*3.1. Microglia*

Microglia was the most abundant cellular type (Figure 2a,d), accounting for an average of 45.4% of the three immunostained cell populations. Based on lesion severity, lambs were classified into cluster A with mild to moderate lesions and cluster B with moderate to severe lesions (Section 2.4). Microglia was significantly more abundant in cluster B (59.3%) than in cluster A (31.5%, *p* < 0.0001; Table 2). Microglia was also significantly more abundant in cortex (55.3%) and hippocampus (55.2%) than in cervical spinal cord (34.4%) or lumbosacral spinal cord (31.6%; both *p* < 0.0001). Additionally, they were significantly more abundant in glial foci (55.2%) than in perivascular cuffing (35.5%, *p* < 0.0001).

**Figure 2.** Immunohistochemistry to detect microglia, T lymphocytes, and B lymphocytes in thalamus (**<sup>a</sup>**–**<sup>c</sup>**) and hippocampus (**d**–**f**). Microglia were identified based on labeling with anti-Iba1 antibodies. Abundant cells are visible within glial foci (**a**) and in the perivascular infiltrate (**d**). T lymphocytes were identified based on labeling with anti-CD3 antibodies. Moderate numbers are visible in the glial focus (**b**) and in the perivascular cuff (**e**). B lymphocytes were identified based on labeling with anti-CD20 antibodies. Few numbers are visible within the glial focus (**c**) and perivascular infiltrate (**f**). Erythrocytes were also immunolabelled (asterisk) but were not included in the counting.


**Table 2.** Proportion (%) of all immunostained cells, i.e., microglia, T or B lymphocytes, or astrocytes, in lambs experimentally infected with SGEV, stratified according to lesion severity, central nervous system (CNS) region, and lesion type.

SE, standard error. *Between clusters:* Proportions of microglia and T lymphocytes were significantly different (*p* < 0.0001), whereas proportions of B lymphocytes (*p* = 0.1213) and astrocytes (*p* = 0.3435) were not. *Among CNS regions*: Proportions of microglia in cortex and hippocampus were different from proportions in cervical and lumbosacral spinal cord (*p* < 0.0001). Proportions of T lymphocytes in pons and cerebellum differed from those in cervical spinal cord (*p* = 0.0139). Proportions of B lymphocytes did not differ significantly among regions (*p* = 0.5056), nor did proportions of astrocytes (*p* = 0.2124). *Between type of lesion*: Proportions of microglia differed (*p* < 0.0001), whereas proportions of T lymphocytes (*p* = 0.8350) or B lymphocytes (*p* = 0.8659) did not.

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Glial foci were significantly more numerous in cluster B, where they covered a significantly larger proportion of affected areas (15.9%) than in cluster A (8.4%, *p* < 0.0001). Glial foci were significantly more numerous in thalamus, hypothalamus, corpus callosum, and medulla oblongata (19.5% of affected areas) than in cervical spinal cord (5.6%, *p* = 0.0682) and lumbosacral spinal cord (6%, *p* = 0.0522).

## *3.2. T lymphocytes*

T lymphocytes were the second most abundant cell type (Figure 2b,e), accounting for 18.6% of the three immunostained cell populations. Like microglia, they were significantly more abundant in cluster B (24.8%) than in cluster A (12.5%; *p* < 0.0001; Table 2). Their proportion was higher in pons and cerebellum (25.1%) than in cervical spinal cord (11.6%, *p* = 0.0139) and was not significantly different between perivascular cuffing (20.6%) and glial foci (16.6%, *p* = 0.8350).

## *3.3. B lymphocytes*

B lymphocytes were the less abundant cell type (4.4%) (Figure 2c,f), and their proportion was similar in cluster B (6.4%) and cluster A (2.4%, *p* = 0.1213; Table 2). All CNS regions contained comparable proportions of B lymphocytes, ranging from 3.4% in cortex and medulla oblongata to 5.8% in midbrain. Their proportions were slightly higher but not significant in perivascular cuffing (6.3%) than in glial foci (2.5%, *p* = 0.8659).

#### *3.4. Proportions of Microglia, T lymphocyte,s and B Lymphocytes According to Lesion Location and Severity*

The proportions of microglia were higher than those of T and B lymphocytes in all CNS regions (Table 2), and the differences were significant in the cortex (microglia vs. B lymphocytes, *p* < 0.0001; microglia vs. T lymphocytes, *p* = 0.0051), thalamus, hypothalamus, and corpus callosum (microglia vs. B lymphocytes, *p* < 0.0001), hippocampus (microglia vs. B lymphocytes, *p* < 0. 0001; microglia vs. T lymphocytes, *p* = 0.0051), midbrain (microglia vs. B lymphocytes, *p* = 0.0047), pons and cerebellum (microglia vs. B lymphocytes, *p* = 0.0002), and medulla oblongata (microglia vs. B lymphocytes, *p* = 0.0001). Proportions of T and B lymphocytes were not significantly different in any region.

The proportion of microglia cells was higher than those of both lymphocyte types in cluster B (both *p* < 0.0001), and the proportion of T lymphocytes was higher than that of B lymphocytes (*p* < 0.0001). Similar results were observed in cluster A. The proportions of microglia were not significantly different between clusters A and B (31.5% vs. 59.3%; *p* = 0.1213), but the proportions of lymphocytes were significantly higher in cluster B (T, 24.8% vs. 12.5%, *p* < 0.0001; B, 6.4% vs. 2.4%, *p* < 0.0001).
