Background: Salmonella enteritidis (S. enteritidis), a zoonotic pathogen with a broad host range, presents a substantial threat to global public health safety. Vaccination stands as an effective strategy for the prevention and control of S. enteritidis infection, highlighting an immediate clinical need for the creation of safe and efficient attenuated live vaccines.Methods: In this study, a S. enteritidis peptidoglycan-associated lipoprotein (pal) gene deletion strain (Δpal), was constructed. To assess its virulence, we conducted experiments on biofilm formation capability, motility, as well as cell and mouse infection. Subsequently, we evaluated the immune-protective effect of Δpal.Results: It was discovered that deletion of the pal gene reduced the biofilm formation capability and motility of S. enteritidis. Cell infection experiments revealed that the Δpal strain exhibited significantly decreased abilities in invasion, adhesion, and intracellular survival, with downregulation of virulence gene expression, including mgtC, invH, spvB, sipA, sipB, ssaV, csgA, and pipB. Mouse infection experiments showed that the LD₅₀ of Δpal increased by 10⁴ times, and its colonization ability in mouse tissue organs was significantly reduced. The results indicated that the pal gene severely affected the virulence of S. enteritidis. Further, immunogenicity evaluation of Δpal showed a significant enhancement in the lymphocyte transformation proliferation capability of immunized mice, producing high titers of specific IgG and IgA, suggesting that Δpal possesses good immunogenicity. Challenge protection tests demonstrated that the strain could provide 100% immune protection against wild-type strains in mice.Discussion: This study proves that the pal gene influences the virulence of S. enteritidis, and Δpal could serve as a candidate strain for attenuated live vaccines, laying the foundation for the development of attenuated live vaccines against Salmonella.

Introdction: Aeromonas veronii is a significant pathogen to various aquatic life. Infections in fish can lead to high mortality rates, causing substantial economic losses in aquaculture. Vaccination is proposed as a substitute for antibiotics in aquaculture to decrease disease-related mortality and morbidity. Our study previously constructed a hisJ-deleted strain of A. veronii, which provided protective effect to Loach.Methods: To further assess the vaccine’s applicability, this study evaluated its genetic stability and safety, and the immune protective effects in Carassius auratus through four distinct administration routes: intraperitoneal injection, intramuscular injection, oral administration, and immersion, to determine the efficacy of these administration routes.Results: The results showed that the vaccine remained genetically stable after 45 generations. Immunization via these administration routes was safe for Carassius auratus, with intraperitoneal and intramuscular injections causing stronger adverse reactions. Immersion immunization resulted in mild adverse reactions, and no significant adverse reactions were observed following oral immunization. Immunizing Carassius auratus at safe concentrations via these routes enhanced the phagocytic activity in serum, increased the levels of non-specific immune-related enzymes (ACP, AKP, C3, C4, LZM, SOD, and IgM), and improved specific serum antibody levels. It also elevated levels of cytokines related to inflammatory responses (IL-1β, IL-10, TNF-α, TGF-β) in organ tissues (liver, spleen, kidney, mid-post intestine, and gills). The survival rates of Carassius auratus were measured after challenging with the virulent strain A. veronii TH0426, resulting in the relative survival rates of 64% for Intraperitoneal vaccine group, 56% for Intramuscular vaccine group, 52% for oral vaccine group, and 48% for immersion vaccine group. Analysis of bacterial load in the liver, spleen, and kidney post-challenge showed a decreasing trend in the control group, indicating that the vaccine strain ΔhisJ could gradually restrict the rapid proliferation of bacteria in these tissues, thereby providing a certain level of immune protection against A. veronii.Discussion: In brief, the vaccine strain ΔhisJ can serve as a safe live attenuated vaccine for Carassius auratus, and this study lays the foundation for the development of live attenuated vaccines against Aeromonas veronii.

Elizabethkingia miricola is an emerging nosocomial pathogen responsible for meningitis, sepsis, urinary tract infection, pneumonia, and joint infection in humans. These pathogens were also reported to be causal agents for meningitis‐like disease in cultured frogs, which displayed high infectivity, mortality, and significant loss. In July 2023, 10 outbreaks of infectious meningitis‐like disease in bullfrogs occurred in Tangshan area. To determine the causal agent, 70 diseased frogs from 10 farms were collected for etiological identification. Gram‐negative bacilli were isolated from the brain and liver of sick bullfrogs and identified as members of E. miricola by biochemical characterization and 16S rRNA sequencing analysis. A total of 42 strains of E. miricola were isolated and further determined as the etiological agent by reproducing neurological symptoms and deaths in an artificial infection test. A representative isolate, HBTS‐1, was picked up for the pathogenicity test, and the data showed that this stain was highly pathogenic to bullfrogs with an LD₅₀ of 3.7 × 10⁵ CFU. Notably, the isolate also showed high pathogenicity to 5‐day‐old suckling mice, with an LD₅₀ of 3.1 × 10⁶ CFU, indicating its potential threat to mammals. Moreover, all the 42 E. miricola isolates showed resistance to multiple antibotics without an apparent inhibition zone observed in the test, making the choice of antimicrobial therapy challenging. These novel findings prioritized E. miricola as an important zoonotic agent, which may provide a reference for human medicine.

Background: Brucellosis is a zoonotic bacterial disease primarily controlled through quarantine, culling, and vaccination. Live attenuated vaccines remain the most effective countermeasure, yet their application is limited by residual virulence and diagnostic interference. This study developed three rough-type attenuated Brucella melitensis mutants (G7, G8, G16) and evaluated their potential as DIVA (Differentiating Infected from Vaccinated Animals) vaccine candidates. Methods: Rough phenotypes were characterized through heat agglutination, acridine orange staining, and immunoblotting. Macrophage cytotoxicity was assessed via LDH release assays, while RT-qPCR analyzed macrophage activation capacity. Mouse infection and immunization-challenge experiments, complemented by histopathology, evaluated residual virulence and protective immunity. Antibody profiles were determined by ELISA, and DIVA capability was verified using LPS-coated ELISA. Results: G7 and G8 exhibited complete rough phenotypes, whereas G16 retained partial O-antigen (semi-rough). All rough mutants induced macrophage cytotoxicity and activation. The strains showed attenuated virulence with no viable bacteria recovered from spleens at 4 weeks post-inoculation. Histopathology revealed no liver lesions at 6 weeks post-inoculation. Immunized mice predominantly produced IgG2a-dominated Th1-type responses. The immune protection levels of G7 and G16 matched the reference vaccine M5–90Δ26, while G8 showed slightly lower efficacy. LPS-ELISA effectively differentiated vaccinated from infected animals via concurrent IgM/IgG detection. Conclusions: This study demonstrates that the rough-type B. melitensis mutants G7 and G16 serve as promising DIVA vaccine candidates, offering strong protection with low residual virulence while enabling serological differentiation between vaccinated and infected animals, highlighting their potential as effective vaccines for brucellosis control.

Background: Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain’s major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model. Results: Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD₅₀ values of C50336 and ΔcheV were 6.3 × 10⁵ CFU and 1.25 × 10⁷ CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection. Conclusion: All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.