The lack of commercial vaccines and treatments for many fish viral diseases remains a major bottleneck in aquaculture production. There is a continuing need to further develop antiviral prophylaxis strategies. The trial compared the effect and modes of action of different h eat-inactivated (HI) Lactobacillus species on the gut barrier , intestinal immunity, and anti-viral responses in the zebrafish (Danio rerio) challenged with viral hemorrhagic septicemia virus (VHSV). Our hypothesis was that postbiotics were able to modulate the innate immune responses in a species and strain-dependent way and exhibit a type-II like interferon response for removal of intracellular pathogens .
The 31-day study used wild-type adult zebrafish (28 fish/tank) subjected to a VHSV challenge by intraperitoneal injection at day 21. Five treatments were tested in triplicate: 1] non-supplemented basal diet (C1), 2] non-supplemented basal diet , challenged (C2) and 3 diets each supplemented prior cold-press at 6 × 106 CFU equivalent / g feed of 3] L. paracasei H108 (T1), 4] L. plantarum HA-119 (T2), or 5] L. helveticus HA122 (T3). Each HI bacterial strain (Lallemand SAS, France) was characterized by electronic and atomic force microscopy. Before the viral challenge , 54 fish/treatment (18 fish /tank) were euthanized and intestinal and kidney collected to perform histomorphometry and immune response profiling using gene expression (qRT -PCR) and immunoblotting. At day 21, 30 fish/treatment (10 fish/tank) were challenged with VHSV. Fish survival and viral clearance (qRT-PCR) were monitored for 10 days post-challenge . Differences between control and experimental groups were estimated by a 2 -way ANOVA and Bonferroni post-hoc test with significance accepted at P < 0.05.
The level (immunoblotting) of TNF-α, IFN-γ, IL-10, and alkaline phosphatase was higher in intestine and kidney compared to that of control with T1, T2 and T3. Interestingly antiviral response genes, such as ifnγ, ifnγ1, mx, and cd8α were up-regulated in both intestine and kidney (except mx in intestine ) by the diet supplemented with T2. A better numerical (T3, P = 0.11) and statistical (T2; P < 0.01) survival was observed in postbiotic groups compared to treatment C2, while a trend (P < 0.10) of a lower number of viral copies was detected in the T1 group compared to C2 . As a result of higher cumulative percent survival and augmentation of immune responses , fish fed the T2 diet presented a robust antiviral defense against VHSV. The T1 group also displayed a positive immune response regardless of survival rate.
In conclusion, th is study shows that heat-inactivated bacteria can be immunogenic by activating a type-II like interferon antiviral defense. Further research to characterize the bacterial cell wall microbial associated molecular patterns will be useful to understand the exact immune functional role(s) of the inactivated bacterial strains, and to optimize their use in aquaculture.