The Clinical Infection with Pigeon Circovirus (PiCV) Leads to Lymphocyte B Apoptosis But Has No Effect on Lymphocyte T Subpopulation

Figures

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  Figure 1. Representative figures of results of the flow cytometry of mononuclear cells isolated from the spleen of pigeons with different PiCV infectious status: (A) the lymphocyte gate; (B) density plots of the extracellular staining for T CD3+ and B IgM+; (C) density plots of the Annexin V and 7-AAD staining. Three states of the cells were identified: viable cells, located lower left/Q3 (Annexin V-APC/ 7-AAD = −/−); early apoptotic cells, located lower right/Q4 (Annexin V-APC/ 7-AAD = +/−); necrotic and late apoptotic cells, located upper part/Q1 and Q2 (Annexin V-APC/ 7-AAD = −/+ and +/+). The samples were standardized to 1 × 106 of mononuclear cells.

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  Figure 2. The results of the flow cytometry analyses. The samples were standardized to 1 × 106 of mononuclear cells. The mean percentage and the ratio of T CD3+ to B IgM+ splenic lymphocytes isolated from the examined pigeons. The different letters (A,B,C) indicate a statistically significant difference between investigated groups (p < 0.01 and p < 0.05, respectively) in the Kruskal–Wallis non-parametric test for independent samples. Error bars represent the standard error of the mean.

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  Figure 3. The results of the flow cytometry analyses. The samples were standardized to 1 × 106 of mononuclear cells. (A) The mean percentage and the ratio of apoptotic (early apoptosis) T CD3+ to B IgM+ splenic lymphocytes isolated from the examined pigeons. (B) The mean percentage and the ratio of necrotic and late apoptotic T CD3+ to B IgM+ splenic lymphocytes isolated from the examined pigeons. The different letters (A,B,C or a,b) indicate a statistically significant difference between investigated groups (p < 0.01 and p < 0.05, respectively) in the Kruskal–Wallis non-parametric test for independent samples. Error bars represent the standard error of the mean.

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  Figure 4. Mean relative expression of the genes encoding CD4 and CD8 lymphocyte receptors and IFN-γ in splenic mononuclear cells of the examined pigeons. The mean relative expression values above 1 (horizontal black line) in groups S and I indicate a higher gene expression compared to the control group (H). The different letters (A,B) indicate a statistically significant difference between investigated groups (p < 0.01) in the Mann–Whitney U test. Error bars represent the standard error of the mean.

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  Figure 5. Detection of anti-pigeon circovirus (PiCV) IgY in sera of the examined pigeons using in-house ELISA. The different letters (A,B or a,b) indicate a statistically significant difference between investigated groups in the Kruskal–Wallis non-parametric test for independent samples. Error bars represent the standard error of the mean.

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  Figure 6. Representative figures of dimensional plots of the ddPCR assay showing PiCV viral loads in the selected samples. The value of y axis (Ch1 amplitude) reflects to amplitude of EvaGreen fluorescence in a detecting channel of a ddPCR device. The sample repetitions are divided by vertical yellow lines. The unbroken pink line is the threshold, above which there are positive droplets (blue) with PCR amplification and underneath which there are negative droplets (gray) without any amplification.