectrum antimicrobial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens. B. velezensis isolates showed typical Bacillus characteristics by endospore staining, physiological and biochemical test, enzyme activity analysis (amylase, protease, and lipase), and molecular identification. Here, Bacillus-containing dietary was orally administrated to Crucian carp for 8 weeks before A. veronii challenge. Immunological parameters and the expression of immune-related genes were measured at 2, 4, 6, 8, and 10 weeks post-administration. The results showed that B. velezensis was found to promote the increase in the phagocytic activities of peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs), as well as the increase in interleukin 1β (IL-1β), IL-10 and tumor necrosis factor α (TNF-α) concentration of serum. Lysozyme levels (113.76 U/mL), ACP activity (25.32 U/mL), AKP activity (130.08 U/mL), and SOD activity (240.63 U/mL) were maximum (P < 0.05) in the B. velezensis C-11 treated group at 8 week. Our results showed that Crucian carp fed with the diet containing B. velezensis C-11 and S-22 developed a strong immune response with significantly higher (P < 0.05) levels of IgM in samples of serum, mucus of skin and intestine compared to B. velezensis L-17 and S-14 groups. Moreover, B. velezensis spores appeared to show no toxicity and damage in fish, which could inhabit the gut of Crucian carp. B. velezensis restrained up-regulation of pro-inflammation cytokines (IL-1β, IFN-γ, and TNF-α) mRNA levels in the intestine and head kidney at final stage of administration, and the expression of IL-10 was increased throughout the 10-week trial. A. veronii infection increased the population of inflammatory cells in the intestinal villi in the controls. In contrast, numerous goblet cells and few inflammatory cells infiltrated the mucosa in the B. velezensis groups after challenge with A. veronii. Compared with A. veronii group, B. velezensis could safeguard the integrity of intestinal villi. The highest post-challenge survival rate (75.0%) was recorded in B. velezensis C-11 group. The present data suggest that probiotic B. velezensis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.IMPORTANCEIn this work, four Bacillus velezensis strains isolated from apparently healthy Crucian carp, which exhibited a broad-spectrum antibacterial activity especially the fish pathogens. Administration of B. velezensis induced the enhancement of the intestinal innate immune response through reducing intestinal colonization by pathogens. The isolation and characterization would help better understand probiotic can be recognized as an alternative of antimicrobial drugs protecting human and animal health.

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-1β, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P

Salmonella Enteritidis is an important foodborne pathogen that can infect a wide range of animal species including human beings, resulting in great losses to commercial husbandry and human health. CirA is an outer membrane receptor involved in iron uptake and colicin1A/B-mediated competitive killing. Although iron uptake is crucial to bacterial virulence, limited literature is available about the role of CirA in infection. In the present work, we aimed to evaluate the role of CirA during S. Enteritidis infection. For this purpose, we generated a CirA-deficient mutant of the S. Enteritidis strain C50336 and examined its biological characteristics. The results showed that cirA gene inactivation caused sharply decreased biofilm formation and apparently impaired antibiotic resistance. Furthermore, the cirA gene deletion mutant showed markedly reduced adhesion and invasion to human epithelial cell line Caco-2 cells and decreased proliferation in mouse macrophage cell line RAW264.7 cells. Moreover, attenuated virulence was determined by a mouse model, with an LD50 increase of approximately 1,000-fold. These data indicated that CirA plays critical roles in the S. Enteritidis infection process.

Minicircle DNA (mcDNA), which contains only the necessary components for eukaryotic expression and is thus smaller than traditional plasmids, has been designed for application in genetic manipulation. In this study, we constructed a novel plasmid containing both the Cre recombinase under the phosphoglycerate kinase (PGK) promoter and recombinant lox66 and lox71 sites located outside the cytomegalovirus (CMV) expression cassette. The strictly controlled synthesis of Cre recombinase in vivo maintained the complete form of the plasmid in vitro, whereas the in vivo production of Cre transformed the parental plasmid to mcDNA after transfection. The newly designed Cre recombinase-mediated in vivomcDNA platform, named CRIM, significantly increased the nuclear entry of mcDNA, followed by increased production of mRNA and protein, using enhanced green fluorescent protein (EGFP) as a model. Similar results were also observed in chickens when the vaccine was delivered by the regulated-delayed-lysis Salmonella strain χ11218, where significantly increased production of EGFP was observed in chicken livers. Then, we used the HN gene of genotype VII Newcastle disease virus as an antigen model to construct the traditional plasmid pYL43 and the novel mcDNA plasmid pYL47. After immunization, our CRIM vaccine provided significantly increased protection against challenge compared with that of the traditional plasmid, providing us with a novel mcDNA vaccine platform. IMPORTANCE Minicircle DNA (mcDNA) has been considered an attractive alternative to DNA vaccines; however, the relatively high cost and complicated process of purifying mcDNA dramatically restricts the application of mcDNA in the veterinary field. We designed a novel in vivo mcDNA platform in which the complete plasmid could spontaneously transform into mcDNA in vivo. In combination with the regulated-delayed-lysis Salmonella strain, the newly designed mcDNA vaccine provides us with an elegant platform for veterinary vaccine development.

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.

Lactobacillus reuteri, a typical intestinal symbiotic bacterium, plays an important role in maintaining intestinal flora stability and host health. However, the effect of Lactobacillus reuteri on peritoneal macrophages has not been thoroughly studied. Our study indicated that Lactobacillus reuteri could activate macrophages and that macrophages treated with Lactobacillus reuteri have an enhanced ability to phagocytose and to kill intracellular Salmonella typhimurium. Lactobacillus reuteri may reduce the inflammatory response caused by Salmonella typhimurium by regulating NO, thus effectively protecting mice against Salmonella typhimurium invasion and dissemination to the liver and spleen. Taken together, these data demonstrated the protective effect of Lactobacillus reuteri on macrophages and mice challenged with Salmonella typhimurium through in vitro and in vivo experiments.

Aeromonas veronii is an important aquatic zoonotic pathogen in humans and animals. In recent years, extracellular proteins from bacteria have been found to be the major pathogenic factors for aquatic animals. The aim of this study was to systematically analyze the extracellular proteins of nine sources of A. veronii and the effects of hisJ on virulence. We screened only the common proteins from nine different sources of A. veronii by liquid chromatography-tandem mass spectrometry and identified the gene hisJ. We then constructed ΔhisJ (deleted) and C-hisJ (complemented) variants of A. veronii TH0426 to assess the biological function of hisJ. While the ΔhisJ strain did not show altered growth (P > 0.05), we observed that it had reduced colony formation and biofilm formation and reduced adhesion to and invasion of epithelioma papulosum cyprini cells by 2.0-, 1.9-, and 10.8-fold, respectively. Additionally, infection experiments on zebrafish and mouse infection experiments showed that the virulence of the ΔhisJ strain was decreased by 865-fold (P < 0.001) compared with the wild-type strain; virulence of the complemented C-hisJ strain was reduced only 2.8-fold. Furthermore, in the context of hisJ deletion, flagella of A. veronii TH0426 were easily detached and the expression of virulence genes was downregulated. A persistence test (of bacterial colonies in crucian carp) showed that the number of bacteria in the immune organs of the ΔhisJ-infected group was lower than that in the wild-type-infected group. Overall, these results show that hisJ affects flagellar shedding, virulence, biofilm formation, adhesion, and invasion of A. veronii TH0426, and that hisJ is closely associated with virulence and plays a crucial role in its pathogenicity of A. veronii TH0426.

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.

Eimeria tenella (E. tenella) has caused severe economic loss in chicken production, especially after the forbidden use of antibiotics in feed. Considering the drug resistant problem caused by misuse of chemoprophylaxis and live oocyst vaccines can affect the productivity of chickens, also it has the risk to reversion of virulence, the development of efficacious, convenient and safe vaccines is still deeply needed. In this study, the EtMic2 protein of E. tenella was anchored on the surface of Lactobacillus plantarum (L. plantarum) NC8 strain. The newly constructed strain was then used to immunize chickens, followed by E. tenella challenge. The results demonstrated that the recombinant strain could provide efficient protection against E. tenella, shown by increased relative body weight gains, percentages of CD4+ and CD8+ T cells, humoral immune response and inflammatory cytokines. In addition, decreased cecum lesion scores and fecal oocyst shedding were also observed during the experiment. In conclusion, this study proves the possibility to use L. plantarum as a vessel to deliver protective antigen to protect chickens against coccidiosis.

Minicircle DNA (mcDNA), which contains only the necessary components for eukaryotic expression and is thus smaller than traditional plasmids, has been designed for application in genetic manipulation. In this study, we constructed a novel plasmid containing both the Cre recombinase under the phosphoglycerate kinase (PGK) promoter and recombinant lox66 and lox71 sites located outside the cytomegalovirus (CMV) expression cassette. The strictly controlled synthesis of Cre recombinase in vivo maintained the complete form of the plasmid in vitro, whereas the in vivo production of Cre transformed the parental plasmid to mcDNA after transfection. The newly designed Cre recombinase-mediated in vivo mcDNA platform, named CRIM, significantly increased the nuclear entry of mcDNA, followed by increased production of mRNA and protein, using enhanced green fluorescent protein (EGFP) as a model. Similar results were also observed in chickens when the vaccine was delivered by the regulated-delayed-lysis Salmonella strain χ11218, where significantly increased production of EGFP was observed in chicken livers. Then, we used the HN gene of genotype VII Newcastle disease virus as an antigen model to construct the traditional plasmid pYL43 and the novel mcDNA plasmid pYL47. After immunization, our CRIM vaccine provided significantly increased protection against challenge compared with that of the traditional plasmid, providing us with a novel mcDNA vaccine platform. IMPORTANCE Minicircle DNA (mcDNA) has been considered an attractive alternative to DNA vaccines; however, the relatively high cost and complicated process of purifying mcDNA dramatically restricts the application of mcDNA in the veterinary field. We designed a novel in vivo mcDNA platform in which the complete plasmid could spontaneously transform into mcDNA in vivo. In combination with the regulated-delayed-lysis Salmonella strain, the newly designed mcDNA vaccine provides us with an elegant platform for veterinary vaccine development.

Trichinellosis is a zoonotic parasitic disease caused by Trichinella spiralis, and it is also a widely prevalent foodborne parasitic disease. At present, albendazole and benzimidazole are the most commonly used therapeutic drugs for the clinical treatment of trichinellosis, but they have many side effects. Sanguinarine is a benzophenanthridine alkaloid that has biological activity, such as antibacterial, antitumour and antiparasitic activities. Therefore, the present study aimed to evaluate the anti-Trichinella effect of sanguinarine in vivo and in vitro. The results showed that sanguinarine had a lethal effect on muscle larvae, adults and new-borne larvae in vitro. The damage to adults treated with sanguinarine was observed by scanning electron microscopy. Sanguinarine could significantly reduce the burden of worms in mice during the pre-adult, migrating larva and encysted larva stages. The ratio of intestinal villus to crypt (V/C) in mice treated with sanguinarine was significantly higher than that in non-treated control mice. Compared with the non-treated control group, the sanguinarine-treated group exhibited a significantly increased number of small intestine goblet cells. The level of reactive oxygen species (ROS) in the serum of mice treated with sanguinarine was significantly higher than that of the control group mice in the pre-adult and encysted larva stages. This study suggests that sanguinarine is a potential drug against trichinellosis.

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. More and more cases have shown that it has become an important zoonotic and aquatic agent. In this study, a A. veronii TH0426 mutant strain (ΔlamB) with an in-frame deletion removed nucleotides 10-1,296 of the lamB gene was firstly constructed to investigate its functions. The results showed that the LD50 value of the mutant ΔlamB to zebrafish and mice was 13.7-fold and 5.6-fold higher than those of the wild-type strain, respectively. The toxicity of wild-type strain to EPC cells was 2.1-fold and threefold higher than those of ∆lamB when infected for 1 and 2 hr. Furthermore, the ability of biofilm formation and the adhesion and invasion to EPC cells of ∆lamB significantly decreased for 5.6-fold and 1.8-fold separately. In addition, motility detection result indicated that ∆lamB lost the swimming ability. The results of flagellar staining and TEM demonstrated that the flagella of ∆lamB were shed. In general, the deletion of lamB gene caused a significant decrease in the virulence and adhesion of A. veronii TH0426, and it can be known that the lamB gene of A. veronii plays a crucial role in the pathogenesis.

Genomes of 24 sequenced Bacillus velezensis strains were characterized to identity shared and unique genes of lignocellulolytic enzymes and predict potential to degrade lignocellulose. All 24 strains had genes that encoded lignocellulolytic enzymes, with potential to degrade cellulose and hemicelluloses. Several lignocellulosic genes related to cellulose degradation were universally present, including one GH5 (endo-1,4-β-glucanase), one GH30 (glucan endo-1,6-β-glucosidase), two GH4 (6-phospho-β-glucosidase, 6-phospho-α-glucosidase), one GH1 (6-phospho-β-galactosidase), one GH16 (β-glucanase) and three GH32 (two sucrose-6-phosphate hydrolase and levanase). However, in the absence of gene(s) for cellobiohydrolase, it was predicted that none of the 24 strains would be able to directly hydrolyse cellulose. Regarding genes for hemicellulose degradation, four GH43 (1,4-β-xylosidase; except strain 9912D), one GH11 (endo-1,4-β-xylanase), three GH43 (two arabinan endo-1,5-α-L-arabinosidase and one arabinoxylan arabinofuranohydrolase), two GH51 (α-N-arabinofuranosidase), one GH30 (glucuronoxylanase), one GH26 (β-mannosidase) and one GH53 (arabinogalactan endo-1,4-β-galactosidase) were present. In addition, two PL1 (pectate lyase) and one PL9 (pectate lyase) with potential for pectin degradation were conserved among all 24 strains. In addition, all 24 Bacillus velezensis had limited representation of the auxiliary activities super-family, consistent with a limited ability to degrade lignin. Therefore, it was predicted that for these bacteria to degrade lignin, pretreatment of lignocellulosic substrates may be required. Finally, based on in silico studies, we inferred that Bacillus velezensis strains may degrade a range of polysaccharides in lignocellulosic biomasses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-018-1270-7) contains supplementary material, which is available to authorized users.

Highlights • The S protein of PDEV was displayed on the surface of a recombinant Lactobacillus plantarum NC8 strain. • NC8-pSIP409-pgsA'-S-DCpep promoted DC activation in the LP. • IgG and sIgA were significant increased in mice orally administered with the NC8-pSIP409-pgsA'-S-DCpep. • The expression of specificity cytokines IFN-γ, IL-4 and IL-17A of MLNs was enhanced in mouse treated with the NC8-pSIP409-pgsA'-S-DCpep. • NC8-pSIP409-pgsA'-S-DCpep might mediate B cell response in mouse., Porcine epidemic diarrhea virus (PEDV) is one of the most important causative pathogens of swine diarrhea, which is widely prevalent throughout the world and is responsible for significant economic losses in the commercial pig industry, both domestic and abroad. The spike (S) protein in the PEDV capsid structure can carry the major B lymphocyte epitope, which induces production of neutralizing antibodies and provides immunoprotective effects. Moreover, the conserved region encoded by the S gene can be considered a target for establishing a new diagnostic method and is a new candidate for vaccine design. In this study, use of anchorin pgsA' allowed the fusion protein of S-DCpep to express on the surface of recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA'-S-DCpep) NC8 strain. Mice were immunized by lavage administration of the recombinant NC8-pSIP409-pgsA'-S-DCpep, which was observed to induce DC activation and high production of sIgA and IgG antibodies in experimental animals, while also eliciting production of significantly more IgA+B220+ B cells. More importantly, secretion of cytokines IFN-γ, IL-4 and IL-17 in mice that were vaccinated with NC8-pSIP409-pgsA'-S-DCpep was remarkably increased. The results of our study suggest that NC8-pSIP409-pgsA'-S-DCpep potently triggers cellular and humoral immune responses. The obtained experimental results can provide a theoretical basis that lays the foundation for production of a novel oral vaccine against PED.

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-β, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora.

Myeloperoxidase (MPO) is a ferrous lysosomal protein with many immune functions that belongs to the heme peroxidase enzyme. In this study, the functions of MPO in the northern snakehead (Channa argus) were investigated by cloning an MPO cDNA sequence with a full length of 3181 bp. Homology analysis showed that northern snakehead MPO gene had the highest (81%) homology with mandarin fish (Siniperca chuatsi). In healthy northern snakehead, the MPO gene was expressed in the head-kidney, kidney, heart, gill, spleen, liver, and muscles but not midgut. After the northern snakehead was infected with Aeromonas veronii, the MPO gene expression varied in different tissues with low level in spleen, liver, gill and muscle, fluctuated in kidney and head-kidney and showed high level in heart. The result indicated that MPO might play an important role in the antimicrobial immune response of the northern snakehead.

Aeromonas veronii is an opportunistic pathogen that is capable of infecting both aquatic livestock and mammals. Natural infection in fishes results in irreparable damage to the aquaculture industry. In this study, we sought to investigate whether recombinant Lactobacillus casei expressing the outer membrane protein W (OmpW) of A.veronii could elicit protective immunity against A.veronii infections. We generated two recombinant Lactobacillus casei (L.casei) strains expressing the OmpW of A.veronii (surface-displayed or secreted) and evaluated the effect on immune responses in a fish model. A 600-bp gene fragment was subcloned into the L.casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secreted). Expression of the recombinant OmpW protein was also confirmed by Western blot and immunofluorescence assays. Common carp immunized with Lc-pPG-1- OmpW and Lc-pPG-2- OmpW via oral administration elicited high serum specific antibody titers and high LZM, ACP, and SOD activities. High levels of the IL-10, IL-β, IFN-γ, and TNF-α genes in different organs indicated that the inflammatory response and cell immune response were triggered. Additionally, when immunized fish were challenged with A.veronii, Lc-pPG1-OmpW and Lc-pPG2-OmpW demonstrated 40% and 50% protective efficacy. These data indicate that the combination of OmpW delivery and the lactic acid bacteria (LAB) approach may be a promising mucosal therapeutic strategy for treatment of A.veronii.