*3.3. Pathology*

#### 3.3.1. Acute Stage

Animals examined at this stage of the disease showed edematous drooping ears, serum exudation and sloughing of the skin in the dorsum of the ears, eyelids, muzzle, lips and forelimbs, characterized histologically by epidermal necrosis and presence of serocellular crusts (Figure 1A). The carcass showed intense jaundice and the liver were bile stained (yellowish) and enlarged (Figure 1B). The extrahepatic bile ducts and the gallbladder were distended by abundant bile and visible brown concretions (pigmented calculi) (Figure 1B). Histologically, in the liver, there was a macrovesicular (large droplet) predominantly periportal fatty change (zone 1). Hepatocytic and canalicular cholestasis with bile granular deposits in the cytoplasm of hepatocytes and presence of bile plugs in canaliculi respectively, confirmed by the Hall histochemical stain, were prominent in centrilobular regions (zone 3). Some of these cells were swollen or necrotic. Small clumps of polimorphonuclear neutrophils (PMNs), Kupffer cells (KCs) with AFIP lipofuscin positive granules, and foreign-body-type giant cells were present surrounding more prominent deposits of bile pigment ('bile lakes') (Figure 1C). Portal tracts were edematous with ductular proliferation at the periphery (bile ducts with a well-defined lumen). There was also a mild (sparse) portal inflammation (scattered lipofuscin-laden macrophages, detected by the AFIP method, and lymphocytes). The epithelial cells in septal bile ducts were shrunken with picnotic nuclei, and larger bile ducts were dilated and contained inspissated bile. The kidney was macroscopically slightly enlarged and showed a greenish discoloration with linear green streaks throughout the cortex and medulla (Figure 1D). Diffuse cellular swelling and green granular pigmented bilirubin, in the proximal tubules, as well as greenyellow acellular tubular bile casts in the distal nephron segments of the renal medulla, both confirmed by the Hall histochemical stain, were observed microscopically (cholemic nephrosis) (Figure 1E,F).

## 3.3.2. Chronic Stage

All the sheep submitted 2 and 6 months after the initial onset of the disease outbreak showed skin lesions limited to areas of the head: dorsum of the head and ears, eyelids, face, lips and nose were alopecic and crusting. In the ears, there was focal necrosis of the epidermis and the underlying cartilage with palisading crust formation. The livers were atrophic, with lesions most marked in the left lobe (2 sheep), yellowish with whitish bands of fibrous tissue and larger lonely nodules of 4 and 8 cm located principally in the visceral surface in the quadrate lobe (2 sheep) (Figure 2A). The intrahepatic and extrahepatic bile ducts and gallbladder were also enlarged and contained biliary sludge and pigmented gallstones.

Histologically, increased biliary fibrosis with an associated inflammatory infiltrate, and prominent ductular proliferation were observed in the liver of all sheep examined. The portal tracts were expanded with proliferating bile ductules with a well-defined lumen (typical cholangiocyte proliferation) and fibrous tissue, which occasionally bridged adjacent portal tracts. Extensive pericellular or subsinusoidal fibrosis was evident using Masson-Goldner trichrome stain. An intense leucocytic portal inflammation (lymphocytes and plasma cells) was noted. At the same time and in the same liver, areas formed by an irregular proliferation of intrahepatic bile ductules at the portal tract margins, with poorly formed lumina that replaces the hepatic parenchyma (atypical ductular reaction) (Figure 2B). Numerous spindle-shaped cells, lymphocytes and macrophages containing brown pigment, were seen throughout the fibrous tissue, as well as in remnants of the hepatic lobules which eventually disappeared, principally in the hepatic left lobe (Figure 2C).

Histochemical studies showed that pigmented macrophages in close association with remnants of hepatocytes were positively red-stained with AFIP method for lipofuscin. This pigmented lipoproteins coexisted in some macrophages with iron deposits (hemosiderin) that reacted with Perls´ Prussian blue stain (Figure 2D). Granular deposits of protein-bound copper salts were observed in periportal hepatocytes and pigmented macrophages as small black granules in their cytoplasm. Collagen fibers were arranged in concentric layers around dilated interlobular bile ducts and proliferating bile ductules ("onion skin" fibrosis). As the result of coalescence of adjacent fibrotic portal tracts, portal-portal fibrous septa were noticed and the surviving parenchyma persisted with a jigsaw pattern characteristic

of the biliary cirrhosis (Figure 2E). Larger bile ducts in sheep examined two months after FE outbreak were dilated and contained inspissated bile plugs and biliary stones. The biliary epithelium was flattened and necrotic and the fibrotic wall contained numerous pigmented macrophages and foreign body type giant cells around bile pigment deposits, admixed with mononuclear inflammatory cells. A marked fibrotic thickening, with a mild lymphocyte infiltrate, was the major histologic finding in large intrahepatic and extrahepatic bile ducts 6 months after the FE outbreak. Canalicular cholestatic changes were only seen in one sheep 2 months after the outbreak. Additionally, this sheep with cholestasis also showed brownish discoloration of the renal cortex and medulla. Intracellular bile pigment, stained green by the Hall histochemical stain, involved proximal tubules in two sheep 2 months after the FE outbreak (cholemic nephrosis).

**Figure 1.** Acute lesions observed in naturally acquired cases of FE in sheep. (**A**) Sloughing of the skin in the ears, eyelids, muzzle and lips. (**B**) The liver was diffusely yellowish in color and the common and cystic bile duct were distended. (**C**) Polimorphonuclear neutrophils were surrounding a hepatic "bile lake". HE. Bar, 20 μm. (**D**) Linear greenish discoloration of the renal cortex and medulla. (**E**) Brown granular pigment in the proximal tubules. HE. Bar, 50 μm. (**F**) Green granular pigment consistent with bilirubin in the proximal tubules. Hall´s bilirubin stain. Bar, 50 μm.

All sheep examined in the chronic stage showed vascular lesions in the liver. In portal hepatic arteries, hepatic arteries in the septa and large hilar hepatic arteries, near severely damaged intrahepatic (interlobular, septal) and extrahepatic (common and cystic) bile ducts, both concentric and eccentric myointimal proliferation was found to some

degree (Figure 2F). Similar occlusive lesions, characterized by intimal cap proliferation, were observed in sublobular veins near damaged bile ducts. Lymphocytic phlebitis and phlebosclerosis consisting, respectively, of chronic inflammatory infiltrate of the wall and perivenular fibrous thickening of central veins and striking fibromuscular hypertrophy of the walls of ectatic hepatic veins were also found.

**Figure 2.** Chronic lesions observed in naturally acquired cases of FE in sheep. (**A**) Marked atrophy in the hepatic left lobe and a large nodule in the hepatic visceral surface. (**B**) Hepatic lobular areas are replaced by proliferated bile ductules (atypical ductular reaction) and fibrous tissue. Masson-Goldner trichome stain. Bar, 50 μm. (**C**) Numerous lymphocytes and pigmented macrophages were seen in the fibrous tissue in association with remnants of hepatic lobes. Masson-Goldner trichome stain. Bar, 50 μm. (**D**) Macrophages were positively red stained for lipofuscin (AFIP stain) that coexisted in some cells with hemosiderin. Perls' Prussian blue stain. Bar, 50 μm. (**E**) Jigsaw pattern characteristic of the biliary cirrhosis. Masson-Goldner trichome stain. Bar, 200 μm. (**F**) Eccentric myointimal proliferation in a hepatic arteriole adjacent to the bile duct. HE. Bar, 20 μm.
