Abstract

Waterborne pathogens are a constant threat to fish. Fish mucosa are the point of entry for the majority of microbes and are key in mounting an effective immune response to block invasion, colonization and spread. In the search for tools to enhance immune responses we have evaluated nanostructured TNF alpha protein nanoparticles (IBs(TNF alpha)) as potential mucosal adjuvants. IBs(TNF alpha) are non-toxic, stable, protein-based biologically active nanomaterials which are cheap to produce in microbial cell factories. Here we test the in vivo uptake of IBs(TNF alpha) in zebrafish larvae by bath immersion and in adult zebrafish by oral gavage. We used flow cytometry, histology and confocal microscopy, to show IBs(TNF alpha) can be efficiently taken up by the intestinal mucosa. Fluorescently labelled nanoparticles were ingested by 5 dpf larvae and accumulated in the intestine. In time course studies in adult zebrafish we observed the nanoparticles could penetrate the intestinal epithelia, traverse the lamina propria and enter the muscle layer. Gene expression analysis of intestine and spleen shows that some immune-related genes were significantly modulated. Further, we show that intraperitoneal injected IBs(TNF alpha) could protect fish against a lethal infection of Mycobacterium marinum administered by intraperitoneal injection. IBs(TNF alpha) therefore can have positive effects on infection outcome and show potential to be developed as a mucosal adjuvant for aquaculture.