Functional and Molecular Immune Response of Rainbow Trout (Oncorhynchus mykiss) Following Challenge with Yersinia ruckeri

Abstract

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30-70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout's short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immune-related genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1β, il-8, il-10, tnf-α1, tnf-α2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.

Keywords: ERM; biomarkers; hematological profile; oxidative stress; yersiniosis.

Figure 1

Hematological parameters of rainbow trout sampled before (0 h) or 3, 6, 9, 24, and 48 h post-intraperitoneal infection with Y. ruckeri (means ± SD, n = 6). (A) WBC (white blood cells); (B) Hematocrit; (C) Lymphocytes; (D) Thrombocytes; (E) Neutrophils; (F) Monocytes. Letters indicate significant differences between the bio-groups (p ≤ 0.05).

Figure 2

Liver SOD (superoxide dismutase) activity of rainbow trout sampled before (0 h) or 3, 6, 9, 24, and 48 h post-intraperitoneal infection with Y. ruckeri (means ± SD, n = 6). Letters indicate significant differences between the bio-groups (p ≤ 0.05).

Figure 3

Normalized gene expression (ΔΔCq) of rainbow trout sampled before (0 h) or 3, 6, 9, 24, and 48 h post-intraperitoneal infection with Y. ruckeri (means ± SD, n = 6). (A) tlr-2 (toll-like receptor 2); (B) il-1β (interleukin 1 beta); (C) tnf-α1 (tumor necrosis factor alpha 1); (D) tnf-α2 (tumor necrosis factor alpha 2); (E) mmp-9 (matrix metalloproteinase 9); (F) socs-3 (suppressor of cytokine signaling 3). Letters denote significant differences between bio-groups. In all cases, a statistical threshold of p ≤ 0.05 was used. The reference gene ef-1α (elongation factor-1 alpha) was used for normalization.

Figure 4

Normalized gene expression (ΔΔCq) of rainbow trout sampled before (0 h) or 3, 6, 9, 24, and 48 h post-intraperitoneal infection with Y. ruckeri (means ± SD, n = 6). (A) il-8 (interleukin 8); (B) hsp-70 (heat shock protein 70); (C) cath (cathelicidin); (D) il-10 (interleukin 10); (E) saa (serum amyloid A); (F) pcb (precerebellin). Letters denote significant differences between bio-groups. In all cases, a statistical threshold of p ≤ 0.05 was used. The reference gene, ef-1α (elongation factor-1 alpha), was used for normalization.

Figure 5

Normalized gene expression (ΔΔCq) of rainbow trout sampled before (0 h) or 3, 6, 9, 24, and 48 h post-intraperitoneal infection with Y. ruckeri (means ± SD, n = 6). (A) fer (ferritin); (B) cd8 (T-cell surface glycoprotein CD8 alpha precursor). Letters denote significant differences between bio-groups. In all cases, a statistical threshold of p ≤ 0.05 was used. The reference gene ef-1α (elongation factor-1 alpha) was used for normalization.

Figure 6

Heat map of gene expression in the head kidney of O. mykiss infected with Y. ruckeri. Lines represent genes and columns represent different time points. (T0: control uninfected; T3: 3 h post-infection; T6: 6 h post-infection; T9: 9 h post-infection; T24: 24 h post-infection; T48: 48 h post-infection). Different colors represent different levels of expression, ranging from low expression (red) to high expression (green).