**4. Discussion**

In this report we described an episode of FE in sheep that occurred in Asturias, Spain in 2003. This mycotoxic disease was first observed in European sheep in France [11] and later in the Basque Country, Spain [15] and the Azores, Portugal [12]. In late 2003, the weather conditions in Asturias were the most favorable for *P. chartarum* growth and sporulation. The autumn was warm (with temperatures above 19 ◦C in September and the first 19 days of October) and very rainy, with total monthly rainfall of 141.4 and 219 L/m<sup>2</sup> in October and November, respectively. Late summer and early autumn with warm temperatures (minimum 16 ◦C) and high humidity (above 90 per cent) were the periods favorable to FE outbreaks in the Azores, Portugal between 1999 to 2001 [12]. The periods and weather conditions in Asturias were similar to those observed in these islands, both with oceanic climate. Spore counts of *P. chartarum* identified in several different grass samples ranged from 0 to 75,000 spores/g of pasture. It has been suggested that spore counts as low as 50,000 spores/g of grass could be dangerous to livestock if grazed for long periods in sunlight and that the greater liver injury occurred when sheep were grazed on pastures having maximum spore counts of 130,000 spores/g of grass [20,21]. Other causes of toxins from fungi or plants were not found around the studied field. Given that FE clinical signs appear 14–18 days after intake of the mycotoxin [20] and, in the present outbreak, sheep with acute signs of photosensitization were observed from the middle of October, plots possibly reached the higher spore numbers in September/October. The low spore counts observed in the present study may be related to the variability between individual sites in the plot. Besides, the grass samples were collected in late November with a low temperature (average of 11.6 ◦C), conditions less favorable to fungal growth and sporulation.

In the present study, a significant rise in the activity of serum GGT associated with histological cholestasis and bile duct damage was demonstrated in sheep in accordance with previous reports in which sporidesmin was administered experimentally [16,30], and also in spontaneous intoxications [10,12,14,15]. GGT and ALP levels were higher in all sheep examined (when compared with reference ranges) and significantly increased (*p* < 0.001 and *p* < 0.01, respectively) in the 11 sheep with skin lesions when comparing with the 62 apparently healthy animals (without skin lesions). In our opinion, in agreemen<sup>t</sup> with previous reports [7,18], the detection of elevated GGT serum levels was the most suitable marker for monitoring FE affected sheep under field conditions because it correlated with the liver lesions, even in apparently healthy animals. In this sense, our results confirmed that a high activity of GGT persists for six months after an FE outbreak, in accordance with other published data [3]. Serum albumin was lower (*p* < 0.01) and TP was increased (*p* < 0.05) in sheep with skin lesions and the serum AST levels did not show statistically significant changes. AST was increased in all the tested animals, even in those without injury 6 months after the outbreak. This fact could be the consequence of a previous exposure and the development of a chronic inflammatory process, so that AST could be a sensitive and long-lived marker of liver damage in sheep, in disagreement with previous observations of natural intoxication with sporidesmin in sheep [18].

The gross lesions in the skin, liver and kidney described in the acute phase of ovine FE agree, in general, with previous descriptions in natural [10,14] and experimental cases [8,30]. Microscopic lesions described in the acute stage have been demonstrated experimentally in sheep dosed pure cultures of *P. chartarum* directly to the stomach, equivalent to approximately 3–4 mg/kg live mass [8,30]. These animals, that became photosensitive on the 9–10th day and died or were euthanized on the 4–10 subsequent days, showed hepatic parenchymal infarcts with leakage of bile between the necrotic hepatocytes and the presence of polymorphonuclear cells, similar to the microscopic findings described in the present study. A moderate bile duct proliferation and mononuclear cell infiltration (including some pigment laden macrophages), as well as necrotic lesions in the bile ducts in the portal tracts, were also observed in this work. Recently, altered cell adhesion and disruption of actin in sheep gallbladder epithelial cells incubated with sporidesmin were

demonstrated, suggesting that the biliary tract pathology in FE may be due to the effects of the toxin on cytoplasmic and cell surface protein networks, affecting the integrity of the epithelial lining of the biliary tract [31]. The special stains used, for the first time in the present study, such as Hall and AFIP stains, were useful to demonstrate the presence of bile and lipofuscin, respectively, in liver. Lipofuscin can accumulate in liver macrophages in natural FE in sheep and may represent the remnants of phagocytosed debris from necrotic hepatocytes. This study also confirmed by the Hall stain the presence of cholemic nephrosis characterized by proximal tubulopathy and intrarenal bile cast formation in ovine FE. Previous studies showed that elevated plasma levels of conjugated bilirubin are related to renal failure associated with obstructive jaundice [32]. The term "bile cast nephropathy", caused by direct bilirubin toxicity and tubular obstruction, has been proposed for this pathologic entity in humans [33].

Liver atrophy, more severe in the left lobe, and large nodules of regeneration in the visceral surface of the liver, as well as alopecic and crusty head skin were the most striking gross findings in chronic forms of FE that are in agreemen<sup>t</sup> with previous descriptions [3,15,34].

The atrophic left hepatic lobe with dilated intrahepatic and extrahepatic bile ducts are conditions that may have been due to compression of the left trunk of the portal vein secondary to hepatolithiasis [35]. In our case, the mechanism of left lobar atrophy might have been due to the occlusion of the ducts by biliary sludge and pigmented gallstones that cause atrophy of the parenchyma served by them [34].

The presence of nodular regeneration and cirrhosis have been considered the most conspicuous pathological changes in natural cases of FE in sheep [8] and may start as early as 2 weeks after toxin insults [7]. Experimentally, this lesion has been observed in sheep challenged with a total of 2.125 mg/kg sporidesmin divided into 17 doses for 144 days [30].

A remarkable histological finding in chronic FE was a strong ductular reaction that replaces the hepatic parenchyma associated with extensive fibrosis and aggregates of lymphocytes and pigment containing macrophages in connective tissue septa. It has been suggested that this ductular reaction can represent regenerative proliferation of bipotential hepatic stem/progenitor cells that have the ability to differentiate into both hepatocytes and cholangiocytes, but there is no definitive evidence for it [23]. Pigmented macrophages contained deposits of lipofuscin, hemosiderin and copper salts, possibly as a result of an increase of their oxidative stress with iron-catalyzed production of reactive oxygen species causing oxidative damage to lipids and proteins [36]. In vitro, the autoxidation of reduced sporidesmin is catalyzed by iron and by copper and generate a dithiol, a superoxide free radical suggesting that any superoxide production from sporidesmin in vivo would be mediated by the intracellular transport pool of copper [37]. Lipofuscin and copper are also deposited in biliary cirrhosis and chronic cholestatic diseases, respectively [38].

In this study liver vascular lesions were constantly seen in the chronic phase of FE near affected bile ducts. It is known that extrahepatic and intrahepatic bile ducts are located with branches of the hepatic artery (their sole blood supply) and portal vein [39]. Eccentric subintimal fibroblastic proliferation on the side adjacent to affected bile ducts were sometimes seen in sheep and goats [1,8] and adult cows [24]. It has been suggested that a high concentration of sporidesmin injures the biliary epithelium and the release of toxin and bile acids produce irritative lesions and coagulative necrosis of blood vessels, both arteries and veins [34].

Ductular reaction (DR) appears to be one of the factors that deteriorate liver function, because gradually replaces the hepatic parenchyma and causes a gradual decrease in mature hepatocytes [40]. In this study DR was recognized as bile duct hyperplasia in extensive areas of the liver in chronic FE cases and the cells present in the lesion, immunostained positively for pancytokeratins but did not express mesenchymal cell markers [23]. DR is observed in cholestatic liver diseases and is closely related to liver fibrosis induced by HSCs and portal fibroblasts and is also an important factor for liver regeneration [41]. According to these last authors, the mechanism responsible for DR is not definitively understood and

cholangiocytes, hepatocytes, or hepatic progenitor cells can be the origin of active cells during DR, depending on specific liver injury.

In FE cirrhotic livers the fibrous septa contain large numbers of HSCs expressing protein α-SMA. It is known that, as HSCs activate, the expression of α-SMA is increased, which confers contractile potential to the cells [25]. HSCs are the major source of type I collagen and other extracellular matrix proteins that characterize the fibrotic liver [25,42].

The sporidesmin elicits biliary insult and an activation of HSCs alongside induction of hepatic inflammation. In the acute exposure to this toxin, an inflammatory response of neutrophils, lysozyme and calprotectin in KCs was observed in areas of cholestasis. This fact could sugges<sup>t</sup> that KC response occurs early in cholestatic injury and bile acids leakage from cholangioles may be involved in this proliferation [27]. Previous studies indicate that HSCs activation also promotes the recruitment of leukocytes in the early phase of liver injury [26]. In chronic FE these macrophages were seen mainly around degenerated bile ducts and, to a lesser extent, in relation to DR. Nevertheless CD206+ macrophages were more numerous in this last location. These results sugges<sup>t</sup> that in the acute and chronic phases of FE, the peribiliar inflammatory infiltrate is dominated by classically activated M1 macrophages. As a detail, in the chronic phase there was an increase of activated M2 macrophages associated with increased fibrogenesis [43]. These data indicated that M1 macrophages prevail during the onset of liver injury, and M2 macrophages, if liver injury becomes chronic, take up a profibrotic role secreting TGF-β. In this work TGF-β expression was observed in cells consistent with macrophages in fibrotic septa in areas of DR. There is evidence supporting an indirect role of M2 macrophages in the development of fibrosis secreting factors like TGF-β, which activate HSCs [44,45]. Besides this, in the present work, TGF-β immunostaining was observed in hepatic arterial vessels with vascular occlusive lesion. This cytokine plays a prominent role in vascular disorders such as the arterial thickening associated with pulmonary hypertension [46] and in other arterial pathologies, with effects on the changes of the vascular smooth-muscle cells during transition from structural to a synthetic phenotype [47]. It has been also documented that both M1 and M2 macrophages accumulate in fibrotic septa of mouse and human end-stage cirrhotic livers, suggesting that both are necessary in fibrotic responses [45]. An interesting finding in the hepatic immune response in the case of FE was the presence of numerous T CD3+ lymphocytes in proximity to HSCs in the fibrous septa and around damaged bile ducts in chronic FE lesions. There is evidence that HSCs secrete cytokines, such as TGF-β, that has lymphocyte chemotactic activity and contributes to recruit and positioning of lymphocytes within the liver stroma in order to maintain an effective immune response [26]. In addition, it has been suggested that T lymphocytes can interact with HSCs and secrete various cytokines to modulate and sustaining fibrotic responses in chronic liver disease [26,48]. In chronic FE sheep an accumulation of CD3+ lymphocytes, and few plasma cells expressing IgG were observed, similarly to alpha-naphthylisothiocyanate (ANIT)- induced biliary pathology in mice and other cholestatic liver diseases in humans [49]. The vascular and ductal system and its macroscopic shape of sheep's liver are very similar to the human organ, so it has a grea<sup>t</sup> potential as an animal model [50].
