Immunological effects of recombinant Lactobacillus casei expressing pilin MshB fused with cholera toxin B subunit adjuvant as an oral vaccine against Aeromonas veronii infection in crucian carp.

Aeromonas veronii is a zoonotic agent capable of infecting fish and mammals, including humans, posing a serious threat to the development of aquaculture and public health safety. Currently, few effective vaccines are available through convenient routes against A. veronii infection. Herein, we developed vaccine candidates by inserting MSH type VI pili B (MshB) from A. veronii as an antigen and cholera toxin B subunit (CTB) as a molecular adjuvant into Lactobacillus casei and evaluated their immunological effect as vaccines in a crucian carp (Carassius auratus) model. The results suggested that recombinant L. casei Lc-pPG-MshB and Lc-pPG-MshB-CTB can be stably inherited for more than 50 generations. Oral administration of recombinant L. casei vaccine candidates stimulated the production of high levels of serum-specific immunoglobulin M (IgM) and increased the activity of acid phosphatase (ACP), alkaline phosphatase (AKP) superoxide dismutase (SOD), lysozyme (LZM), complement 3 (C3) and C4 in crucian carp compared to the control group (Lc-pPG612 group and PBS group) without significant changes. Moreover, the expression levels of interleukin-10 (IL-10), interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß) genes in the gills, liver, spleen, kidney and gut of crucian carp orally immunized with recombinant L. casei were significantly upregulated compared to the control groups, indicating that recombinant L. casei induced a significant cellular immune response. In addition, viable recombinant L. casei can be detected and stably colonized in the intestine tract of crucian carp. Particularly, crucian carp immunized orally with Lc-pPG-MshB and Lc-pPG-MshB-CTB exhibited higher survival rates (48% for Lc-pPG-MshB and 60% for Lc-pPG-MshB-CTB) and significantly reduced loads of A. veronii in the major immune organs after A. veronii challenge. Our findings indicated that both recombinant L. casei strains provide favorable immune protection, with Lc-pPG-MshB-CTB in particular being more effective and promising as an ideal candidate for oral vaccination.

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

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

Great effect of porin(aha) in bacterial adhesion and virulence regulation in Aeromonas 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.