Alterations of Plasma Biochemical and Immunological Parameters and Spatiotemporal Expression of TLR2 and TLR9 in Gibel Carp (Carassius auratus gibelio) after CyHV-2 Infection

Abstract

Cyprinid herpesvirus II (CyHV-2), a highly contagious pathogen of gibel carp (Carassius auratus gibelio), causes herpesviral hematopoietic necrosis disease (HVHND) and enormous financial losses. However, there is limited information available regarding the changes in plasma biochemical and immunological parameters and the response characteristics of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9) in gibel carp after CyHV-2 infection. To address this knowledge gap, a sub-lethal CyHV-2 infection was conducted in gibel carp, and the sample was collected daily from 1 to 7 days post infection. The plasma biochemical analyses showed significant decreases in the content of glucose, total cholesterol (TCHO), and total protein (TP), along with marked increases in the level of uric acid, urea, creatinine (CREA), Complement 3 (C3), immunoglobulin D (IgD), and immunoglobulin M (IgM) as well as in the activity of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in the infected group. Compared with the control group, the concentration of cortisol, triglyceride (TG), and Complement 4 (C4) had no noticeable alterations in the infected group. Real-time quantitative PCR analysis showed significant upregulation of TLR2 and TLR9 mRNA expression in the spleen, kidney, brain, liver, intestine, and gill post CyHV-2 infection. Interestingly, a time- and tissue-dependent expression profile has been comparatively observed for TLR2 and TLR9 in the above tissues of gibel carp after CyHV-2 infection, suggesting distinct roles between TLR2 and TLR9 in antiviral response to CyHV-2 infection. Overall, our results demonstrated that CyHV-2 infection led to the disruption of the physiological metabolic process and damage to the liver and kidney, and induced different spatiotemporal expression patterns of TLR2 and TLR9, ultimately stimulating antiviral response via innate and adaptive immune system. These findings may provide a deeper understanding of the host immunity response to CyHV-2 infection and offer novel perspectives for the prevention and treatment and therapeutic drug development against CyHV-2.

Keywords: CyHV-2; TLR2; TLR9; biochemical indicators; gibel carp.

Figure 1

Clinical signs of main tissues of infected gibel carp. Gibel carp in infected group presented hyperemia in the regions of the submaxilla, abdomen, and liver, brain hyperemia, gill hemorrhages, splenomegaly, renal hypertrophy, and intestine swelling.

Figure 2

PCR products of helicase gene of CyHV-2 in six tissues of infected gibel carp. bp: base pair; lane M: DL 2000 DNA marker; lane B: no template as blank control; lane N: DNA template extracted from spleen of healthy gibel carp as negative control, lane P: DNA template extracted from CyHV-2 suspension as positive. Lane 1–6: DNA template extracted from brain, gill, liver, kidney, intestine, and spleen, respectively.

Figure 3

Content of cortisol (A), glucose (B), total cholesterol (C), and triglyceride (D) in plasma of the infected and healthy gibel carp. TCHO: total cholesterol; TG: triglyceride. The asterisk (*) indicates significant differences at the same point of time between the infected group and control group (n = 6, p < 0.05); bars with different upper and small capital letters at different points of time have significant differences in control group and infected group (one-way ANOVA, p < 0.05).

Figure 4

Content of total protein (A), urea (B), uric acid (C), and creatinine (D) in plasma of the infected and healthy gibel carp. TP: total protein; CREA: creatinine. The asterisk (*) indicates significant differences at the same point of time between the infected group and control group (n = 6, p < 0.05); bars with different upper and small capital letters at different points of time have significant differences in control group and infected group (one-way ANOVA, p < 0.05).

Figure 5

The enzyme activities of lactate dehydrogenase (A), alkaline phosphatase (B), alanine aminotransferase (C), and aspartate aminotransferase (D) in plasma of the infected and healthy gibel carp. LDH: lactate dehydrogenase; ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate transaminase. The asterisk (*) indicates significant differences at the same point of time between the infected group and control group (n = 6, p < 0.05); bars with different upper and small capital letters at different points of time have significant differences in control group and infected group (one-way ANOVA, p < 0.05).

Figure 6

Content of complement 3 (A), complement 4 (B), IgM (C), and IgD (D) in plasma of the infected and healthy gibel carp. C3: Complement 3; C4: Complement 4; IgD: immunoglobulin D; IgM: immunoglobulin M. The asterisk (*) indicates significant differences at the same point of time between the infected group and control group (n = 6, p < 0.05); bars with different upper and small capital letters at different points of time have significant differences in control group and infected group (one-way ANOVA, p < 0.05).

Figure 7

Relative expression of Toll-like receptor 2 (TLR2) in brain (A), gill (B), kidney (C), intestine (D), liver (E), and spleen (F) of the infected and healthy gibel carp. The asterisk (*) indicates significant differences at the same point of time between the infected group and control group (n = 6, p < 0.05); bars with different upper and small capital letters at different points of time have significant differences in control group and infected group (one-way ANOVA, p < 0.05).

Figure 8

Relative expression of Toll-like receptor 9 (TLR9) in brain (A), gill (B), kidney (C), intestine (D), liver (E), and spleen (F) of the infected and healthy gibel carp. The asterisk (*) indicates significant differences at the same point of time between the infected group and control group (n = 6, p < 0.05); bars swith different upper and small capital letters at different points of time have significant differences in control group and infected group (one-way ANOVA, p < 0.05).

Figure 9

Heatmap showing fold induction of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9) gene expression between infected gibel carp and healthy gibel carp following normalization to 18S rRNA at the same time point. Significant differences in the relative fold change between the infected group and control group at each time points are indicated with an * (p <0.05).