The in-house ELISA developed in the study seems suitable for monitoring the specific humoral response in tilapia against the columnaris disease. == 2. anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, assisting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after demanding fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation ofIgM,IgT,TNF , andIL1-genes in the spleen and kidney of vaccinated fish. Significant upregulation ofIgMandIgTgenes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish. Keywords:reddish tilapia, systemic immune response, columnaris disease, nano-immersion vaccine == 1. Intro == Flavobacterium columnare, a gram-negative, filamentous, thin pole bacterium, with or without yellow rhizoid colony formation, is a serious pathogenic bacterium causing columnaris KX1-004 disease in intensively farmed tilapias worldwide [1,2].Flavobacterium columnareinfections can lead to skin lesions, fin decay, and gill tissue damage, contributing to significant economic deficits and a high mortality rate [3,4]. The virulence ofF. columnareis shown by its ability to abide by mucosal surfaces, gliding motility, biofilm formation, and capsule production, which have been associated with its rhizoid morphotype [5]. The colonization of the bacterium to the mucosal surfaces of the fish (pores and skin and gills) is an important step in initiating the infection, disease severity, and progression, and the typical pathological KX1-004 characteristics associated with columnaris disease. Vaccination against columnaris disease has been trialed in a variety of fish species. However, only low or partial protection has been reported for columnaris vaccines given by injection or immersion Rabbit polyclonal to Caspase 7 using formalin-killed whole cell preparations in coho salmonOncorhynchus kisutch[6], channel catfishIctalurus punctatus[7,8], eels [9], carpCyprinus carpio[10], and tilapiaOreochromis niloticus[11,12]. Among the vaccination delivery routes used to administer vaccines to fish, immersion vaccination is considered to be the most suitable for delivering columnaris vaccines to the mucosal cells to confer a protecting mucosal immune response to protect fish against the disease. Nevertheless, this approach has been impeded by the fact that the effectiveness of antigen absorption by mucosal cells is limited and the potency of induction of protecting immune responses can be low and short in period. Our earlier study demonstrated the use of a biomimetic-mucoadhesive nanovaccine that allows better adsorption of antigens to the mucosal surfaces of fish [13,14]. Strong mucosal immunity was induced from the vaccine, inducing an immune cascade in the mucosal site and in the mucosal connected lymphoid cells (MALT) following immersion immunization [4]. However, the ability of this vaccine to activate a systemic humoral immune response has not yet been elucidated. The aim of the present study was to investigate the specific humoral immune response stimulated in tilapia from the biomimetic-mucoadhesive nanovaccine againstF. columnare,using an indirect-enzyme linked immunosorbent assay (ELISA) to measure serum antibody reactions, serum bactericidal activity (SBA), and the manifestation of immune-related genes within the head-kidney and spleen. The in-house ELISA developed in the study seems suitable for monitoring the specific humoral response in tilapia against the columnaris disease. == 2. Materials and Methods == The use of animals in experimentation for this study was KX1-004 officially authorized by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee of Faculty of Veterinary Technology, Chulalongkorn University or college (IBC1831052; IACUC1831020). All methods were carried out in accordance with university or college recommendations and regulations as well as plans governing biosafety methods. == 2.1. Fish and Experimental Conditions == Six hundred reddish tilapia (Oreochromissp.) with an average excess weight of 100 g, were acclimatized for 10 days and randomly placed in four 200-L fiberglass tanks (150 fish per tank) for the four treatments explained below. The tanks were maintained under continuous aeration at 2528 C, 5.86.8 ppm dissolved oxygen (DO), pH 7.58 and less than 0.1 mg/L of total ammonia throughout the experiment. Experimental fish were fed twice each KX1-004 day and water was changed up to 50% every second time. == 2.2. Bacterias and Vaccine Planning == Flavobacterium columnareisolate (F-K17/1, GenBank accession no.MW362353), found in our prior research, was selected predicated on its capability to form rhizoid colonies, its high virulence in clinical outbreaks and belonging.