Investigation of two different PACAP-38 (Pituitary Adenylate Cyclase Activating Polypeptide) formulated feeds on Atlantic salmon (Salmo salar) immune responses with Enteric Red Mouth disease (Yersinia ruckeri)

• ELSA detection of antigen-specific IgT and IgM levels revealed that infected fish receiving both non-amidated and amidated PACAP had significantly lower IgT levels in their serum compared to those on a placebo diet. • Additionally, a significant difference was noted between the amidated and non-amidated groups within the non-infected category after quantifying IL-1β serum protein concentrations at 1 day post-infection (dpi). • Notably, in the gut, there were marked differences in il-1β expression among the three diets at both 1 dpi and 3 dpi. Furthermore, in infected fish, il-10 expression showed significant downregulation in the gut tissue at 1 dpi in the non-amidated PACAP group. • When comparing the expression of hepcidin between infected and non-infected groups in the spleen and head kidney at 1 dpi, significant upregulation was observed in the infected groups for both amidated and non-amidated PACAP in the spleen. Moreover, the spleen demonstrated a significant increase in tgf-β expression following infection with Yersinia ruckeri in the amidated PACAP group. • The amidated PACAP-38 feed elicited greater immunological responses in all three tissues at all time points and resulted in decreased mortality rates. In salmonids, as in other teleosts, Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) has been demonstrated to have anti-inflammatory activity, anti-oxidative stress, and cytoprotective impacts against several aquatic pathogens, including Yersinia ruckeri , which is the causative agent of Enteric Red Mouth (ERM) disease. This study aimed to evaluate the impacts of feed formulated with two different forms of PACAP-38 on the immunological response of Atlantic salmon ( Salmo salar ) to infection with Y. ruckeri . Atlantic salmon ( n = 318, 45±5.6 g) were randomly assigned to three replicate tanks per diet, with a commercial base diet (Placebo), an amidated form of PACAP added to the commercial diet, and a non-amidated form of PACAP added to the commercial diet. After 28 days of feeding these diets, two tanks per diet underwent bath exposure (80 L tank for 60 min with 72 fish; water temperature was 16.6 ± 0.2 °C) to Y. ruckeri (isolate U27451–11; at 7.8 × 10 8 CFU/ml) and one tank per diet was unexposed. Fish (five fish per tank) were sampled prior to exposure, 1 day, 3 days, 7 days, and 20 days post-infection (dpi). Infected salmon that received amidated PACAP feed showed a significantly higher survival rate ( P = 0.006 ) than the Placebo group. Bacteriological samples collected from the kidney and intestine identified Y. ruckeri as the cause of death. Acute mortality coincided with strong inflammatory responses that were variable across fish and groups. Furthermore, PACAP diets induced some inflammatory gene expression in the head kidney, intestine and spleen, mostly at 3 and 7 dpi ( il10 and hepc ). These data suggest that both amidated and non-amidated PACAP had induced il1ß, hepc and il10 , and the timing suggests protection against infection and stress, possibly by inhibiting inflammatory pathways and reducing oxidative stress. Due to the anti-inflammatory effects of il10 and its pleiotropic nature, gene expression results in response to PACAP feeds may suggest a more complex regulation of inflammation in the PACAP-fed fish. Further, mmp9 gene expression and assessment of IgT and IgM in the serum were observed to be stimulated earlier and maintained longer in the amidated diet, contributing to the resolution of infection and reduction of mortality.

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