*2.3. Histopathology*

Microscopic lesions appeared as early as 3 dpi but were more severe and widespread at 6 dpi (Table 2). At that time, the most affected tissues were the heart, lung, spleen, liver, and bursa of Fabricius (Table 2). The main microscopic finding was the presence of inflammatory infiltrates from 3 dpi onwards. These were mainly composed of lymphocytes, macrophages, and plasma cells. Heterophils were also detected in the liver, spleen, and intestine and, to a lesser extent, in the heart, feather follicles (Figure 1A), and skin of both groups. Cellular degeneration and/or necrosis were especially severe in the heart and liver at 6 dpi and in the kidney at 14 dpi (Figure 1B–C, Table 2). On those days, the liver and spleen showed hemosiderosis. In the brain, endothelial cell swelling was observed throughout the experiment in both groups; however, the development of a nonsuppurative encephalitis and neuronal necrosis occurred earlier in partridges infected with Morocco/2003 (Figure 1D, Table 2).

**Table 1.** Viral genome load in tissues of experimentally WNV-infected red-legged partridges. The presence of WNV genome was analyzed in different tissues at different days post-inoculation (dpi) by real-time reverse transcription polymerase chain reaction (RRT-PCR). Data are presented as Ct values. SP07: Spain/2007, MO03: Morocco/2003, NA: tissue sample not analyzed, −: tissue sample with Ct ≥ 40.


\* Tissues from individual No. 9 were not available for analysis by RRT-PCR; however, they were used for histopathology and immunohistochemistry analyses.

**Table 2.** Microscopic lesions in tissues of experimentally WNV-infected red-legged partridges. Lesions were graded according to their distribution and severity at different days post-inoculation (dpi) in both groups, Spain/2007 (SP07) and Morocco/2003 (MO03). −: no lesion, +: focal and mild or moderate/multifocal and mild, ++: focal and marked/multifocal and moderate/diffuse and mild, +++: multifocal and marked/diffuse and moderate or marked. NA: tissue sample not analyzed.



**Table 2.** *Cont*.

**Figure 1.** Microscopic lesions in tissues of experimentally WNV-infected red-legged partridges. (**A**) Feather follicle; partridge inoculated with Morocco/2003, 14 dpi. Multifocal infiltration of inflammatory cells in the feather pulp. Inset: detail of an inflammatory nodule composed of lymphocytes, macrophages, and granulocytes. (**B**) Heart; partridge inoculated with Spain/2007, 6 dpi. Diffuse and marked necrosis and degeneration of cardiac myofibers, and infiltration of inflammatory cells. Inset: detail of necrotic cardiac myofibers which show degeneration, fragmentation, and accumulation of hyaline material in the cytoplasm. Infiltration of mononuclear inflammatory cells is also observed. (**C**) Liver; partridge inoculated with Morocco/2003, 6 dpi. Necrosis of hepatocytes characterized by cellular detachment, lysis of the cytoplasm and pyknosis and fragmentation of the nucleus. There is also a mild infiltration of lymphocytes and plasma cells (black arrowheads) and heterophils (white arrowheads). (**D**) Cerebrum; partridge inoculated with Morocco/2003, 6 dpi. Focal necrosis with degeneration of the neuropil and infiltration of lymphocytic and glial cells. Scale bars = 100 μm.

> As an incidental finding, there were coccidian oocysts and gametes in the enterocytes of the large intestine. These parasites were much more numerous in WNV-infected birds as compared to control individuals (Figure 2A), especially in partridges euthanized at 6 dpi and in those infected with Morocco/2003. In fact, one bird of this group showed an associated severe necrosis of the epithelium of intestinal mucosa and crypts (Figure 2B).

**Figure 2.** Coccidiosis in the large intestine of WNV-infected and control red-legged partridges. (**A**) Graph showing the mean numbers ± standard deviation of log transformed nos. of coccidian forms in transverse sections of the large intestine at the level of cecal tonsils in red-legged partridges experimentally infected with WNV Morocco/2003 (MO03) and Spain/2007 (SP07) strains. (**B**) Cecum; partridge inoculated with Morocco/2003, 6 dpi. Presence of gametes and coccidian oocysts in the epithelium of the mucosa and crypts (arrows). There is also inflammation in the lamina propria and severe necrosis of the epithelium of both the mucosa (arrowheads) and crypts (stars). Scale bar = 100 μm.
