Assessment of the Live Attenuated and Wild-Type Edwardsiella ictaluri-Induced Immune Gene Expression and Langerhans-Like Cell Profiles in the Immune-Related Organs of Catfish

Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines

Phagocytic and Bactericidal Properties of Channel Catfish Peritoneal Macrophages Exposed to Edwardsiella ictaluri Live Attenuated Vaccine and Wild-Type Strains

Edwardsiella ictaluri (E. ictaluri), a Gram-negative, intracellular, facultative bacterium, is the causative agent of enteric septicemia of catfish (ESC), which is one of the most significant diseases of farmed channel catfish. Macrophages have a critical role in major defense mechanisms against bacterial infections by migrating to the site of infection, engulfing and killing pathogens, and priming adaptive immune responses. Vaccination of catfish with E. ictaluri live attenuated vaccine (LAV) strains increased the efficiency of phagocytosis and bacterial killing in catfish peritoneal macrophages compared in vitro with macrophages from non-vaccinated fish. Recently, our group developed several protective LAV strains from E. ictaluri. However, their effects on the antigen uptake and bacterial killing in catfish macrophages have not been evaluated. In this study, we assessed the phagocytic and bactericidal activity of peritoneal macrophages in the uptake of E. ictaluri wild-type (WT) and two LAV strains. We found that phagocytosis of LAV strains was significantly higher compared to their WT counterpart in peritoneal macrophages. Moreover, the uptake of E. ictaluri opsonized with sera from vaccinated catfish was more efficient than when opsonized with sera from sham-vaccinated fish. Notably, catfish macrophages did not lose their phagocytic properties at 4°C, as described previously in mammalian and zebrafish models. Also, opsonization of E. ictaluri with inactivated sera from vaccinated and sham-vaccinated catfish decreased significantly phagocytic uptake of bacteria at 32°C, and virtually suppressed endocytosis at 4°C, suggesting the important role of complement-dependent mechanisms in catfish macrophage phagocytosis. In conclusion, our data on enhanced phagocytic capacity and effective killing ability in macrophages of vaccine strains suggested the LAVs’ advantage if processed and presented in the form of peptides to specific lymphocytes of an adaptive immune system and emphasize the importance of macrophage-mediated immunity against ESC. Furthermore, we showed the role of complement-dependent mechanisms in the phagocytic uptakes of E. ictaluri in catfish peritoneal macrophages at 4 and 32°C. Finally, LAV vaccine-induced bacterial phagocytosis and killing properties of peritoneal macrophages emphasized the importance of the innate immune responses in ESC