Fig 1.
Skin sections sampled in this study.
Illustration showing the distribution of the different skin sections used to study immune gene transcription in this study.
Table 1.
Primers used in this study.
Fig 2.
Constitutive levels of expression of chemokines and genes related to B cell activity in the different skin sections.
The skin sections indicated in Fig 1 were sampled from naïve fish for RNA extraction and analysis of immune gene transcription through real time PCR. Data are shown as the mean relative gene expression normalized to the transcription of the house-keeping gene EF-1α ± SD (n = 10). Relative expression values were lowest in section 7 for most genes analyzed and were set as 1 for comparative reasons. Asterisks indicate significant differences (P < 0.05) relative to values in section 7.
Fig 3.
Constitutive levels of expression of genes related to T cell activity in the different skin sections.
The skin sections indicated in Fig 1 were sampled from naïve fish for RNA extraction and analysis of immune gene transcription through real time PCR. Data are shown as the mean relative gene expression normalized to the transcription of the house-keeping gene EF-1α ± SD (n = 10). Relative expression values were lowest in section 7 for most genes analyzed and were set as 1 for comparative reasons. Asterisks indicate significant differences (P < 0.05) relative to values in section 7.
Fig 4.
Distribution of trout CD3+ cells in anterior and posterior skin regions.
Inmunohistochemical detection of trout CD3+ cells in different skin areas of anterior (A) and posterior (B) body sections. Counterstained with Mayer’s haematoxylin. Scale bar represents 100μm. Total numbers of CD3+ cells for each skin section were calculated using the imaging analysis Image J software (NIH). Then, relative number of cells per mm2 were calculated and plotted. Data are shown as mean ± SD (n = 10). Asterisks indicate mean values in posterior sections significantly lower than values in anterior areas (P < 0.001).
Fig 5.
Quantification of trout CD8+ cells in anterior and posterior skin regions.
Flow cytometry analysis of rainbow trout leukocytes isolated from anterior (A) and posterior (B) sections of the skin were stained with anti-CD8α mAb. For each individual tissue, FSC/SSC profiles are shown (top panels) and gates for lymphoid cells were defined. Bottom panels depict the CD8+ cells among the lymphoid gate (percentages of CD8α+ cells are shown). (C) Mean percentages of CD8α+ cells from three independent experiments are shown (n = 9 fish, mean ± SD). Asterisks indicate mean values in posterior sections significantly lower than values in anterior areas (P < 0.001).
Fig 6.
Comparative transcription of genes related to T cell activity in skin and spleen.
The levels of transcription of different genes related to T cell activity were studied in skin samples corresponding to section 3 as indicated in Fig 1 and spleen in unstimulated fish through real time PCR. Data are shown as the mean relative gene expression normalized to the transcription of the house-keeping gene EF-1α ± SD (n = 5). “a” indicate values in skin significantly lower to values obtained in spleen whereas “b” indicate skin values significantly higher than those found in spleen (P < 0.05).
Fig 7.
Transcription levels of immune genes characteristic of T lymphocytes in skin in response to VHSV.
Rainbow trout were infected by bath with VHSV (5 x 105 TCID50/ml) or mock-infected. At days 1 (A) and 3 (B) post-infection, six trout from each group were killed and the two sections in the skin corresponding to those indicated as 3 and 7 in Fig 1 sampled to determine the levels of expression of a selection of immune genes related to T cell immunity by real-time PCR. Data are shown as the mean gene expression relative to the expression of endogenous control EF-1α ± SD (n = 5). * Levels of expression in VHSV-infected animals significantly different to those observed in mock-infected fish (p < 0.05).
Fig 8.
Transcription levels of immune genes characteristic of T lymphocytes in spleen in response to VHSV.
Rainbow trout were infected by bath with VHSV (5 x 105 TCID50/ml) or mock-infected. At days 1 and 3 post-infection, six trout from each group were killed and the spleen sampled to determine the levels of expression of a selection of immune genes related to T cell immunity by real-time PCR. Data are shown as the mean gene expression relative to the expression of endogenous control EF-1α ± SD (n = 5). * Levels of expression in VHSV-infected animals significantly to those observed in mock-infected fish (p < 0.05).