**3. Results**

#### *3.1. Total Number of Inflammatory Cells*

Samples from all species showed high numbers of inflammatory cells, with the fox showing the highest number. Inflammatory infiltrate was significantly higher in the fox than in the wolf and chamois (Table 2). On the other hand, while intra-species variation in the number of inflammatory cells was low for the fox and red deer, two of the five wolf samples showed lower numbers, and one chamois sample showed a much higher number.

> **Table 2.** Total numbers of inflammatory cells in skin mange lesions from four species.


\* SD, standard deviation. IQR, interquartile range. a,b Statistical analysis by pairwise comparisons showed significant differences between means followed by same letters in the same column (*p* < 0.05).

#### *3.2. Relative Proportions of Inflammatory Cell Types within Each Species*

In all species, the most abundant cells in the inflammatory infiltrate were macrophages, while the least abundant were T or B lymphocytes (Figure 1).

**Figure 1.** Intra-species differences in the numbers of macrophages (based on Iba1 immunostaining), plasma cells (lambda chain), T lymphocytes (CD3) and B lymphocytes (CD20) in skin mange lesions from wild carnivores and ruminants. For each animal species, the same letters above different rectangular bars indicates significant differences between means. For wolf: (a, b, c) *p* < 0.001. For fox: (a, b, c, d, e) *p* < 0.05. For chamois: (a, b) *p* < 0.05. For red deer: (a, b, c) *p* < 0.001. A Tukey test for multiple comparisons was applied for statistical analysis.

In the wolf and red deer, macrophages were significantly more abundant than plasma cells, T lymphocytes and B lymphocytes, which were similarly abundant (Figures 1 and 2). In the fox, the proportion of macrophages was significantly higher than the proportions of other cell types, and the proportion of plasma cells was significantly higher than those of T and B lymphocytes (Figure 1). In chamois, macrophages and T lymphocytes were more abundant than B lymphocytes and plasma cells (Figures 1 and 2), although the differences were significant only in the case of macrophages.

**Figure 2.** Comparative immunohistochemistry of cellular response in the dermis of wolves, foxes, chamois and red deer naturally infested with *Sarcoptes scabiei*. Skin biopsies were stained with primary antibodies against the indicated markers and the avidin-biotin complex kit. Macrophages were the predominant type of inflammatory cell in all species (top row). Plasma cells were present in all species, but less abundant than macrophages (second row). T lymphocytes (third row) and B lymphocytes (bottom row) were scarce in all species. Arrows indicate examples of stained cells when they are scarce (even with no B lymphocytes in red deer). Tissue sections in each column came from the same animal, and the results shown are representative of all five animals of each species. Bars = 50 microns.

#### *3.3. Relative Proportions of Inflammatory Cell Types across Species*

Wolves showed more abundant macrophages than the other species, although the difference was significant only with red deer (Table 3). Plasma cells were similarly abundant in the wolf, fox and red deer; the abundance in the wolf and fox was significantly higher than in chamois. Abundance of T and B lymphocytes was similarly low across all four species. Of all species, chamois showed the highest abundance of T lymphocytes and wolf showed the highest abundance of B lymphocytes, though the differences across the four species were not significant.

**Table 3.** Percentages of cells staining positive for inflammatory cell biomarkers in skin lesions from four species.



**Table 3.** *Cont.*

\* SD, standard deviation. Means followed by same letters in the same column differ significantly (Tukey test): a *p* = 0.038, b *p* = 0.004 and c *p* = 0.017.
