Interleukin-1 beta (IL-1ß) as adjuvant enhances the immune effects of Aeromonas veronii inactivated vaccine in largemouth bass (Micropterus salmoides).

Largemouth bass (Micropterus salmoides) has emerged as a significant economic fish species, with a rise in Aeromonas veronii infections in farming. However, research on adjuvants for vaccines against A. veronii in largemouth bass remains scarce. In present study, recombinant largemouth bass IL-1ß (LbIL-1ß) was expressed to explore its adjuvant effect on the A. veronii inactivated vaccine. Following vaccination with recombinant LbIL-1ß (rLbIL-1ß) and the inactivated A. veronii, higher serum SOD levels and lysozyme activities were observed in largemouth bass from inactivated A. veronii + rLbIL-1ß vaccinated group. Furthermore, it was discovered that rLbIL-1ß was able to boost the serum-specific antibody levels induced by the inactivated A. veronii. The qRT-PCR analysis revealed that rLbIL-1ß also enhanced the expression of IgM, CD4, and MHC II in largemouth bass triggered by the inactivated A. veronii. After challenged with live A. veronii, the outcomes demonstrated that the relative percentage survival (RPS) for largemouth bass resulting from the inactivated A. veronii in combination with rLbIL-1ß was 76.67 %, surpassing the RPS of 60 % in the inactivated A. veronii group. Collectively, these findings indicate that rLbIL-1ß enhances the protective effect of the A. veronii inactivated vaccine on largemouth bass, showcasing potential as an adjuvant for further development.

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

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

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.

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.

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge.

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.

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.

Oral Administration of Lactobacillus Casei Expressing Flagellin A Protein Confers Effective Protection against Aeromonas Veronii in Common Carp, Cyprinus Carpio.

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-1ß, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 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.

OmpW expressed by recombinant Lactobacillus casei elicits protective immunity against Aeromonas veronii in common carp.

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-ß, 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.

Characterization, evolution, and expression analysis of TLR7 gene subfamily members in Mastacembelus armatus (Synbranchiformes: Mastacembelidae).

TLR7 subfamily members are important pattern recognition receptors participating in the recognition of pathogen-associated molecular patterns. In this study, we successfully identified 3 members of TLR7 subfamily from the spiny eel Mastacembelus armatus (MaTLR7, MaTLR8 and MaTLR9). The amino acid sequence identities of MaTLR7 and MaTLR8 with Monopterus albus TLR7 were 87.2% and 76.5%, respectively and the identity of MaTLR9 with Seriola lalandi TLR9 was 74.7%. The phylogenetic analysis revealed MaTLRs showed close relationship to other species in Synbranchiformes or Perciformes. Quantitative real-time PCR analysis revealed that they were expressed in all tested tissues and higher expression was found in spleen or gill. After infection with Aeromonas veronii, expression of MaTLR7, MaTLR8 and MaTLR9 were all significantly downregulated in spleen and kidney. Evolutionary analysis suggested that the ancestral lineages of teleost TLR8 and TLR9 had been subject to positive selection pressures and multiple Maximum likelihood methods recovered 3 positively selected sites in teleost TLR7, 4 in TLR8 and 8 in TLR9. Domain distribution revealed most positively selected sites were located in leucine-rich repeat domain. Our results will contribute to better understanding the antibacterial mechanism of TLRs and their co-evolution with pathogens.

Efficacy of synbiotic Jerusalem artichoke and Lactobacillus rhamnosus GG-supplemented diets on growth performance, serum biochemical parameters, intestinal morphology, immune parameters and protection against Aeromonas veronii in juvenile red tilapia (Oreochromis spp.).

Synbiotics, a synergistic combination of probiotics and prebiotics, are currently regarded as one of the most practical nutritional supplements in tilapia farms. In this study, the effect of supplementing the diet of red tilapia (Oreochromis spp.) with Jerusalem artichoke (Helianthus tuberosus) and Lactobacillus rhamnosus GG (LGG) was evaluated. Growth performance, serum biochemical parameters, intestinal morphology, goblet cell counts, immune parameters and protection against Aeromonas veronii challenge were determined. The results showed that fish fed with synbiotic-supplemented diets had a significantly higher (P < 0.05) feed conversion ratio (FCR), specific growth rate (SGR), and average daily gain (ADG) than fish fed with a control diet. The synbiotic-supplemented diet increased glucose, total protein and the total cholesterol levels. The absorptive area of the proximal and distal intestine of fish fed on the synbiotic diet was significantly higher (P < 0.05) than in those fed with probiotics (LGG), prebiotic-supplemented diets (JA), and the control diet. Goblet cell counts revealed that the numbers of acid mucous cells, neutral mucous cells and double-staining mucous cells of fish fed the synbiotic-supplemented diet (JA + LGG) were significantly higher (P < 0.05) in the proximal and distal intestine. Fish fed the synbiotic-supplemented diets also exhibited significantly higher (P < 0.05) lysozyme activity. The cumulative mortalities of fish fed with a synbiotic-supplemented diet were significantly lower than those of fish fed other diets. The results suggested the beneficial effect of JA and LGG synbiotic diet on growth performance and health status of red tilapia. Direct administration of JA and LGG in fish feed can be used as a practical nutritional supplement in red tilapia.

Generation and immunity effect evaluation of biotechnology-derived Aeromonas veronii ghost by PhiX174 gene E-mediated inactivation in koi (Cyprinus carprio koi).

Aeromonas veronii is a conditional pathogen causing high mortality in many freshwater fish species worldwide. Bacterial ghosts are nonliving Gram-negative bacteria devoid of cytoplasmic contents, which induce protective immunity against microbial pathogens. The aims of this study were: a) to produce A. veronii ghost (AVG) constructed by PhiX174 gene E; b) to evaluate the specific, non-specific immune effects and protective immunity of AVG against A. veronii in koi. The lysis plasmid pBBR-E was constructed by cloning PhiX174 gene E into the broad-host-range vector pBBR1MCS2, and then transformed into A. veronii 7231. AVG was generated by increasing the incubation temperature up to 42 °C. Lysis of A. veronii occurred 3 h after temperature induction and completed in 12 h. The efficiency of ghost induction was 99.9998 ±â€¯0.0002%. Koi were immunized intraperitoneally with AVG, formalin-killed bacteria (FKC) or phosphate buffered saline (PBS) respectively, and then respiratory burst (RB), myeloperoxidase (MPO), lysozyme (LZM), malondialdehyde (MDA), complement 3 (C3) and antibody activities were examined in serum. Compared with negative control of PBS, the RB, MPO, LZM activities were significantly higher in koi immunized with AVG (P < 0.05). Nevertheless, the MDA activities of AVG treatment were significantly lower than those of PBS treatment (P < 0.05). The serum agglutination titers and IgM antibody titers in AVG group were significantly higher than those in FKC or PBS groups. After challenged with the parent strain A. veronii 7231, the average mortality of AVG group was significantly lower than that of FKC and PBS groups (P < 0.05) and the relative percent survival (RPS) of AVG group (73.92%) was higher than that of FKC group (43.48%). Therefore, AVG have the potential to induce protective immunity and they may be ideal vaccine candidates against A. veronii in koi.

Immunogenicity of extracellular products from an inactivated vaccine against Aeromonas veronii TH0426 in koi, Cyprinus carpio.

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-1ß, 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.

Oral immunization with recombinant Lactobacillus casei expressing OmpAI confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio.

Aeromonas veronii is a gram-negative pathogen capable of infecting both fish and mammals, including humans, and natural infection in fish results in irreparable damage to the aquaculture industry. Lactic acid bacteria (LAB) have a number of properties that make them attractive candidates as delivery vehicles for presentation to the mucosa sites of compounds with pharmaceutical interest, in particular vaccines. In this study, we generated two recombinant Lactobacillus casei (surface-displayed or secretory) expressing the OmpAI of A.veronii and evaluated the effect on immune responses in fish model. A 1022 bp gene fragment of the 42 kDa OmpAI antigen of A.veronii was cloned into pPG-1 (surface-displayed) and pPG-2 (secretory) and electrotransformed into Lactobacillus casei CC16. The recombinant plasmid in L.casei could be stably inherited over 50 generations, and production of OmpAI protein had slight limited effects on cells growth. 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-ß, IFN-γ, TNF-α 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. Further, viable recombinant L.casei strains were directly delivered and survive throughout the intestinal tract, the recombinant OmpAI was also detected in intestine mucosal. The results showed that common carp received Lc-pPG1-OmpAI (66.7%) and Lc-pPG2-OmpAI (50.0%) had higher survival rates compared with the controls after challenge with A.veronii, indicating that Lc-pPG1-OmpAI and Lc-pPG2-OmpAI had beneficial effects on immune response and enhanced disease resistance of common carp against A.veronii infection. Our study here demonstrates, for the first time, the ability of recombinant L.casei as oral vaccine against A.veronii infection in carps. The combination of OmpAI delivery and LAB approach may be a promising mucosal therapeutic agent for treating and controlling A.veronii.