Boosting tilapia (Oreochromis niloticus) mucosal/systemic immune responses to Flavobacterium columnare using PACAP (pituitary adenylate cyclase activating polypeptide)

Antimicrobial peptides have been isolated from variety of organisms and play an essential role in defense against infections. They are small peptides (less than 60 amino acids) with broad spectrum antibacterial activity. In teleosts, pituitary adenylate cyclase-activating polypeptide (PACAP) has been demonstrated to have direct antimicrobial activity against several aquatic pathogens, including those from the genus Flavobacterium. Our goal was to examine the in vivo immunostimulatory capabilities of PACAP in tilapia and whether this was impacted by administration route. Over the course of two studies, tilapia (416.06+-116.66 g) randomly assigned to replicate tanks were administered either PACAP-38 or a modified form of PACAP38, via intraperitoneal (i.p.) injection, bath, nares flush, or gill flush, and compared to PBS controls. Following individual treatments, tilapia underwent a bath exposure (40 L tank for 45 min) to F. columnare (isolate ALG-00-530; at 2.1 x 108 CFU/ml) or sham exposure without the addition of the bacterial culture. Fish were sampled prior to exposure, and 48 h after stimulation, at 1 day after the onset of mortality in exposed tanks, and at resolution of mortality. Tilapia that received i.p. injection of PACAP-38 showed significantly lower mortality (10%) than those receiving PBS i.p. (25%) from F. columnare. However, bath immersion of fish both with and without F. columnare resulted in significant mortality due to secondary infections with Edwardsiella tarda. Modified PACAP-38 however, initiated greater mortality in nares/gill flushed or i.p. injected fish compared to sham/PBS. Furthermore, the same modified form of PACAP-38 also induced inflammatory gene expression in the spleen, and eoisinophilic granule cell aggregation in the nares, following flushing. These data suggest that the different forms of PACAP-38 induce different mucosal and systemic immune responses, which may result in differing clinical outcomes when exposed to F. columnare.