The enteric redmouth disease causes significant failures in rainbow trout farms. This highly contagious disease is caused by Yersinia ruckeri, a common bacterial infection in Peru. Although vaccination is the best strategy in terms of fish survival, no commercial vaccine is currently available or authorized in Peru, perhaps due to incomplete clearness about native Y. ruckeri populations. Recently, vaccination (immersion and intraperitoneal) experiments against Y. ruckeri were conducted by IMARPE in rainbow trout, and its effect was evaluated by measuring the expression of five immune response genes, in different tissues (kidney and spleen).
Alevines from Peruvian farms (Juli, Puno) were selected and vaccinated by immersion (treatment T1) considering 10^9 CFU/ml, and after 30 days organisms were vaccinated intraperitoneally for a second time (T2). PBS for the control (no-vaccinated) groups of each treatment was considered (T1-co and T2-co). Also, organisms before the first vaccination (T0) were collected and considered as a normalization group. cDNAs (n=8 per treatment per tissue) were synthesized from high quality RNA samples, from the five groups. Primers and probes for Efact-1a, bAct, Cath-2, IgM, IgT, IL-1B, and SAA genes were selected from literature, while primers for bAct amplification were designed in our laboratory. Gene expression analysis was conducted using qRT-PCR, and ??Ct method was used to obtain the relative fold change. The average of two reference genes (Efact-1a and bAct) was used as the internal calibrator. Non-parametric test was performed for media comparisons.
The expression of adaptive immune effector molecules genes were up-regulated in both tissues for IgM, and in kidney for IgT. Both vaccinated (T1 and T2) fish groups showed significant up-regulation (p<0.05 and log fold change > 2-fold) of IgM gene in both tissues, while IgT only registered significance in T2. Probably, both Ig were stimulated by antigens in vaccine formulation. On the other hand, the pro-inflammatory interleukin IL-1B, and effector molecules Cath-2 and SAA showed significant down-regulation in T1, in both tissues. In particular, IL-1B gene expression was also significant in vaccinated fish against their respective non-vaccinated groups. These observations were expected, since these molecules are active when an infection is ongoing. Although these immune responses patterns are influenced by several factors (such as temperature, type of vaccine, days post vaccination, and infection status after vaccination), it seems that the majority of target genes tested in our experiment serve as proper molecular markers for vaccine evaluation. A further immune response post challenge could improve our understanding of immune related genes in this study. However, testing more immune-related genes in different important tissues is crucial to propose an accurate gene panel for experimental Y. ruckeri vaccines in the O. mykiss cultivation.
Acknowledgment: This work was funded by PpR-2022, Meta 03 Desarrollo Tecnológico, IMARPE, Ministerio de la Producción, and the National Council of Science and Technology - CONCYTEC, Contract 128-2020-FONDECYT.