A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum

Abstract

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM⁺ and IgM⁺⁺ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT⁺ and/or IgT⁺⁺ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT⁺ B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT⁺ B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM⁺ B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT⁺ B lineage cells may have a protective function in the immune response to Fp.

Figure 1

Flow cytometric analysis for the in vivo Fp-challenge experiment, using two-color, HCtau and HCmu staining. Shown here are PBL, spleen (SPL), and peritoneal cavity fluid (PCF). Illustration of patterns in the form of contour graphs, on day 5 post-challenge, using MR samples for PBL and SPL tissue, and an FpR sample for PCF. Gate 1, IgM⁺⁺ ISCs; gate 2, IgM⁺/IgT⁻ B cells, gate 3: IgM⁻/IgT⁺ B cells; gate 4: IgT⁺⁺ ISC, gate 5: IgM⁺/IgT⁺ cells (see Figure 2).

Figure 2

Characterization of IgT⁺ cells in the AK using flow cytometry. The anterior kidney sample used here is pooled from four FpS fish. A–E: 3-color flow cytometry. Grey arrow: control antibody/non-staining population. Black arrow: IgM⁺/IgT⁺ cells. White arrow: IgT⁺ B cells. A. Example of a contour graph with HCtau and HCmu staining shown. Gates numbered as in Figure 1: gate 1, IgM⁺⁺ ISCs; gate 2, IgM⁺/IgT⁻ B cells, gate 3: IgM⁻/IgT⁺ B cells; gate 4: IgT⁺⁺ ISC, gate 5: IgM⁺/IgT⁺ cells. B. Pax5.PD expression of selected subpopulations from Figure 2A. Grey filled line: all cells; black-white dotted line: IgM⁺/IgT⁻ cells (gate 2); black solid line: IgM⁻/IgT⁺ cells (gate 3); black-striped: IgM⁺/IgT⁺ cells (gate 5). C. Similar to B., but without IgM⁺/IgT⁻ cells shown to enlarge the IgT⁺/IgM⁺ population. Grey line represents negative control stain. Black arrow indicates a small population of IgM⁺/IgT⁺ cells (gate 5) with Pax5.PD⁺⁺ staining. Grey arrow: control antibody. D. Contour of FSC and HCtau staining, showing an IgT⁺ population of Late developing B cells (low FSC), and an unknown, IgT⁺ population with high FSC. E. Histogram with Pax5.PD staining of the two gated populations from Figure 2D: Late developing B cell (grey filled, with white arrow), and an unknown population with Pax5.PD⁺⁺ staining (black line). Grey arrow: control antibody F. HCtau and RAG1 staining using 2-color flow cytometry.

Figure 3

Strip plot showing changes in gene expression for secHCmu and secHCtau transcripts in PBLs. The mean fold change values of gene expression (± SE) are shown on the Y-axis. Each of the four groups is shown on the X-axis. Day 5 post Fp-challenge (Fp2016). A. secHCtau. B. secHCmu. ^*, <0.05, ^**<0.01.