Liver and Biliary System

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Abstract

Acknowledgments The authors acknowledge the major contributions of Dr. W. Roger Kelly and Dr. M.A. (Tony) Hayes as previous authors of this section. In addition, the authors thank Drs. W. Roger Kelly and Jeremy Allen for critical review, discussions, and provision of images. General Considerations From a contemporary perspective, the liver is a marvel of biology. It is the guardian of homeostasis, the epicenter of the body's metabolic capability, a massive filter detoxifying the portal blood releasing cleansed blood to the systemic circulation, and a lymphoid organ protecting against infection. However high our regard for the liver, it is dwarfed by the perspective of ancient civilizations that regarded the liver as the seat of life and window to the future. In ancient Mesopotamia and Babylonia, the liver was used to divine the future using a technique termed hepatoscopy. This interest is captured in a Biblical quote from Ezekiel 21 : 21, “For the king of Babylon stands at the parting of the way, at the head of the two ways, to use divination; he shakes the arrows, he consults the household idols, he looks at the liver.” Hepatoscopy was continued by the Greeks, Etruscans, and the Romans. The practice is continued in a fashion today. Liver injury or neoplasia foretells a poor future for many pharmaceuticals in development. The liver plays a central role in processing dietary carbohydrates, lipids, amino acids, and vitamins; in the synthesis and turnover of most plasma proteins; and in the detoxification and biliary excretion of endogenous wastes and xenobiotic compounds. The liver also functions as an important organ of the innate immune system, integrated into the complex system of defense against foreign macromolecules. As such, hepatic disorders have far-reaching consequences, given the dependence of other organs on the metabolic function of the liver. Origin, structure, and function The embryonic origin of the liver is an out-pouching of the embryonic endoderm forming the duodenum termed the hepatic diverticulum or the liver bud. Primitive epithelial cells of the hepatic diverticulum extend into the adjacent mesenchymal stroma of the septum transversum, a sheet of cells that incompletely separates the pericardial and peritoneal cavity and that will develop into the connective tissue of the liver. The primitive epithelial cells are arranged in close approximation with the vessels that form the vitelline venous plexus, a complex of vessels that drain the yolk sac. Thus the essential sinusoidal arrangement of the liver is established very early in development. The gallbladder and the cystic duct arise from the caudal part of the hepatic diverticulum. The hepatic diverticulum is also the origin of the biliary epithelium. Development of the biliary tree begins at the hilus and spreads outward to reach the subcapsular zone over time. Intrahepatic bile ducts develop from the ductal plate, a structure that is composed initially of a single row of hepatoblasts that surround the portal vein branches and ensheath the mesenchyme of the primitive portal tract. The development of the hepatoblasts adjacent to the portal tract mesenchyme is altered by interactions with the mesenchyme. Initially, these cells form the ductal plate, a single row of cells surrounding the portal tract. The cells of the ductal plate can be identified by expression of cytokeratin 7, differentiating them from the hepatoblasts (Fig. 2-1 ). A second discontinuous outer layer of cells forms subsequently, and the 2-cell–thick regions remodel into tubules. Most undergo apoptosis, but 1 or 2 ducts become incorporated in the developing portal tract. At about the time the bile ducts are forming, the hepatic artery branches appear in the developing portal tract. Nerves and lymphatics eventually invest the portal tracts as well, completing the mature portal tract. The wave of development from the hilus to the subcapsular region is imperfect, as the veins, arteries, and bile ducts reach their terminal ends separately. Consequently, in human liver, up to 30% of subcapsular portal tracts are “dyads” containing only bile duct and artery profiles. Sinusoidal lining cells other than the endothelium likely arise in the bone marrow and populate the liver via a hematogenous route. Figure 2-1 The ductal plate of a fetal dog with 2-cell–thick rows of cytokeratin 7–positive cells formed at the edge of the developing portal tract. The liver is the largest internal organ in the body. In adult carnivores, the liver constitutes ~3% of body weight. In adult omnivores, it is ~2% of body weight and ~1% of body weight in adult herbivores. In neonates of all species, the liver is a larger percentage of body weight than in the adult. The liver has a smooth capsular surface, and the parenchyma consists of friable red-brown tissue that is divided into lobes. The number and shape of the liver lobes of the major domestic mammals vary among species. In monogastric animals, the liver abuts the diaphragm and occupies the central area of the cranial abdomen. In ruminants, and to a lesser extent in horses, the liver is displaced to the right side of the cranial abdominal cavity. A series of ligaments maintains the liver in its position. The coronary ligament attaches the liver to the diaphragm near the esophagus. The falciform ligament attaches the midline of the liver to the ventral midline of the abdomen. The round ligament, a remnant of the umbilical vein, is embedded within the falciform ligament. The liver receives ~25% of the cardiac output, but is also supplied by the portal vein. The valveless portal vein drains the digestive tract, forestomachs, glandular stomach, and intestines, as well as the spleen and pancreas. Portal vein flow provides 70-80% of the total afferent hepatic blood flow and ~50% of the oxygen supply. The hepatic artery provides the remainder of hepatic blood flow. Portal blood flow is not regulated, but hepatic arterial flow is regulated, primarily by adenosine, and responds to changes in portal flow. When portal blood flow is reduced, less adenosine is washed out of the hepatic circulation, and this increased concentration drives hepatic arterial dilation, creating a hepatic arterial buffer effect whereby a consistent hepatic blood flow is maintained. Portal blood flow is important for the rapid clearance of nutrients, xenobiotics, microorganisms, and potentially immunogenic materials that enter the circulation from the gastrointestinal tract. Hepatic arterioles disperse into a peribiliary capillary plexus, a perivenous plexus surrounding the portal vein, or join terminal hepatic arterioles before entering the sinusoids, lowering pressure and preventing reversal of portal venous inflow. Portal and arterial blood eventually mix in the low-pressure hepatic sinusoids. Both terminal portal venules and hepatic arterioles flow into the sinusoids, but flow is closely regulated by a series of inlet sphincters formed by endothelial cells for the venules and smooth muscle for the arterioles. Thus, at any particular time, sinusoidal blood could be entirely venous, mixed arterial and venous, or arterial. This blood flow pattern may account for the interlobular and intralobular heterogeneity of lesions following various toxicities. Blood leaves the liver via the hepatic vein, which is very short, and enters the caudal vena cava. Hepatic sinusoids have an average diameter of 10 µm, but can expand up to 30 µm. The periportal sinusoids are more tortuous than those in the centrilobular region. Hepatic sinusoids are lined by specialized endothelial cells. Hepatic sinusoids differ from vascular structures elsewhere in that they lack a typical basement membrane, and are supported by a specialized, discontinuous or loose extracellular matrix (ECM). Hepatic sinusoidal endothelial cells are fenestrated, and these 100-nm diameter sieve-like pores control fluid, solute, and particulate interchange between blood and the perisinusoidal space, regulated by the action of the cellular cytoskeleton. Sinusoidal endothelial cells are actively pinocytotic and internalize and degrade various endogenous glycoproteins, glycosaminoglycans, and immune complexes. Kupffer cells are fixed macrophages attached to the inner sinusoidal wall in direct contact with blood moving at a relatively low velocity. This arrangement facilitates phagocytic removal of particulates, especially bacteria that enter the portal blood via the lower alimentary tract. Kupffer cells also participate in the regulation of inflammatory and repair responses by secretion of various cytokines into the circulation and perisinusoidal space. Natural killer cells (formerly referred to as pit cells) are large granular lymphocytes with natural killer activity that adhere to the sinusoidal endothelium, where they are also well situated to participate in various innate immune defenses, for example, targeting infected cells that enter the liver via the blood. The sinusoids are separated from the adjacent hepatocellular plates by an extracellular space, known as the space of Disse, that contains hepatic stellate cells (also termed lipocytes or Ito cells), reticulin fibers, and nerves. The space of Disse is not readily visible by light microscopy unless there is fluid retention, such as can occur with impediment to venous outflow. Although hepatic sinusoids in the normal liver lack a conventional basement membrane, the perisinusoidal space contains a low-density ECM consisting of collagen type IV; laminin; fibronectin; minor amounts of collagen types I, III, V, and VI; nonfibrillar collagen XVIII; tenascin; and various proteoglycans. A conventional ECM composed of fibrillar collagen types I, III, V, and fibronectin is found in the external capsule (Glisson's capsule), septa, and around portal triads and central veins. “Reticulin fibers” are the components of the ECM that are stainable by silver impregnation techniques, consisting mainly of collagen type III with attached fibronectin and other glycoproteins. The fenestrated sinusoidal endothelium, coupled with the loose subendothelial matrix, allows for exchange of various macromolecules between hepatocytes and the sinusoidal blood. After hepatic injury, a denser, less permeable matrix resembling a true basement membrane may form, and sinusoidal endothelial cells may lose their fenestrae (so-called “capillarization” of sinusoids), reducing uptake and secretion of plasma proteins and other metabolically important substances. The terminal hepatic venules (“central veins”) collect the outflow blood from the sinusoids. These venules converge into the larger hepatic veins that empty into the caudal vena cava. In most species, increased pressure in the vena cava during right-sided heart failure or hepatic vein thrombosis causes passive congestion and distension of the hepatic veins and sinusoids. However, the large and small hepatic veins in dogs have a prominent spiral circumferential smooth muscle that can also affect the central venous pressure on the sinusoids. Fluids from the perisinusoidal space drain into lymphatics in the extracellular connective-tissue spaces of the liver capsule, the portal tracts, and the connective tissue of the terminal veins. These flow out the portal hilus to the hepatic lymph nodes and eventually enter the thoracic duct. In some species, such as the dog, there are also lymphatics around the larger hepatic veins; these cross the diaphragm into the mediastinum. The liver is the largest lymph producer in the body, contributing 20-50% of the thoracic duct flow. Hepatic lymph is high in protein, containing 85-95% of the protein of plasma and a high cell count composed of lymphocytes and macrophages. In sheep, more lymphocytes pass through the liver than any typical lymphoid organ, and ~2 × 108 macrophages leave the liver in lymph daily. Hepatic nerves contain both sympathetic and parasympathetic fibers. The fibers invest major blood vessels and also extend along the sinusoids. These may modulate function of hepatocytes, endothelial cells, and hepatic stellate cells. The vasculature of the liver parenchyma defines its functional microanatomy, but debate continues as to what best represents the hepatic structural-functional unit. Mammalian hepatocytes are organized in plate-like monolayer arrays among the sinusoids and in 3 dimensions; plates, sinusoids, and tracts anastomose in a complex pattern. Currently, a somewhat baffling array of models exists, each with their own adherents. These include the well-known lobular and acinar patterns (discussed later) and several others. Matsumoto's primary lobule is based on detailed reconstructions of human liver sections, and considers the penetrating venule extending from the portal tract as a “vascular septum” and the origin of the primary lobule's blood flow as it is a starting place for the radially arranged sinusoids flowing to the terminal hepatic vein. In this model, there is a series of branches formed by the portal vein. The first branches provide a conducting portal flow, and the next level of branches drain directly into the sinusoids forming the distributing portal flow. The choleohepaton, related to the concept of the nephron, is composed of an isosceles triangle of hepatocytes with its apex in contact with the terminal hepatic venule and drained by a single bile ductule/canal of Hering at the base of the triangle. More detail is available in the cited references. Arrangements of hepatocytes in the most commonly used nomenclature systems are referred to as either acini or lobules. • The classic hepatic lobule is a 6-sided anatomic arrangement of hepatocytes centered on the terminal hepatic venule, also termed the “central vein” in this context. Peripherally, lobules are outlined by fibrovascular septa extending from the portal tracts. In the pig liver, septa form obvious lobular perimeters, but in most mammalian species, the lobules are less pronounced because connective tissue is more restricted to portal tracts. The terms periportal and centrilobular are mainly used for pathologic conditions that are centered on the hepatocytes surrounding the portal tracts or the central veins of the classic lobule. • The hepatic acinus of Rappaport is a functional diamond-shaped subunit divided into zones in relation to blood supply: • Zone 1 hepatocytes are arranged around an axis formed by the portal tract and the distributing vascular branches that leave the portal tract and are closest to the oxygen- and nutrient-rich arterial and portal inflow. • Zone 2 is the transitional midzone. • Zone 3 (periacinar) hepatocytes form the apex of the diamond-shaped acinus, are nearest the outflow (terminal hepatic venule), and are exposed to reduced oxygen and nutrients. The functional activity of hepatocytes is heterogeneous, and virtually all liver functions have a zonal gradient. Periportal hepatocytes, exposed to the blood with the highest concentration of oxygen, insulin, glucagon, and amino acids, are the principal site of gluconeogenesis, protein synthesis, aerobic metabolism, urea cycle, and lipid and cholesterol metabolism. In the centrilobular region, glycolysis, lipogenesis, and the major biotransformation functions are more active, including the expression of most cytochromes P450, glucuronyl transferases, glutathione S–transferases, and other biotransformation/detoxification enzymes. Centrilobular hepatocytes are therefore more susceptible to hypoxic injury as well as injury by toxic substances that are metabolically activated by cytochromes P450. By comparison, hepatocytes in periportal hepatocytes are more susceptible to direct-acting toxicants, such as ingested metal salts, given their proximity to the vascular inflow. Under the influences of various inducers, the patterns of enzyme expression can extend beyond the resting limits. Lobular variation is not restricted to parenchymal cells, but is also apparent in the structure and function of sinusoidal endothelial cells, Kupffer cells, perisinusoidal stellate cells, and the composition of the matrix in the space of Disse. The portal tract, or portal triad, is a well-defined structure containing at least one small arterial branch, a portal vein branch, and a bile duct, surrounded by connective tissue composed primarily of type I collagen (Fig. 2-2 ). Because of the pattern of progressive branching of the portal tract system, individual tracts exhibit a range of sizes and shapes, from round to triangular or branching. In larger portal tracts, lymphatic channels and autonomic nerve fibers may be seen. The bile duct system is a branching outflow that ultimately enters the proximal duodenum. Most species, with the exception of the horse and rat, have a bile storage diverticulum (gallbladder). Cats occasionally have divided or bipartite gallbladders. The bile duct joins the pancreatic duct before entry into the duodenum in some species and has a separate entry in others. Intrahepatic bile ducts range in size from the larger septal or trabecular ducts (internal diameter of >100 µm in humans) to the smaller interlobular ducts, and tend to be adjacent to a hepatic artery branch of approximately the same size. Bile ducts are lined by cuboidal to low columnar bile duct epithelial cells, subtended by a periodic acid–Schiff (PAS)-positive basement membrane. Bile ductules are smaller yet (lumen size of <20 µm) and are located at the periphery of portal tracts. Figure 2-2 The normal portal tract contains a branch of the portal vein and hepatic artery, as well as a bile duct. The first row of hepatocytes adjacent to the portal tract connective tissue is termed the limiting plate. Cells of the liver Hepatocytes (referred to as parenchymal cells) constitute ~70-80% of the liver mass. However, >50% of liver DNA is found in smaller nonparenchymal cells (bile duct epithelium, hepatic stellate cells, sinusoidal endothelium, Kupffer cells) and itinerant cells (such as leukocytes). The hepatocyte is a polygonal epithelial cell, ~30-40 µm in diameter, arranged in single-cell–thick anastomosing plates, separated by hepatic sinusoids. Each hepatocyte is therefore exposed to sinusoidal blood on 2 sides. A discontinuous line of hepatocytes, termed the limiting plate, is found at the interface with the collagenous ECM of the portal tract. Normal hepatocytes have abundant eosinophilic cytoplasm, and most have a single, round, centrally placed nucleus with finely dispersed chromatin and at least one nucleolus. Some binucleate hepatocytes are present normally in mammals and can become more numerous in response to various stimuli and injuries that induce or affect regeneration. Hepatocytes are metabolically highly active cells, containing an array of organelles, including smooth and rough endoplasmic reticulum, mitochondria, lysosomes, peroxisomes, Golgi complexes, and transport vesicles. These organelles support a variety of hepatocellular functions, including the synthesis and secretion of plasma proteins, coagulation factors, and acute-phase proteins. Hepatocytes store nutrients in times of adequate energy and release glucose when needed. They are key modulators of lipid metabolism, and they synthesize and secrete lipoproteins. In addition, they are the only cells capable of bile acid synthesis, and they can absorb and secrete them into bile. Finally, hepatocytes detoxify the large majority of xenobiotics and secrete them into the bile. Because of this central role in metabolism, the liver is subjected to a variety of nutritionally based insults as well as toxin-related damage. A greater proportion of the genome is expressed in the normal liver than has been observed in any other tissue, an indication that brief surveys of liver functions are necessarily oversimplified. However, those constituents that are most abundant have most influence on the microscopic appearance of the liver. The normal hepatocyte contains abundant glycogen, which varies depending on food intake, and which can be demonstrated by PAS staining, as well as variable amounts of stored triglycerides and various proteins, such as ferritin, an iron-binding protein. The cytoplasm of the centrilobular hepatocytes may also contain uniform, golden-brown granules of lipofuscin, particularly in older cats. This so-called “wear-and-tear” pigment becomes more prominent with age, and progressively accumulates in midzonal and periportal hepatocytes. Hepatocytes have a cytoskeleton composed of microtubules, microfilaments, and intermediate filaments. Microtubules are found throughout the cytoplasm, and are involved in the movement of secreted proteins into the extracellular perisinusoidal space; accordingly, microtubule inhibitors such as colchicine and Vinca alkaloids may reduce hepatic protein secretion. Microfilaments, composed of actin and myosin, are concentrated around the bile canaliculus, where they are involved in canalicular peristalsis and bile secretion; microfilament inhibitors result in cholestasis. Intermediate filaments (predominantly cytokeratins 8 and 18) form an irregular meshwork extending from the plasma membrane to the perinuclear zone, and are responsible for spatial organization of the hepatocyte. There are 3 morphologically and functionally distinct surfaces of the hepatocyte plasma membrane. 1. The sinusoidal domain faces the space of Disse and has numerous irregular microvilli, increasing hepatocyte surface area by ~6-fold (considerably less that that seen in enterocytes). This specialized membrane is modified to facilitate an exchange of substances with the blood. There are ultrastructurally evident pits between the villi, some of which represent secretory vacuoles in the process of exocytosis, sending various products into the plasma, and others are clatharin-coated pits involved in selective receptor-mediated endocytosis. Numerous membrane receptors for glycoproteins, asialoglycoproteins, peptides, hormones, growth factors, immunoglobulin A, and other endocytotic or signaling ligands are found at the sinusoidal pole. In addition, transmembrane proteins involved in plasma exchange of small ionic substances with the sinusoidal plasma, and transmembrane proteins responsible for matrix recognition, are concentrated on the sinusoidal surface. 2. The lateral domain extends from the sinusoidal surface to the edge of the canaliculus. This portion of the cell membrane is specialized for adhesion via junctional complexes, including desmosomes, tight junctions, and intermediate junctions, as well as for intercellular communication via gap junctions. 3. The canalicular domain is the beginning of the bile drainage system of the liver. The canaliculus is an intercellular space between 2 adjacent hepatocytes, isolated by junctional complexes. The canalicular surface is covered with an irregular array of microvilli. Canalicular diameter increases as it approaches the periportal region, enlarging from ~0.5-2.5 µm. Bile is propelled along the canaliculi by a web of contractile microfilaments. This specialized membrane contains various adenosine triphosphate (ATP)-dependent carriers that export many products, including leukotrienes, bile salts, xenobiotics, and their metabolites into the bile. The canal of Hering is partly lined by biliary epithelium and partly by hepatocytes and connects the bile canaliculus to the cholangioles and eventually the interlobular bile ducts. Cholangiocytes (biliary epithelium) account for ~3-5% of the liver cell population. Although derived from common embryologic progenitor cells, cholangiocytes differ from hepatocytes in both phenotype and function. They contain a strongly developed network of intermediate filaments, including cytokeratins 7 and 19. They also express marked heterogeneity along the anatomic course of the biliary system. Functionally, bile duct epithelial cells actively modify the composition of bile. Secretion is primarily under the control of secretin and somatostatin. Secretin released from the duodenum triggers secretion of bicarbonate-rich fluids that buffer acids released from the stomach. Cholangiocytes secrete immunoglobulin A (IgA) and IgM, but not IgG. Absorption involves the sodium-dependent glucose transporter and aquaporins responsible for glucose and water uptake, as in the renal proximal tubule. They express γ-glutamyltranspeptidase, which removes glutamic acid from glutathione conjugates. Hepatic progenitor cells (HPCs), formerly known as oval cells in rodents, reside in the region of the canal of Hering. These cells are bipotential and can mature into either biliary epithelium or hepatocytes. They can proliferate and form a type of ductular reaction. In cases of massive hepatic necrosis in which the animal survives for a few days after the initial injury, HPCs can proliferate dramatically, forming cords or small caliber ducts lined by cuboidal basophilic cells with abundant mitochondria. These cells can mature and replace lost hepatocytes and bile ducts. HPCs in humans and rats contain both markers of hepatocyte phenotype (i.e., albumin) and biliary phenotype (i.e., cytokeratin 7). Similar markers have been described in dogs and cats as well. Bone marrow–derived pluripotential stem cells also appear to have the ability to differentiate into hepatocytes. Other types of ductular reaction are discussed in the section on Responses of the liver to injury. Hepatic endothelial cells are specialized, perforated by numerous fenestrations, ~175 nm in diameter, and often clustered together forming sieve plates. Larger, but less frequent, fenestrations are more common at the periportal end of the sinusoid, but opening size is dynamic, responding to endogenous mediators and toxins. The endothelial cells rest on a very thin and discontinuous ECM. The fenestrations allow direct contact between the sinusoidal lumen and the space of Disse. Only larger particles, such as chylomicrons, and cells are excluded. Sinusoidal endothelial cells differ from normal vascular endothelium in several additional ways, including the absence of factor VIII–related antigen (except in inflammatory conditions) and high endocytic activity. Endocytosis of immune complexes and some proteoglycans are major functions. They also synthesize molecules that affect vascular tone such as nitric oxide, endothelins, and prostaglandins. Kupffer cells are specialized macrophages located in sinusoidal lumens, mainly at branch points. Once thought to be “fixed,” it is now known that they can migrate along the sinusoid and into areas of tissue injury (Fig. 2-3 ). Kupffer cells may have a dual origin as they are derived, at least in part, from blood-borne monocytes, but they are also capable of local proliferation, particularly in inflammation. They are not efficient antigen presenters, but they are proficient phagocytes of apoptotic and necrotic cells, particulates, and microorganisms; consequently, the liver is a major “filtering organ” for the body. There are species differences in the efficiency of this process. Clearance of particulates, and endotoxin in particular, is accomplished more effectively by Kupffer cells in dogs, humans, and laboratory rodents than in ruminants, horse, pig, cats, and whales, species that have a significant population of intravascular macrophages in the pulmonary vasculature. Kupffer cells can phagocytose a variety of gut-derived materials; bacteria, various biologically active bacterial components, including lipopolysaccharides, lipoteichoic acids, and peptidoglycans, without stimulating inflammation. Activated Kupffer cells can secrete tumor necrosis factor-α and other cytokines, and nitric oxide; these contribute to peripheral vasodilation and hypotension in systemic inflammatory response syndromes initiated by bacterial components. Other secreted cytokines, such as interleukin-1 (IL-1) and IL-6, mediate the acute-phase response and some aspects of the immune and liver regenerative responses. However, there can be a balance in proinflammatory and anti-inflammatory signaling as there are distinct differences in the signaling repertoire of different Kupffer cells. Some Kupffer cells are more likely to secrete IL-10, which can suppress macrophage activation and cytokine secretion. Cytokine responses of Kupffer cells are believed to be important in regulating the extent of adaptive immune response or tolerance to potentially antigenic macromolecules that can reach the liver through the portal blood. Figure 2-3 Kupffer cells, stained with antibodies against myeloperoxidase, line the sinusoids at regular intervals. The liver contains large numbers of lymphocytes, comprising ~5% of the entire cell population of the liver, with an organ-specific lymphocyte distribution characterized by the enrichment of elements of the innate immune system, including natural killer T lymphocytes (NKT cells), natural killer (NK cells), and innate lymphocytes, in addition to the Kupffer cells previously mentioned. Elements of the acquired immune system, CD8+ T cells, are also increased when compared with peripheral blood. The majority of intrahepatic lymphocytes are involved in innate immune responses rather than acquired immunity. Hepatic NK cells constitute ~40% of hepatic lymphocytes and are distinct phenotypically and functionally from blood NK cells. Hepatic NKT cells reside in the space of Disse, are considered large granular lymphocytes, and were previously referred to as pit cells. Intrahepatic NK cells have important functions in defense against foreign antigens released from the gut, viral infections, metastatic tumors, hepatocellular carcinoma, and modulation of hepatic fibrosis. The liver also contains the largest population of γδ T cells in the body. Although the precise function of these diverse lymphocyte types is not currently understood, their large number alone suggests that they must be involved in immunologic homeostasis and respond to immunologic challenges, indicating that the liver can be considered a lymphoid organ. Hepatic dendritic cells play an important role in the induction and regulation of immune responses. These antigen-presenting cells have only recently been studied in the liver. There are several other antigen-presenting cells in the liver, including the sinusoidal endothelial cells and Kupffer cells. Unlike cells that reside within the sinusoid, hepatic dendritic cells are found within the portal tract. Hepatic dendritic cells are considered to be functionally immature compared to the dendritic cells of the spleen and bone marrow, and they may be involved in immune tolerance in the liver. Hepatic stellate cells (HSC), originally described by Boll and von Kupffer in the 1870s, were neglected until the 1950s, when they were described in detail by Ito. They have also been known as lipocytes, Ito cells, or fat-storing cells. HSC reside in the space of Disse, but there are other populations of similar cells with the ability to produce ECM and to transform into a myofibroblast phenotype within the connective tissue of the portal tract and centrilobular veins. There are 4 key functions of HSC: (1) storage and homeostasis of retinoids, including vitamin A; (2) maintenance and remodeling of the sinusoidal ECM in health and disease; (3) production of growth factors, such as hepatocyte growth factor and various cytokines; and (4) regulation of sinusoidal diameter by contraction of cellular processes. This may be in response to adrenergic stimulation, as all HSC are in contact with autonomic nerve fibers. HSC can become greatly distended with lipid in carnivores on some diets. Activation of HSC has been extensively studied because of their importance in hepatic fibrosis. In the transition from quiescence to activation, HSC lose their characteristic lipid droplets, possibly catabolizing the lipid to support their activation. They then develop a myofibroblast phenotype characterized by the expression α–smooth muscle actin. Proinflammatory cytokines released primarily by Kupffer cells, such as transforming growth factor-β released in response to tissue injury, stimulate HSC to increase the deposition of ECM, including collagen type I, III, and IV, and laminin. This new ECM transforms the sinusoid to a less permeable capillary, lined by a basement membrane–like layer and without fenestrations in the sinusoidal endothelium, reducing transfer of macromolecules between hepatocytes and the blood. The acquired contractility of the HSC during fibrogenesis is increased because of an increase in the contractile stimulus of endothelin-1 and a reduction in vasodilation driven by diminished nitric oxide generation. In severe chronic injury leading to cirrhosis, the increased expression of contractile proteins within activated stellate cells can further restrict sinusoidal blood flow as a primary effect, rather than a consequence of nodule formation and fibrosis. Peribiliary fibrosis arises from activation of the circumferential fibroblasts of the bile ducts that undergo a phenotypic transformation similar to that of the HSC along the sinusoids. Fibrosis of the portal tracts and the central vein connective tissue develops from activation of myofibroblasts resident in these areas as well. It is also possible that epithelial-mesenchymal transition of hepatocytes, biliary epithelial cells, or HSC can contribute to hepatic fibrosis during chronic injury. Mast cells are abundant in the liver, particularly in dogs. They typically occupy a perivenous location, where they may influence vascular tone and respond to various potentially injurious substances or organisms. Degranulation of mast cells in the liver leads to contraction of the spiral smooth muscle, restricting blood outflow from the canine liver. This is a feature of shock in dogs. Hematopoiesis in the fetal life of mammals occurs mainly in the perisinusoidal compartment of the liver sinusoids. Postnatally, hepatic hematopoiesis declines but can return as extramedullary hematopoiesis in conditions of increased demand (Fig. 2-4 ). Because the liver is an early site of hematopoiesis, the environmental conditions and cells, including resident populations of appropriate stromal cells and, possibly hematopoietic stem cells, remain supportive for the initiation or reactivation of a stem cell niche. The degree of hepatic extramedullary hematopoiesis in larger species can have diagnostic significance, but in laboratory rodents and other small animals, it can be an incidental observation. Figure 2-4 Hepatic extramedullary hematopoiesis in a dog. Further reading Crawford JM, Burt AD. Anatomy, pathophysiology and basic mechanisms of disease. In: Burt AD, et al., editors. Macsween's Pathology of the Liver. 6th ed. New York: Churchill Livingstone; 2012. p. 2-77. Desmet VJ. Ductal plates in hepatic ductular reactions. Hypothesis and implications. I. Types of ductular reaction reconsidered. Virchows Arch 2011;458:251-259. Desmet VJ. Ductal plates in hepatic ductular reactions. Hypothesis and implications. III. Implications for liver pathology. Virchows Arch 2011;458:271-279. Dixon LJ, et al. Kupffer cells in the liver. Compr Physiol 2013;3:785-797. Ekataksin W, Wake K. The anatomy and physiology of the liver. In: Boyer JL, Ockner RK, editors. Progress in Liver Disease. Philadelphia: WB Saunders; 1997. p. 1-30. Johns JL, Christopher MM. Extramedullary hematopoiesis: a new look at the underlying stem cell niche, theories of development, and occurrence in animals. Vet Pathol 2012;49:508-523. Malarkey DE, et al. New insights into functional aspects of liver morphology. Toxicol Pathol 2005;33:27-34. Matsumoto T, Kawakami M. The unit-concept of hepatic parenchyma—a re-examination based on angioarchitectural studies. Acta Pathol Jpn 1982;32(Suppl. 2):285-314. Oda M, et al. Regulatory mechanisms of hepatic microcirculatory hemodynamics: hepatic arterial system. Clin Hemorheol Microcirc 2006;34:11-26. Winkler GC. Pulmonary intravascular macrophages in domestic animal species: review of structural and functional properties. Am J Anat 1988;181:217-234. Yamamoto K. Electron microscopy of mast cells in the venous wall of canine liver. J Vet Med Sci 2000;62:1183-1188. Developmental Disorders Hepatic cysts Serous cysts are occasionally found attached to the capsule on the diaphragmatic surface in calves, lambs, and foals (Fig. 2-5 ). These cysts are usually small and multiple, but some are isolated and very large. Cyst walls are composed of connective tissue lined by flattened or cuboidal epithelium. The content is clear and serous. Their origin is not known, but it is variously postulated that they are serosal inclusion cysts, part of congenital polycystic biliary anomalies, or of endodermal origin. They do not contain bile. The declining incidence of these anomalies with age suggests that a large proportion of them involute in the early postnatal period. Figure 2-5 Serous cyst attached by a stalk to the hepatic capsule in a 3-day-old Holstein calf. (Courtesy J.L. Caswell.) Solitary biliary cysts—single round cysts lined by a flattened single layer of biliary epithelium—are uncommon and may be congenital or acquired. Multiple hepatic peribiliary cysts putatively arising from peribiliary glands have been reported in a 6-month-old pig. Hamartomas Von Meyenburg complexes (biliary microhamartomas) are developmental malformations arising from persistent embryonic ductal plate remnants. These are discrete, usually subcapsular, fibrotic areas containing small, irregularly shaped, often dilated, U-shaped or branching, bile duct–like structures lined by low cuboidal epithelium (Fig. 2-6 ). Figure 2-6 Von Meyenburg complexes in a dog liver. Mesenchymal or mixed liver hamartomas, rare benign tumor-like lesions characterized by disorganized hepatocellular and/or biliary structures embedded in a mucinous primitive mesenchyme have been reported in 2 equine fetuses. Ductal plate malformations Persistence and/or aberrant remodeling of the embryonic ductal plate can give rise to a spectrum of cystic biliary diseases. Congenital hepatic fibrocystic diseases, part of the group of hepatorenal fibrocystic disease that includes the polycystic kidney diseases, are a product of ductal plate malformations occurring at different levels of the biliary tree. Analysis of the underlying genetic basis of the human hepatorenal fibrocystic diseases has identified defective protein components in primary cilia and associated basal bodies. These mechanotransducer organelles are involved in environmental monitoring, signal transduction, and cell proliferation, and are important in the normal development of the biliary system in the liver, as well as renal tubules. As such, many of these diseases are now considered “ciliopathies.” In human hepatopathology, the hepatorenal fibrocystic diseases can be grouped into 3 descriptive categories: (1) polycystic liver disease (often seen in association with autosomal dominant polycystic kidney disease of adults), characterized by isolated microscopic to macroscopic unilocular or multilocular cysts in a fibrous stroma, with no continuity with the intrahepatic biliary tree, thought to originate from von Meyenburg complexes in the most peripheral branches of the biliary tree; (2) congenital hepatic fibrosis (often seen in association with autosomal recessive polycystic kidney disease of childhood), characterized by defective remodeling of the ductal plate at the level of interlobular ducts, with excess abnormally shaped embryonic bile ducts retained in the primitive ductal plate configuration, abnormal portal veins, and progressive fibrosis of the portal tracts; and (3) Caroli disease, characterized by non-obstructive saccular or fusiform dilation of medium- and large-sized intrahepatic bile ducts, with maintenance of continuity with the biliary system. Caroli syndrome refers to Caroli disease co-occurring with congenital hepatic fibrosis. In veterinary medicine, a similar classification of the liver lesions has been proposed: adult polycystic disease (including von Meyenburg complexes), juvenile polycystic disease/congenital hepatic fibrosis, and congenital dilation of the large and segmental bile ducts (resembling Caroli disease). Congenital cystic lesions involving the hepatic biliary system and kidneys have been reported in juvenile dogs, cats, pigs, goats, and foals, and have been compared to congenital hepatorenal fibrocystic disorders of humans. Cysts may also be found in the pancreas or other organs. Animals may die from progressive renal insufficiency, and/or from hepatic dysfunction and portal hypertension associated with hepatic fibrosis. Hepatic fibrosis and cysts are present in a significant proportion of cats with polycystic kidney disease, inherited in Persian cats, exotic shorthaired, and other related breeds as an autosomal dominant C→A transversion mutation in exon 29 of the feline PKD1 gene, resembling the adult form of polycystic kidney disease in humans. The liver lesions have been more difficult to classify, and may appear as multiple large cysts resembling adult-type polycystic disease, as congenital hepatic fibrosis characterized by portoportal bridging fibrosis with excess abnormally formed bile ductules (Fig. 2-7 ), or as combinations of both lesions. Polycystic kidney and liver disease reported in West Highland White and Cairn Terrier litters resembles the autosomal recessive polycystic kidney disease of children. Congenital hepatic fibrosis has been described in dogs. Affected animals are presented at or before a year of age with clinical signs of liver disease, including ascites, microhepatica, and extrahepatic portosystemic shunts. Histologically, these dogs had livers with extensive bands of portal bridging fibrosis containing numerous small irregular, tortuous bile ducts, often accompanied by absent or hypoplastic portal veins and compensatory arteriolar proliferation, and with no evidence of nodular regeneration and minimal inflammation, allowing differentiation of this congenital condition from acquired chronic liver disease. Congenital hepatic fibrosis has also been reported in aborted and neonatal calves, in the latter case accompanied by cyst formation in the kidney and lung. Congenital hepatic fibrosis with cystic bile ducts has been described in Swiss Freiberger foals (Fig. 2-8 ) and is seen occasionally in other breeds, with generalized portal bridging fibrosis containing many small, irregularly formed and occasionally cystic bile ducts. Macroscopic congenital dilation of the large and segmental bile ducts and diffuse cystic kidney disease, resembling Caroli disease, has been reported in dogs. Figure 2-7 Congenital hepatic fibrosis in a Himalayan cat with polycystic kidney disease. Figure 2-8 Congenital hepatic fibrosis in a Swiss Freiberger foal. Extrahepatic biliary anomalies Cats occasionally have divided or bipartite gallbladders. Reduplication of the gallbladder has also been reported in swine. Other anomalies of the extrahepatic biliary system include agenesis of the gallbladder reported in dogs, and absence or atresia of one or more ducts, reported in lambs, calves (eFig. 2-1), foals, a cat, a dog, and a pig. In carnivores, bile duct atresia may lead not only to jaundice, but also to vitamin D–deficiency rickets, because of their inability to absorb fat-soluble vitamins. Congenital atresia may be associated with defects in the developmental morphogenesis of bile ducts, or in utero vascular, inflammatory, or toxic insults to the biliary tree that culminate in the obliteration of the lumen. eFigure 2-1 Congenital biliary atresia and gallbladder agenesis in a calf. (Courtesy B. Njaa.) Choledochal cysts arising from the cystic or common bile duct have been described in cats. Congenital vascular anomalies These include congenital portal vein aneurysms, hepatic arteriovenous malformations, congenital portosystemic shunts between the portal vein and other systemic veins, and primary hypoplasia of the portal vein. Extrahepatic congenital portosystemic shunts are readily distinguished from shunts that are acquired during portal hypertension, as acquired shunts are typically multiple, thin-walled, tortuous collateral venous connections between the portal vein or its tributaries and caudal vena cava, renal vein, or azygos vein (Fig. 2-9 ). Although multiple acquired shunts do not develop in the presence of congenital PSS, they can arise with other congenital abnormalities, such as arteriovenous malformations or hypoplasia or dysplasia of portal veins, as a consequence of portal hypertension. Acquired shunts resulting from portal hypertension secondary to liver injury and repair are discussed later in the section Vascular factors in hepatic injury and circulatory disorders. Figure 2-9 Multiple acquired portosystemic vascular shunts in a dog with chronic liver disease. Portal vein aneurysms, both congenital and acquired as a consequence of concurrent liver disease, have been described in dogs. Extrahepatic aneurysms were always located at the level of the gastroduodenal vein insertion. All were asymptomatic, although predisposed to portal vein thrombosis. Hepatic arteriovenous malformations have been reported in dogs and cats. These are congenital or, in some instances, acquired communications between branches of the hepatic artery, and more rarely, the gastroduodenal artery and left gastric artery and portal vein. Mixing of higher-pressure arterial blood with venous blood results in retrograde flow into the portal vein, arterialization of the portal circulation, and development of portal hypertension, with the opening of vestigial, low-resistance, collateral, extrahepatic portosystemic communications (acquired extrahepatic shunts). The fistulae may be macroscopic or microscopic, are typically multiple, and may involve one or more lobes of the liver. The hepatic parenchyma of affected lobes may be atrophied, with dilated, tortuous, pulsatile vessels visible on the capsular surface. Histopathologic findings include hyperplasia and anastomoses of arterioles and venules (Fig. 2-10 ). Affected vessels have irregularly thickened walls with intimal hyperplasia consisting of smooth muscle proliferation and deposition of elastin fibers, focal subintimal fibromuscular proliferation, and smooth muscle hyperplasia of the tunica media. Degenerative changes characterized by deposition of mucinous material and mineral in the intima and media of arterioles, as well as thrombosis and recanalization of portal veins, are also observed. Adjacent hepatic parenchyma may be atrophic, with periportal fibrosis, and bile duct hyperplasia, arteriolar proliferation, and relative collapse of portal vein branches within portal tracts. Arteriovenous fistulae may also be acquired, developing subsequent to abdominal trauma, rupture of hepatic artery aneurysms, and secondary to hepatic vein obstruction or cirrhosis with extreme portal hypertension. Figure 2-10 Congenital intrahepatic arterioportal fistulae with thick-walled anastomosing vessels and atrophy of adjacent parenchyma in a dog. Congenital portosystemic vascular anomalies are typically single anomalous vessels that directly connect the portal venous system with the systemic venous circulation, bypassing the hepatic sinusoids and hepatic parenchyma. They occur in dogs and cats, and, rarely, in pigs, foals, goats and calves. These portosystemic shunts (PSS) may be either intrahepatic or extrahepatic in location. The most common intrahepatic shunt, located in the left hepatic division, is a persistent patent ductus venosus (Fig. 2-11 ). Central and right divisional intrahepatic shunts have also been described in dogs and cats. The major types of extrahepatic shunts include direct shunting from the portal vein or major tributary (typically left gastric or splenic veins, less commonly the gastroduodenal or mesenteric veins) to the caudal vena cava (portocaval shunt) (Fig. 2-12 , eFig. 2-2) or to the azygos vein (portoazygos shunt), or connection of the portal vein to the caudal vena cava, which itself shunts to the azygos vein. Extrahepatic portosystemic shunts may also have hypoplasia of the portal vein distal to the origin of the shunt. Large-breed dogs typically have intrahepatic shunts, usually a patent ductus venosus, but sometimes other large intrahepatic communications. Small-breed dogs and cats usually have single large extrahepatic shunts between the portal vein and vena cava or azygos vein. An inherited basis is suspected for several breeds, including Irish Wolfhounds, Maltese, Yorkshire Terriers, and Australian cattle dogs. Figure 2-11 Congenital intrahepatic shunt, persistent patent ductus venosus in a dog. (Courtesy J.L. Caswell.) Figure 2-12 Congenital extrahepatic portocaval shunt in a cat. eFigure 2-2 Congenital extrahepatic portocaval shunt in a dog. (Courtesy University of Guelph.) Affected dogs are usually presented in adolescence with failure to thrive or with the neurobehavioral manifestations of hepatic encephalopathy. Often, there is a clinical history of depression, convulsions, and other nervous signs that are exacerbated by a high-protein diet, and may be alleviated by dietary control. Because there is no cause of portal hypertension, these dogs do not develop ascites. The liver that has been bypassed by a congenital shunt is hypoplastic, largely because of diversion of hepatotrophic factors, including insulin, glucagon, and epidermal growth factor that originate in the intestine and pancreas. Affected livers may be smooth surfaced with normal color and texture. Histologically, hepatocytes and hepatic lobules are small with close and irregular spacing of portal triads. Larger portal veins may be inapparent or appear collapsed and empty of circulating blood elements; portal veins in smaller triads may be small, collapsed, absent, or indistinguishable. Hepatic arterioles are often more prominent, and may be multiple and tortuous (Fig. 2-13 ), related to increased compensatory arterial perfusion. Numbers of arteriolar structures within triads may also appear increased, as small caliber and usually inapparent arterioles become evident histologically after compensatory hypertrophy. A proliferation of small caliber bile ducts (ductular reaction) has been confirmed in some cases by cytokeratin 19 immunohistochemistry. Dilated vascular structures devoid of blood, presumably small- and large-caliber lymphatics, are often prominent in the periphery of some portal triads. Dilated lymphatics may also be present in the connective tissue surrounding hepatic veins. In dogs, the spiral smooth muscle in the wall of the hepatic vein may be more prominent in dogs with shunts than normal dogs. There may be increased deposition of fibrous connective tissue surrounding portal triads and hepatic veins. Hepatocytes may contain cytoplasmic lipid droplets, and multiple small lipogranulomatous foci with hemosiderin and ceroid in Kupffer cells, and macrophages are typically present throughout the liver, especially in animals >1 year of age. Figure 2-13 Histology of the liver of a dog with a congenital portocaval shunt. Closely spaced portal triads contain multiple sections of hepatic arterioles and lack discernable portal veins. Primary portal vein hypoplasia (PVH) has been reported in dogs, particularly Cairn and Yorkshire Terriers, and occasionally in cats, affecting either the extrahepatic or intrahepatic portal vein, or both. Intrahepatic portal vein hypoplasia is considered to be the underlying lesion in conditions previously described as microvascular dysplasia, hepatoportal fibrosis, and idiopathic noncirrhotic portal hypertension in some young dogs. Depending on the level of the abnormality and extent of involvement of the lobes of liver, PVH may be accompanied by portal hypertension, ascites and the development of multiple collateral portosystemic shunts. Histologically, there is hypoplasia or absence of portal vein radicles, secondary arteriolar proliferation, and atrophy of hepatocytes. Moderate to marked portal fibrosis may also be present, with biliary hyperplasia. These changes represent stereotypic sequelae to under-perfusion and thus can be indistinguishable histologically from congenital portosystemic shunts; however, development of portal hypertension is a distinguishing feature of PVH. Macroscopic PSS and microscopic portosystemic vascular anomalies may co-occur in dogs, as evidenced by a lack of resolution of clinical signs, and persistence of histologic changes in additional liver biopsies subsequent to macroscopic shunt ligation. Decreased tolerance of complete surgical shunt attenuation has been associated with lack of identifiable portal veins and the presence of a ductular reaction in biopsies taken during the initial surgical shunt attenuation procedure, although an earlier study showed no association of severity of several histologic findings, such as arteriolar proliferation, biliary hyperplasia, and fibrosis, with survival time after shunt attenuation. Further reading Awasthi A, et al. Morphological and immunohistochemical analysis of ductal plate malformation: correlation with fetal liver. Histopathol 2004;45:260-267. Baade S, et al. Histopathological and immunohistochemical investigations of hepatic lesions associated with congenital portosystemic shunt in dogs. J Comp Pathol 2006;134:80-90. Berent AC, Tobias KM. Portosystemic vascular anomalies. Vet Clin North Am Small Anim Pract 2009;39:513-541. Bertonlini G, Caldin M. Computed tomography findings in portal vein aneurysm of dogs. Vet J 2012;193:475-480. Best EJ, et al. Suspected choledochal cyst in a domestic shorthair cat. J Feline Med Surg 2010;12:814-817. Bosje JT, et al. Polycystic kidney and liver disease in cats. Vet Q 1998;20:136-140. Bourque AC, et al. Congenital hepatic fibrosis in calves. Can Vet J 2001;42:145-146. Brown DL, et al. Mesenchymal hamartoma of the liver in a late-term equine fetus. Vet Pathol 2007;44:100-102. Brown DL, et al. Congenital hepatic fibrosis in 5 dogs. Vet Pathol 2010;47:102-107. Buczinski S, et al. Portacaval shunt in a calf: clinical, pathologic, and ultrasonographic findings. Can Vet J 2007;48:407-410. Bunch SE, et al. Idiopathic noncirrhotic portal hypertension in dogs: 33 cases (1982-1998). J Am Vet Med Assoc 2001;218:392-399. Center SA, et al. Hepatoportal microvascular dysplasia. In: Bonagura J, editor. Kirk's Current Veterinary Therapy: XIII. Small Animal Practice. Philadelphia: WB Saunders; 2000. p. 682-686. Christiansen JS, et al. Hepatic microvascular dysplasia in dogs: a retrospective study of 24 cases (1987-1995). J Am Anim Hosp Assoc 2000;36:385-389. Cullen JM. Summary of the World Small Animal Veterinary Association standardization committee guide to classification of liver disease in dogs and cats. Vet Clin North Am Small Anim Pract 2009;39:395-418. Cullen JM, et al. Morphological classification of circulatory disorders of the canine and feline liver. In: Rothuizen J, et al., editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Philadelphia: Saunders Elsevier; 2006. p. 41-59. DeMarco J, et al. A syndrome resembling idiopathic noncirrhotic portal hypertension in 4 young Doberman Pinschers. J Vet Intern Med 1998;12:147-156. Desmet VJ. Pathogenesis of ductal plate abnormalities. Mayo Clin Proc 1998;73:80-89. Görlinger S, et al. Congenital dilatation of the bile ducts (Caroli's disease) in young dogs. J Vet Intern Med 2003;17:28-32. Grand JG, et al. Cyst of the common bile duct in a cat. Aust Vet J 2010;88:268-271. Gunay-Aygun M. Liver and kidney disease in ciliopathies. Am J Med Genet Part C Semin Med Genet 2009;151C:296-306. Haechler S, et al. Congenital hepatic fibrosis and cystic bile duct formation in Swiss Freiberger horses. Vet Pathol 2000;37:669-671. Harper P, et al. Congenital biliary atresia and jaundice in lambs and calves. Aust Vet J 1990;67:18-22. Hunt GB. Effect of breed on anatomy of portosystemic shunts resulting from congenital diseases in dogs and cats: a review of 242 cases. Aust Vet J 2004;82:746-749. Hunt GB, et al. Evaluation of hepatic steatosis in dogs with congenital portosystemic shunts using oil red O staining. Vet Pathol 2013;50:1109-1115. Isobe K, et al. Histopathological characteristics of hepatic lipogranulomas with portosystemic shunt in dogs. J Vet Med Sci 2008;70:133-138. Kamishina H, et al. Gallbladder agenesis in a Chihuahua. J Vet Med Sci 2010;72:959-962. Komine M, et al. Multiple hepatic peribiliary cysts in a young pig. Vet Pathol 2007;44:707-709. Lamb CR, White RN. Morphology of congenital intrahepatic portacaval shunts in dogs and cats. Vet Rec 1998;142:55-60. Last RD, et al. Congenital dilatation of the large and segmental intrahepatic bile ducts (Caroli's disease) in two Golden retriever littermates. J S Afr Vet Assoc 2006;77:210-214. Lee KCL, et al. Association between hepatic histopathologic lesions and clinical findings in dogs undergoing surgical attenuation of a congenital portosystemic shunt: 38 cases (2000-2004). J Am Vet Med Assoc 2011;239:638-645. Lyons LA, et al. Feline polycystic kidney disease mutation identified in PKD1. J Am Soc Nephrol 2004;15:2548-2555. McAloose D, et al. Polycystic kidney and liver disease in two related West Highland white terrier litters. Vet Pathol 1998;35:77-81. Mochizuki S, Makita T. Double gallbladder of swine. Kaibogaku Zasshi 1996;71:650-655. Moore PF, Whiting PG. Hepatic lesions associated with intrahepatic arterioportal fistulae in dogs. Vet Pathol 1986;23:57-62. Parker JS, et al. Histologic examination of hepatic biopsy samples as a prognostic indicator in dogs undergoing surgical correction of congenital portosystemic shunts: 64 cases (1997-2005). J Am Vet Med Assoc 2008;232:1511-1514. Payne JY, et al. The anatomy and embryology of portosystemic shunts in dogs and cats. Semin Vet Med Surg 1990;5:76-82. Schermerhorn T, et al. Characterization of a hepatoportal microvascular dysplasia in a kindred of Cairn terriers. J Vet Intern Med 1996;10:219-230. Tisdall PLC, et al. Congenital portosystemic shunts in Maltese and Australian cattle dogs. Aust Vet J 1994;71:174-178. van den Ingh TSGAM, et al. Congenital portosystemic shunts in three pigs and one calf. Vet Pathol 1990;27:56-58. van den Ingh TSGAM, et al. Circulatory disorders of the liver in dogs and cats. Vet Q 1995;17:70-76. van den Ingh TSGAM, et al. Portal hypertension associated with primary hypoplasia of the hepatic portal vein in dogs. Vet Rec 1995;137:424-427. van den Ingh TSGA, et al. Morphological classification of biliary disorders of the canine and feline liver. In: Rothuizen J, et al., editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Philadelphia: Saunders Elsevier; 2006. p. 61-76. Yoshikawa H, et al. Congenital hepatic fibrosis in a newborn calf. Vet Pathol 2002;39:143-145. Zandvliet MM, et al. Acquired portosystemic shunting in 2 cats secondary to congenital hepatic fibrosis. J Vet Intern Med 2005;19:765-767. Displacement, Torsion, and Rupture The position of the liver should be observed as soon as the abdomen is opened at postmortem examination. Caudal displacements resulting in extension of the margins of the liver beyond the costal arch may be the result of hepatic enlargement or of displacement of the diaphragm secondary to pleural effusion or other space-occupying lesions in the thorax. Congenital or acquired displacements associated with ventral and diaphragmatic hernias are common. Individual lobes or the entire organ may be displaced into the subcutis, pleural cavity, or pericardial sac, often along with other viscera; lobar blood supply may not always be compromised; however, individual displaced lobes may be severely congested and may rupture, or, given time, become indurated. Partial or complete liver lobe torsions have been reported in pigs, dogs, cats, and horses. The left lateral lobe may be predisposed because of its mobility, large size, and relative separation from other lobes; however, torsions of other lobes, in particular the left medial lobe, as well as double-lobe torsions have been reported in dogs and horses. Other predisposing causes include absence of or damage to the ligamentous attachments that provide spatial support for the liver, trauma, or the presence of a mass lesion in the affected lobe. Torsed lobes undergo various degrees of ischemia, culminating in infarction caused by venous occlusion or venous and/or arterial thrombosis, and affected animals may die because of shock, hemorrhage, or development of septic peritonitis. Ischemia may favor overgrowth of Clostridium spp. with development of necrosis and emphysema. Subacute cases may develop hepatic abscessation, and if the animal survives, fibrosis and chronic inflammation. Rupture of the liver occurs commonly as the result of trauma because the organ is fragile relative to its mass. Fatal liver rupture may be produced by the sudden accelerations and pressures of vehicle collisions without much evidence of trauma to other parts of the body. Large tears may be obvious in the liver capsule and hepatic parenchyma after trauma; however, anastomosing linear patterns of fine shallow capsular fissures may be concealed in part by clotted blood. Liver rupture is often clinically occult, because quite large ruptures may not disturb liver function unless severe enough to cause rapid exsanguination, or unless the biliary tract is involved. Intrahepatic bile duct rupture results in bile extravasation into the hepatic parenchyma or beneath the hepatic capsule, forming bile lakes or bile infarcts, areas of hepatocyte degeneration, and necrosis surrounded by reactive macrophages; larger accumulations of bile may be walled off by a pseudocapsule, forming biliary pseudocysts. Rupture of major bile ducts or the gallbladder results in yellow-stained bile peritonitis, which may remain sterile and become chronic, or may be fatal, particularly if infected by enterohepatic circulation of bacteria such as clostridia. The liver is more likely to rupture after trauma in young animals. Fatal ruptures occur in foals during parturition, sometimes concurrently with costal fractures, and in the smaller species subject to energetic emergency resuscitation. Diffuse hepatic conditions with enlarged friable parenchyma (e.g., acute hepatitis, amyloidosis, severe congestion, severe lipidosis, and infiltrating neoplasms) are more likely to rupture, sometimes spontaneously, and the clinical consequences are related to the extent of hemorrhage. Parasites that penetrate the capsule cause numerous small ruptures but seldom lead to significant hemorrhage. Further reading Banz AC, Gottfried SD. Peritoneopericardial diaphragmatic hernia: a retrospective study of 31 cats and eight dogs. J Am Anim Hosp Assoc 2010;46:398-404. Bentz KJ, et al. Hepatic lobe torsion in a horse. Can Vet J 2009;50:283-286. Bhandal J, et al. Spontaneous left medial liver lobe torsion and left lateral lobe infarction in a Rottweiler. Can Vet J 2008;49:1002-1004. Boerboom D, et al. Duodenal obstruction caused by malposition of the gallbladder in a heifer. J Am Vet Med Assoc 2003;223:1475-1477. Downs MO, et al. Liver lobe torsion and liver abscess in a dog. J Am Vet Med Assoc 1998;212:678-680. Hinkle SG, et al. Liver lobe torsion in dogs: 13 cases (1995-2004). J Am Vet Med Assoc 2006;228:242-247. Scheck MG. Liver lobe torsion in a dog. Can Vet J 2007;48:423-425. Swann HM. Hepatic lobe torsion in 3 dogs and a cat. Vet Surg 2001;30:482-486. Tennent-Brown BS, et al. Liver lobe torsion in six horses. J Am Vet Med Assoc 2012;241:615-620. von Pfeil DJF, et al. Left lateral and left middle liver lobe torsion in a Saint Bernard puppy. J Am Anim Hosp Assoc 2006;42:381-385. Hepatocellular Adaptations and Intracellular Accumulation The liver must be highly adaptable to balance function with changing demand. Increases in the size of hepatocytes (hypertrophy) and their numbers (hyperplasia) collectively bring a larger mass of hepatocytes into service. Such adaptations in hepatic volume and function result from alterations in the expression of many genes. These responses are more evident in smaller species, notably in laboratory rodents that have a very pronounced liver growth response after exposure to various xenobiotics. The liver can also adapt to reduced demand or oxygen supply by a combination of cellular atrophy and apoptosis. Hepatocytes can be lost by apoptosis in substantial numbers, with minimal elevation in serum enzymes of hepatic origin such as alanine aminotransferase. Hepatocellular atrophy Hepatic mass readily adapts to metabolic demands, and the liver can undergo marked atrophy during illness and/or starvation without much evidence of impaired hepatic function. During prolonged starvation, some hepatocytes are removed by apoptosis without replacement, but most of the atrophy is explained by loss of cytoplasmic mass. Atrophic livers, as seen, for example, in old grazing herbivores with poor teeth, are dark and small, and the capsule may appear too large for the organ, showing fine wrinkles on handling. These livers may even appear to be firmer than normal because of condensation of normal stroma. Histologically, portal triads and hepatic venules are closer together, and lobules contain increased numbers of smaller hepatocytes with scanty cytoplasm (Fig. 2-14 ). Hepatocellular mass can be rapidly lost by autophagy and apoptosis (see later section on Types and patterns of cell death in the liver). Figure 2-14 Hepatocellular atrophy in a dog with chronic right-sided heart failure. Hepatic atrophy, rather than hepatocellular atrophy, can also result from impaired replication of hepatocytes. Most adult hepatocytes are replicatively competent, although mitotic figures are infrequent because healthy hepatocytes have a relatively long life-span of several months. Diminished portal blood flow not only limits oxygen, but also trophic factors that act to regulate replication and mass of the liver. These trophic factors include several polypeptide growth factors, including hepatocyte growth factor and insulin-like growth factors, and many hormones, including insulin, glucagon, and catecholamines. Atrophy of only a part of the liver may be a response to pressure or to impairment of blood or bile flow. The histologic features of this atrophy are similar to those of starvation atrophy. However, the functional consequences of focal hepatic atrophy are minor because the remaining liver can compensate and adapt by a process that involves replication and enlargement of hepatocytes. Local pressure atrophy occurs adjacent to space-occupying lesions in the liver, or as a result of chronic pressures from neighboring organs, such as distended rumen in the ox. It has been suggested that right hepatic lobe atrophy, reported in horses, results from long-term compression from abnormal distension of the right dorsal colon and base of the cecum (eFig. 2-3). Chronic diffuse diseases of the biliary tract, such as sporidesmin poisoning and fascioliasis, are likely to cause atrophy of the left lobe in ruminants, possibly as a result of the greater difficulty in maintaining adequate biliary drainage from this lobe, whose bile ducts are longer than those of the right in these species. The atrophy of biliary obstruction is complicated by some degree of superimposed inflammation and fibrosis. eFigure 2-3 Atrophic right lobe of the liver of a horse. (Courtesy A.P. Loretti.) Hepatocellular hypertrophy Hypertrophy is the term used for the increase in liver size caused by an increase in hepatocyte volume that may result from expansion of one or more organellar components of the hepatocytes. Exposure to various xenobiotics can induce the expression of many genes, leading to expansion of the smooth endoplasmic reticulum (SER), resulting in hepatocyte hypertrophy. Agents that elicit this response act via nuclear receptors, such as the arylhydrocarbon-activated receptor, the constitutive androstane receptor, or the pregnane X receptor. Phenobarbital, for example, is a potent inducer of the various enzyme systems of the SER, including several cytochromes P450 (CYPs). Hypertrophy may occur in defined lobular regions, typically the centrilobular region or may affect the entire lobule in more advanced cases, depending upon the activity and dose level of the xenobiotic (Fig. 2-15 ). Even when it is restricted to the centrilobular region, hypertrophy usually enlarges the entire liver. Although hepatocellular hypertrophy is most often associated with preferential increase in SER, proliferation of peroxisomes, or mitochondria can also cause hepatocellular hypertrophy. The light microscopic appearance of hypertrophy upon routine hematoxylin and eosin (H&E) staining will sometimes suggest the selective involvement of one organelle. If total SER volume is increased, the cytoplasm will typically have an eosinophilic ground-glass appearance upon light microscopy. If total peroxisomal volume is increased, the cytoplasm is often noted to have an eosinophilic granular appearance. The response can be seen within a few days after exposure to various drugs and other xenobiotic compounds. Accordingly, the liver becomes grossly enlarged. This induction of SER or peroxisomes is reversible, and after discontinuation of exposure to the inducing agent, the expanded SER or excess peroxisomes are removed by autophagy, and many hepatocytes undergo apoptosis. Although these changes are considered physiologic adaptations, there are potential adverse sequelae; accordingly, this response has toxicologic significance, and will be dealt with later in the section on Toxic hepatic disease. Figure 2-15 Increased cytoplasmic volume resulting from smooth endoplasmic reticulum induction in a dog treated chronically with phenobarbital. Polyploidy and multinucleation Most mature mammalian hepatocytes are tetraploid or octaploid, whereas many immature and replicating hepatocytes are diploid. The relative proportion of polyploid hepatocytes varies among species, and polyploidy increases with age. Polyploidy is more common in rodents and is believed to result from asynchrony of cell division in which binucleated diploid cells undergo a second round of DNA replication, giving rise to 2 tetraploid daughter cells. Impaired replication can also increase the number of polyploid cells. The term megalocytosis was first used to describe the changes of liver cell cytoplasm and nucleus that occur in pyrrolizidine alkaloid poisoning. This form of megalocytosis has some specific features and is described in the later section on Chronic hepatotoxicity. Impaired regeneration, atrophy and >4N polyploidy can also be produced by other DNA-damaging agents such as aflatoxins. A feature of these patterns of atrophy with polyploidy is the persistence of larger replicatively impaired polyploid hepatocytes amid regenerating smaller diploid hepatocytes, hepatocellular nodules, and hyperplastic bile ductules. Most hepatocytes are mononuclear, but a variable proportion is binucleated, especially in young or regenerating livers of small animals. Multinucleation by more than 2 nuclei of non-neoplastic hepatocytes is a rare phenomenon in domestic mammals, and its diagnostic or pathogenetic significance is usually unclear. Multinucleation can result from incomplete cell division or cell fusion, but this distinction is difficult to determine. Hepatocytes can fuse during severe steatosis, but the degree of multinucleation in fatty livers is hard to discern because the plasma membrane perimeters of fatty hepatocytes are ill defined. Syncytial multinucleation of hepatocytes has been observed in various degenerative and regenerative conditions. In protoporphyria of Limousin cattle, small clusters of hepatocytes contain 4-10 or more closely packed nuclei, but it is not clear whether this represents fusion or multiple nuclear divisions. Syncytial hepatocytes are a characteristic of postinfantile giant cell hepatitis of children, and similar hepatocyte multinucleation can be observed in association with some forms of hepatitis in newborn cats, foals, and piglets (see Inflammatory diseases of the liver and biliary tract). Multinucleated hepatocytes have been described in young cats with thymic lymphomas, and in cats with experimental dioxin poisoning. Intranuclear inclusions In addition to the various nuclear inclusions associated with some viral infections, 3 types of inclusions may be found in hepatocyte nuclei. 1. Spherical, apparently hollow globules within the body of the nucleus are membrane-bound entrapped nuclear membrane invaginations that ultrastructurally contain cytoplasmic components such as glycogen and mitochondria. These inclusions are infrequent in otherwise normal livers but are more often seen in chronically injured livers, especially in chronic pyrrolizidine alkaloid poisoning, in which polyploid nuclei are more likely to indent and invaginate. 2. Eosinophilic block-like intranuclear inclusions with a regular crystal lattice (“brick inclusions”) are common in hepatocytes and renal proximal tubular epithelium. They are more numerous in old animals, particularly dogs. Their composition and pathogenesis are still unknown, but they evidently have little effect on the health of the cells in which they occur, even when they are large enough to distort the nucleus. They do not contain heavy metals and can be distinguished from the acid-fast, noncrystalline intranuclear inclusions observed in renal epithelial cells and occasionally in hepatocytes in lead poisoning. 3. Lead inclusions consist of a lead-protein complex and have a characteristic furry electron-dense ultrastructure. Pigmentation Congenital melanosis occurs in calves and occasionally in lambs and swine. The melanin deposits may be numerous, and vary in size from flecks, to irregular, blue-black areas 2 cm or more in diameter. The melanin is confined to the capsule and the stroma. These deposits are sharply defined in young animals, but become more diffuse and fade with age (eFig. 2-4). eFigure 2-4 Congenital melanosis in a calf. A condition known as acquired melanosis is the massive accumulation of black pigment (not melanin) in hepatocytes and Kupffer cells of mature sheep and, less frequently cattle, after prolonged grazing on extensive unimproved pastures in inland eastern Australia, the Falkland Islands, and Scandinavia. The condition in Norway has also been described as hepatic lipofuscinosis. The color of the affected livers ranges from a dull gray to uniform black, and there is usually a prominent acinar pattern. In severe cases, there is also pigmentation of the hepatic lymph nodes, lungs, and renal cortex. Histologically, the pigment is present as granules in lysosomes in periportal and midzonal hepatocytes and macrophages of the liver, the proximal tubular epithelium of the kidneys, and in alveolar and interstitial macrophages in the lung. There is no evidence of liver dysfunction, even in the blackest livers. The source of this pigment is not known, but the epidemiologic features of its occurrence indicate that it is derived from a component of the diet that is sequestered within lysosomes. Bile pigmentation may impart an olive-green color to the liver in diffuse or segmental obstructive biliary disease or intrahepatic cholestasis. Histologically, conjugated bile pigments may distend bile canaliculi, visible microscopically as golden-brown linear streaks arrayed in a chicken wire-like pattern between the hepatocytes (Fig. 2-16 ). In this case, the identity of the pigment is obvious, but when it is present in granular form in Kupffer cell cytoplasm, it may easily be confused with hemosiderin and hematin. Bile pigment is encountered infrequently in hepatocyte cytoplasm, almost never in dogs, except in rare cases with dramatic cholestasis. Death of individual hepatocytes releases the canalicular plugs into the space of Disse and the sinusoids, where they may be phagocytosed by Kupffer cells. Figure 2-16 Bile plugs distend bile canaliculi in a horse with intravascular hemolysis following ingestion of red maple leaves. Lipofuscin is the term given to small, golden, granular cytoplasmic deposits derived from the lipid component of membranous organelles, more obvious in hepatocytes near the centrilobular areas. Lipofuscin accumulates in hepatocellular lysosomes and indicates senility, atrophy, or increased turnover of membrane lipids. The pigment is particularly common in the centrilobular regions of the liver of cats after they reach maturity. Ceroid is a yellow pigment similar to lipofuscin and is associated with peroxidation of fat deposits. Black pigment also accumulates in hepatocellular lysosomes of mutant Corriedale sheep with hyperbilirubinemia. These sheep have a condition resembling the human Dubin-Johnson syndrome, in which there is mutation in the canalicular transporter in the organic anion-transporting polypeptide family. This suggests that retention of the pigment might be a sequel to a hepatic excretory defect. In congenital erythrocytic protoporphyria (ferrochelatase deficiency) of Limousin and Blonde d'Aquitaine cattle, a dark golden-brown lipofuscin-like lysosomal material is present in portal areas, Kupffer cells, and sinusoidal endothelial cells, and is heavily concentrated in the cytoplasm of hepatocytes. Acquired protoporphyria (Fig. 2-17A, B ) with liver injury has been described in German Shepherd dogs as an idiopathic syndrome and in a group of Beagle dogs treated with an experimental drug. Grossly, affected German Shepherd dogs had dark livers that contained multiple regenerative nodules. Histologically, there was abundant bridging portal fibrosis and typical orange birefringent protoporphyrin crystals in hepatocytes. Photosensitization was not evident. Hepatic vacuolation and nodule formation has also been described in a cat with porphyria. Figure 2-17 A. Dark golden-brown protoporphyrin crystals in the liver of a dog. B. Protoporphyrin crystals with bright red Maltese cross birefringence when viewed under polarized light. Hemosiderin deposits are seldom sufficient to give gross discoloration, but when this occurs, the color is dark brown. The pigment is detected microscopically as yellow or brown crystals chiefly in the Kupffer cells, although small amounts may be found in hepatic cells. The ferric iron component of this pigment can be demonstrated by staining with Prussian blue; otherwise, it can easily be confused with lipofuscin. Most hemosiderin deposits are punctate Prussian blue staining aggregates in Kupffer cells but can also be seen as finer particles in hepatocytes. Diffuse hemosiderin deposits in Kupffer cells occur quite commonly in all species, and its presence is usually suggestive of excess hemolytic activity relative to the rate of reutilization of iron. Thus it is seen in hemolytic anemia, anemia of copper deficiency, in cachexia, and after blood transfusions or iron injections. It may be seen in Kupffer cells of the centrilobular zones in severe chronic passive congestion of the liver. Localized hemosiderin deposition occurs in areas of hemorrhage. Hemosiderin is normally present in the liver in the early neonatal period, when fetal hemoglobin is being replaced by mature hemoglobin. Hemosiderin should be distinguished from hematin, which is produced by the action of formic acid on hemoglobin following a prolonged postmortem interval, and is usually regarded as a histologic artifact. Hematin is also an iron-containing pigment, but the iron is in the reduced ferrous state and does not stain with ferricyanide. It takes the form of crystalline brown deposits that are birefringent under polarized light, mainly within hemoglobin-rich areas such as blood vessels. Hematin is darker than hemosiderin and occurs in irregular clumps, often extracellularly. Hematin may, however, be found in Kupffer cells and macrophages in small amounts. Hepatic iron overload has previously been divided into hemosiderosis when there is only an excess accumulation of hepatocellular iron and hemochromatosis when the excess iron storage has produced fibrosis and inflammation and hepatic injury, although this terminology is not used consistently. Hemochromatosis, as an inherited disorder is relatively common in humans with specific mutations. Iron overload is common in some birds, such as mynahs, and in lemurs, but rare in domestic mammals. A form resembling heritable hemochromatosis has been reported in Salers or Salers-cross cattle. Affected animals develop a wasting disease at ~1-2 years of age, have a 30-100 fold increase in liver iron content, with dark-brown discolored, firm livers, hemosiderin accumulation in hepatocytes, and periportal and perivenular bridging fibrosis with nodular regeneration. Hemosiderin also accumulates in Kupffer cells, lymph nodes, kidney, pancreas, spleen, and other organs. Iron overload from dietary excess has been observed in sheep and cattle exposed to high levels of iron in pasture and water. The liver is enlarged and brown with diffuse fine nodularity, and the hepatic and adjacent lymph nodes are also darkened. Large amounts of iron are present in the hepatic parenchyma, the biliary epithelium, and the cortex of lymph nodes, and lesser amounts are present in the broad fibrous septa. The iron is stored predominantly in lysosomes. Brown discoloration of bone marrow resembles the osseous pigmentation of porphyria. The pathogenesis of nutritional iron overload is unknown. Iron overload has also been described in horses, with animals displaying signs of liver failure and neurologic impairment. The microscopic lesions in the livers of these animals are similar to those for other species. Brown crystalline deposits of 2,8-dihydroxyadenine (2,8-DHA) have been described in hepatocytes and in other tissues in slaughtered cattle with no evidence of other disease. These accumulations are strongly birefringent under polarized light, and are seen in the cytoplasm of hepatocytes and macrophages of hepatic lymph nodes and as extracellular deposits in portal stroma and renal tubular lumens. Grossly, the portal stroma stands out as a green network, and affected lymph nodes were enlarged, and the medullary sinusoids were distended with green pasty material. The crystals were identified as 2,8-DHA by a panel of crystallographic methods and mass spectrometry. The pathogenesis is unknown. Pigments of parasitic origin are particularly associated with flukes. Heavy deposits of black iron-porphyrin compound are formed around the cysts and migratory pathways of Fascioloides magna. Lesser amounts of similar pigment are deposited in bile ducts infested by Fasciola hepatica (see later section on Helminthic infections). The presence of this pigment in the hilar nodes should suggest otherwise inapparent infestations by flukes. In schistosomiasis, the liver may be gray because of the accumulation of black pigment in Kupffer cells. Vacuolation The term “vacuolar hepatopathy” has been used to denote multifocal or diffuse zonal hepatocellular cytoplasmic vacuolation before a more specific term can be applied, for example, before contents of vacuoles are identified. Specific forms of hepatocyte vacuolar change include hydropic swelling, glycogenosis, and steatosis (lipidosis). Hydropic degeneration can only be appreciated in carefully controlled experimental circumstances and is an early cytoplasmic ballooning seen after various toxic and metabolic insults, hypoxia, and cholestasis. Water and sodium ion influx expands membranous compartments of mitochondria, lysosomes, and endoplasmic reticulum (ER). The term feathery degeneration is applied to the type of hydropic change that occurs in hepatocytes in which there has been prolonged cholestasis. The cells are swollen and vacuolated and crisscrossed by a fine protoplasmic network that is brown with bile pigments (Fig. 2-18 ). This change is uncommon in cats and dogs. Figure 2-18 Feathery degeneration in a horse with cholestasis. Hepatic glycogenosis or glucocorticoid hepatopathy, often referred to as steroid-induced hepatopathy, is probably the most severe example of hepatocellular vacuolar change in dogs. It involves glycogen accumulation induced by hyperadrenocorticism, because of either functional adrenocortical or pituitary tumors, or to treatment with glucocorticoids. The liver is enlarged and pale tan. Hepatocytes are swollen, and the cytoplasm appears as fine, diaphanous strands that enclose multiple spaces with poorly demarcated edges. Vacuolar change may range from mild to severe, with swelling of hepatocytes from 2-10 times normal size and displacement of the nucleus and organelles to the cellular periphery (Fig. 2-19 ). The zonal distribution may be very variable and may become diffuse in long-standing cases or with higher doses. Single-cell drop-out, multifocal small aggregates of neutrophils along sinusoids, and scattered small foci of extracellular hematopoiesis are also commonly observed. The ill-defined vacuolar boundaries in steroid hepatopathy are readily distinguishable from spherical lipid vacuoles in hepatic steatosis. The pathogenesis of this condition is uncertain, because glycogen storage alone does not fully explain the influx of fluid, and possible perturbations in hepatocellular ion channels or aquaporins have not been assessed. The amount of glycogen remaining in affected cells is widely variable, a function of the original glycogen concentration, recent catabolism, and postmortem dissolution. Glycogen content can best be demonstrated in frozen section, followed by staining with periodic acid–Schiff, and can be confirmed as glycogen by its sensitivity to digestion with diastase. Livers affected by glucocorticoid hepatopathy maintain normal hepatocyte functions, but the condition can be confirmed by the induction and serum increase of glucocorticoid-inducible alkaline phosphatase, with minor or negligible increases in alanine aminotransferase. Similar vacuoles may be created by other adrenocortical steroids, possibly progestins, as occasionally seen in older, female dogs that appear to have overproduction of adrenal cortical hormones other than cortisol and alkaline phosphatase elevation, although the specific pathogenesis is unknown. Some drugs, such as the chelator D-penicillamine, can produce similar vacuolation. Figure 2-19 Prominent vacuolation of hepatocytes following glucocorticoid administration in a dog. Loss-of-function mutations in hepatic and renal glucose-6-phosphatase resulting in increased hepatic glycogen storage have been reported in Maltese puppies or related crossbred dogs, and these dogs serve as an animal model of glycogen storage disease (glycogenosis) type Ia. These dogs have severely debilitating problems in maintaining their blood glucose because dephosphorylation of glucose-6-phosphate is a key step in both glycogenolysis and gluconeogenesis. The condition is typified by severe hepatocellular glycogenosis. Livers are markedly enlarged, pale, and have diffuse vacuolation of hepatocytes with large amounts of glycogen and small amounts of lipid. Hepatic fibrosis and nodular regeneration can develop with time. Renal tubular epithelium is also vacuolated. Glycogen storage disease type III, with hepatic glycogen storage, has been reported in dogs, predominantly German Shepherd dogs. Type IV glycogenosis has been reported in Norwegian Forest cats, although this disorder causes pale blue granules in hepatocellular cytoplasm rather than clear vacuoles. Characteristic hepatocellular vacuolation is also described in dogs with end-stage nodular livers presented clinically with hepatocutaneous syndrome. Hepatocellular steatosis (lipidosis) Hepatocellular steatosis is the term used to describe fatty livers of animals. However, the terms steatosis, lipidosis, and fatty change tend to also be used interchangeably. All of these terms refer to the visible accumulation of triglycerides (triacylglycerols) as round globules in the cytoplasm of hepatocytes. The threshold for application of these terms is vague because triglyceride storage and transport are normal hepatic functions, but they are appropriate when the amounts are greater than would normally be seen. Hepatocellular steatosis can be physiologic or pathologic. Any circumstance in which hepatic uptake of lipids exceeds oxidation or secretion can lead to hepatocellular steatosis. Increased mobilization of triglycerides during late pregnancy or heavy lactation in ruminants is associated with hepatocellular steatosis. The lipid represents increased transit of triglycerides in an otherwise healthy liver, so there is little diagnostic significance in mild degrees of hepatocellular steatosis in lactating cows. In some of these animals, severe energy deficiency can also lead to clinical ketosis with metabolic acidosis. In addition, severe steatosis occurs in high-producing dairy cows fed diets in which either the mix of available fatty acids is incorrect or lipids are oxidized and rancid. Hepatocellular steatosis is common in injured hepatocytes because the normal high throughput of fatty acids and triglycerides can be readily impeded at various points in the complex pathway of hepatic lipid metabolism and secretion of very-low-density lipoproteins (VLDLs). Hepatocytes obtain some fatty acids from albumin and other carrier proteins in the portal blood, but most is hydrolyzed by sinusoidal endothelial hepatic lipase from triglyceride in chylomicrons or VLDL in plasma. Very-long-chain fatty acids are initially oxidized by acyl–coenzyme A oxidase in peroxisomes to shorter acyl–coenzyme A that can be transported via a carnitine-dependent process into mitochondria for further oxidation. Although some fatty acid is used for energy in hepatic mitochondria, most from dietary or adipose sources is converted to triglycerides that are further processed into lipoproteins in the hepatocyte ER and actively secreted into the plasma as VLDL. Endothelial lipoprotein lipase in muscle and adipose tissue hydrolyzes triglycerides in the VLDL, and the fatty acids released are used locally by β-oxidation in mitochondria of muscle or re-esterified into triglyceride in adipocytes. The depleted VLDLs are returned by receptor-mediated endocytosis to hepatocytes, where their constituents are catabolized and recycled. The synthesis and export of VLDL in hepatocytes are energy- and resource-dependent, so any disturbance of the supply of apoproteins (apoprotein B primarily), phospholipids, cholesterol, or ATP, or physical disruption of the organelles involved in synthesis, assembly, and secretion, has the potential to inhibit lipoprotein synthesis or secretion. Fatty acids can continue to enter hepatocytes, allowing triglyceride globules to accumulate in the hepatocyte cytoplasm. In some species, damage to peroxisomes reduces the initial peroxisomal oxidation step for catabolism of long-chain fatty acids and also upregulates various genes regulated by peroxisome proliferator–activated receptor-α. Lipoproteins in the ER, Golgi, and membrane-bound secretory vesicles can also accumulate if there is selective damage to the distal secretory apparatus. Damage by various toxic and hypoxic insults will not lead to triglyceride accumulation in hepatocytes if supplies of mobile triglycerides from adipose tissue or the diet are low. The microscopic appearance of triglyceride globules in hepatocytes ranges from small discrete microvesicles to large coalescing macrovesicles. Microvesicular lipid vacuoles are smaller than the nucleus and tend not to displace the nucleus (Fig. 2-20 ). Microvesicular steatosis can be a hallmark of more severe hepatic dysfunction than macrovesicular steatosis. This pattern can occur in several toxic hepatopathies causing mitochondrial injury, including some drug toxicities, such as antiviral nucleosides, aspirin in Reye's syndrome, and excessive tetracycline administration, all of which can be fatal. These toxicities tend to target: mitochondria, disrupting energy production; the normal electron transfer chain, causing oxidative injury; and interfere with β-oxidation of lipids. Partially oxidized lipids typically have a lower surface tension than triglycerides and therefore form smaller vesicles in the aqueous medium of the cytoplasm. Uncontrolled diabetes mellitus and feline fatty liver syndrome can produce microvesicular hepatic steatosis or a mixture of microvesicular and macrovesicular steatosis. Acute steatosis with predominantly microvesicular accumulation tends to result in a modestly enlarged pale liver without much change in texture, whereas macrovesicular hepatic steatosis more often produces an enlarged liver. Figure 2-20 Microvesicular lipid is characterized by multiple round, clear vacuoles that are smaller than the nucleus and do not displace the nucleus. In some more protracted toxic injuries, smaller lipid globules can coalesce into large central macrovesicles that displace the nucleus. The pathogenesis of the larger globules is not well understood, but it involves some alteration of the globule-cytoplasm interface that normally prevents coalescence of the small micellar globules, possibly the greater hydrophobicity of the triglycerides. These fatty livers tend to be more yellow and enlarged, and the texture is more friable than livers with microvesicular steatosis. Each hepatocyte usually contains one large globule (macrovesicle) that alters the contour of the cell and displaces the nucleus (Fig. 2-21 ). The sinusoids are compressed and appear under-perfused, and the tissue at low magnification resembles adipose tissue. In severe degeneration, the liver is moderately or greatly enlarged, with a uniform light yellow color. The edges are rounded, and the surface is smooth (eFig. 2-5). The cut surface has a diffuse greasy appearance or a red and yellow lobular pattern if there is also hepatic congestion or zonal necrosis. In severe diffuse hepatic steatosis, the parenchyma is less dense, and portions will float in water or fixative. Figure 2-21 Macrovesicular lipid vacuoles in the liver of a donkey. Vacuoles are larger than the nucleus and tend to displace it to the periphery of the cell. eFigure 2-5 Hepatomegaly, steatosis associated with equine hyperlipemia in a donkey. Assessment of the functional significance of steatosis depends on the differentiation of physiologic steatosis caused by increased mobilization by otherwise normal hepatocytes from pathologic changes that represent some degenerative change in hepatocytes. However, fat accumulation is a sensitive response to hepatocellular injury and can occur in the absence of other obvious alterations in hepatic structure or function. The triglyceride globules themselves are not harmful to hepatocytes, so the amount of fat present is more an indicator of the duration of insult and triglyceride supply than of the severity of hepatic injury. Steatosis is usually reversible, although a liver that has been fatty for some time is more likely to have concurrent damage, including fibrosis, pigment accumulation, and nodular hyperplasia, mostly attributable to ongoing peroxidative damage, cell turnover, and activation of stellate cells. Hepatocellular steatosis is now recognized as a more significant indicator of potential, more severe liver injury than previously appreciated. In ruminants, the association between hepatic steatosis, ketosis, and displaced abomasum are well recognized. Infectious diseases, such as metritis and mastitis, may be more likely or prolonged. Neutrophil function is suppressed, as is interferon production by lymphocytes, and endotoxin clearance is also reduced. Hepatocytes become more sensitive to injury following exposure to cytokines such as tumor necrosis factor-α or endotoxin as well. Consequently, fatty livers are more vulnerable to a wide range of toxic and nutritional insults, so other necrogenic insults and responses can be concurrent. Increased levels of fatty acids in the liver increase the oxidative stress and contribute to membrane lipid peroxidation, reduced hepatocyte life-span, and some local repair responses. When lipid accumulates in large amounts, there is a tendency for groups of the fat-laden cells to rupture or fuse and eventually form a multinucleated rim about a foamy mass of lipid. This epithelial structure is known as a fatty cyst (Fig. 2-22 ). The next stage, which occurs when released lipid is picked up by macrophages that form foamy aggregations in sinusoids, in the stroma of portal tracts, and hepatic venules is termed a lipogranuloma. Subsequent peroxidation of the less saturated fatty acids and covalent modification and polymerization of oxidized lipids form a complex of lysosomal residues known collectively as ceroid. This material is only slightly soluble in lipid solvents and is periodic acid–Schiff positive, diastase resistant, variably acid fast, and autofluorescent. In histologic sections, ceroid pigment appears as colorless or yellow irregular fragments associated with the lipid globules in macrophages and, to a lesser extent, hepatocytes. Affected portal lymph nodes become slightly enlarged, yellow-green, and rather oily on section. If iron accumulates along with the ceroid, the lesion is more correctly termed a pigment granuloma (Fig. 2-23 ). These are discrete perisinusoidal clusters of macrophages, containing cytoplasmic lipid vacuoles, lipofuscin, and hemosiderin, and often contain lymphocytes or plasma cells. They accumulate and congregate with age and hepatocellular turnover, but have no known clinical significance. Figure 2-22 Fatty cyst in the liver of a dog. Figure 2-23 Pigment granuloma (lipogranuloma) in the liver of a dog. Some chronic changes commonly seen in the livers of old dogs can be associated with fatty liver; these include lipogranulomas, fatty cysts, and ceroid accumulation. Occasionally, there can be found sharply defined, fibrous, stony, hard masses, usually close to the surface, sometimes as much as 3-4 cm in diameter. These masses are usually sufficiently mineralized to show up distinctly on clinical radiographs. The mineral appears to be deposited on a matrix of degenerate collagen, laid down about perivascular foci of foamy macrophages and accumulations of cholesterol. The fibrous tissue may be laid down in response to the continued presence of fat, ceroid, or cholesterol, but no reason is apparent for the strictly localized distribution of the reaction. There are no recognizable hepatocytes in these lesions. Physiologic steatosis (fatty liver) occurs in late pregnancy and heavy lactation, particularly in ruminants and llamas. In this circumstance, the lipid is typically macrovesicular, with large, round, clear vacuoles that tend to displace the nucleus. Obvious steatosis is also seen in neonates, especially in those species whose milk is relatively rich in fat. These livers are fatty enough to be pale to the naked eye. The high rate of mobilization of triglycerides from body fat stores is mainly responsible for fatty liver in lactating ruminants. However, fatty liver is a concern because lipid mobilization also increases when the dietary energy intake is insufficient relative to the production demands that are greatest in early lactation of high-producing cows. Insufficient dietary intake by an animal with adequate fat reserves depletes hepatocellular glycogen and initiates a heavy demand for triglycerides from adipose tissue. When hepatic triglyceride concentrations exceed 10% on a wet weight basis, severe or clinical fatty liver ensues. At this time, urinary ketones are elevated, and body weight loss and appetite depression can occur. In severe cases, cattle can suffer from hepatoencephalopathy. It is estimated that in the first month after parturition 5-10% of dairy cattle severe fatty liver, and 30-40% have moderate fatty liver (5-10% liver triglyceride by wet weight basis). The liver depends primarily on fatty acid oxidation for its own considerable energy needs. It must also synthesize a large amount of protein and phospholipids for lipoprotein export to other tissues, and this can be rate limiting, resulting in accumulation of triglyceride in the cytoplasm. In starvation, the reduced availability of protein and lipotrope cofactors, such as choline, can exacerbate the bottleneck. The liver is the main supplier of glucose for the brain and milk saccharides, and it adapts by converting fatty acid metabolites to glucose (gluconeogenesis) and ketones. Acute ketosis of lactating dairy cows with intake insufficiency or secondary to abomasal displacement is usually associated with fatty liver with a predominantly diffuse microvesicular pattern. Cows are more tolerant of ketosis associated with lactation than are pregnant ewes that can die from starvation-induced pregnancy toxemia and ketoacidosis. In cows, hepatic steatosis is predominantly centrilobular, whereas it is most severe in the periportal zone in pregnancy toxemia of sheep. The hepatic changes reflect increased mobilization of triglycerides from adipose tissue rather than hepatic disease per se. In cows and ewes with increased mobilization of triglycerides, there may be indistinct foci of white discoloration of abdominal fat that tend to be obscured when adipose tissue solidifies postmortem. Fatty liver of diabetes occurs when insulin is deficient or inactive because of lack of functioning receptors. Reduced insulin-dependent glucose uptake by cells leads to accelerated lipolysis from adipose tissue in much the same way as when energy intake is limiting. The liver is thus presented with a large load of fatty acids, and the rate by which this moves through the liver can be impeded in various ways. Insulin deficiency alone will produce fatty liver. Some cases in carnivores are also complicated by concurrent exocrine pancreatic insufficiency, so protein malabsorption can be a contributing influence on diabetic hepatic steatosis. The centrilobular hepatocytes usually show the greatest degree of steatosis, but in advanced long-standing diabetes, the change is often diffuse and marked. Lipoprotein synthesis and transport are dependent on oxidative metabolism, so hypoxia of hepatocytes leads to triglyceride accumulation. The 2 most common causes of hepatocellular hypoxia are anemia and reduced sinusoidal perfusion in passive venous congestion. In these situations, hepatic steatosis is most severe in the centrilobular zone, provided that the adipose and dietary supply of triglyceride is sufficient. Hepatic steatosis can be grossly visible as a yellow zonal pattern in chronic passive congestion of the bovine liver, but is less obvious in carnivores with passive hepatic congestion. Local hypoxia is probably the basis for another example of fatty liver. Small, sharply demarcated patches of intense fatty infiltration are often seen in bovine livers at or adjacent to sites of capsular fibrous adhesions—so-called “tension lipidosis.” These patches are neither swollen nor shrunken, usually extend <1 cm into the parenchyma, and are of the same consistency as normal liver (Fig. 2-24 ). The acinar structure of these lesions is undisturbed, but the hepatocytes therein show pronounced steatosis, presumably related to interference with local perfusion caused by tensions transmitted to the parenchyma by the adhesion. Figure 2-24 Subcapsular focal fatty change (“tension lipidosis”) associated with capsular ligamentous attachment in an ox. (Courtesy A.P. Loretti.) Hepatic steatosis caused by intoxication is common. There are several stages of the cycle of hepatic lipid metabolism that can be affected selectively by various toxins to produce fatty liver. For example, it is possible experimentally to cause triglyceride accumulation by interfering with mitochondrial fatty acid oxidation with sublethal doses of cyanide, or by inhibiting apolipoprotein synthesis by administration of orotic acid. Most toxins that cause fatty liver in naturally occurring situations, however, also produce a greater or lesser degree of hepatocellular necrosis. Fatty liver occurring as a manifestation of toxic hepatic disease will be further discussed in the later section on Toxic hepatic disease, but the generalization may be made here that most important veterinary hepatic intoxications cause widespread membrane damage and/or disturbance of protein synthesis. These cause lipid accumulation in the hepatocyte by interfering with lipoprotein synthesis and export, as well as with fatty acid oxidation. Steatosis requires time and a negative energy balance to develop, so it is more likely to occur in toxicoses with a longer clinical course. However, if adipose reserves are depleted, there is less lipid available to accumulate in the liver. Although fatty liver in domestic animals is more frequently associated with generalized interferences with energy metabolism, there are some specific nutritional deficiencies that will produce fatty liver. These have usually been defined under experimental conditions. Choline deficiency, in conjunction with deficiency of other lipotropic factors, such as L-methionine and vitamin B12 , rapidly produces fatty liver, largely as a result of reduced synthesis of phosphatidylcholine, a component of secreted lipoproteins. Fatty liver in experimental choline deficiency involves lipid peroxidation and increased hepatocellular turnover, leading to cirrhosis and neoplasia. It is unlikely that primary choline deficiency occurs in domestic animals, but other lipotrope deficiencies have been reported. Ovine white-liver disease, first described in lambs in New Zealand, also occurs in southern Australia, the United Kingdom, and continental Europe. Goats are also susceptible. Ovine white-liver disease is characterized by a syndrome of ill-thrift to emaciation, anorexia, and mild normocytic normochromic anemia, occasionally with photosensitization and icterus. The condition is associated with low liver cobalt levels and low plasma concentrations of vitamin B12, and the disease has been shown to be cobalt and vitamin B12 responsive. Lambs up to 1 year of age are more commonly affected than ewes, and pastures are likely to be adequate at the times of peak incidence in late spring and early summer. In the early stages, the liver changes consist of vacuolar accumulation of triglyceride in hepatocytes, usually most severe in the centrilobular zones. In addition, ceroid pigment is present in all cases, early in hepatocytes and later also in sinusoidal cells and macrophages. The fatty change may be very severe in the early stage, the liver being grossly swollen. A moderate degree of bile ductular proliferation is also a consistent feature, and the epithelium of the smaller ductules in the triads is dysplastic. Spongy degeneration of cerebral white matter, typical of the hyperammonemia of hepatic failure, is present in some cases. Experimental feeding of a diet low in cobalt to sheep resulted in reduced growth rate, anorexia, lacrimation, alopecia, emaciation, and marked reduction in plasma and liver vitamin B12 concentrations. At autopsy, livers were pale, swollen, and fatty. Histologically, livers with severe fatty degeneration were characterized by widespread hepatocyte disassociation, accumulation of lipid droplets, eosinophilic inclusions and lipofuscin in hepatocyte and Kupffer cell cytoplasm, nuclear lipid inclusions, ductular proliferation, and hepatocyte apoptosis. These lesions are characteristic of spontaneous cases of ovine white-liver disease. Ultrastructurally, degeneration of mitochondria and proliferation of the smooth endoplasmic reticulum are evident. The disease can be produced in cobalt-deficient sheep fed diets high in propionate precursors, which may help explain the explosive nature of outbreaks on lush pasture. Hepatic lesions similar to lipotrope-deficient forms of experimental nutritional cirrhosis have been reported in sheep, goats, cattle, deer, and pronghorn antelope from Texas, New Mexico, and northeastern Mexico. Hard yellow-liver disease, or hepatic fatty cirrhosis, is a progressive, chronic disease characterized by weight loss and hepatic encephalopathy (eFig. 2-6). The disease typically appears in years following above-average winter rains, followed by drought conditions in the summer months. Grossly visible liver lesions in sheep begin in the subcapsular hepatic parenchyma along the porta hepatis as pale yellow, firm areas, spreading peripherally to involve ~80% of the liver in the final stages of the disease. Microscopic changes include accumulation of fine cytoplasmic lipid droplets in centrilobular hepatocytes, later involving the entire lobule, with rupture and formation of fatty cysts. Centrilobular fibrosis accompanies the ruptured fatty cysts, progressing to widespread bridging centrilobular fibrosis, with islands of regenerating hepatocytes. Kupffer cells and macrophages in regional lymph nodes, spleen, and lung contain abundant ceroid. The etiology of this condition is unknown, although unidentified hepatotoxins, possibly altering lipoprotein synthesis and secretion, combined with nutritional stress, have been postulated. eFigure 2-6 Hard yellow-liver disease in a goat. (Courtesy P. Stromberg.) Equine hyperlipemia is almost exclusively a disease of donkeys, miniature horses, and ponies, and among these, the Shetland breed predominates. The syndrome is characterized by marked elevation of serum triglyceride concentration, predominantly very-low-density lipoprotein (VLDL), but other lipid fractions are also elevated, with visible prominent lipemia and hepatic steatosis. The condition is usually fatal after about a week. A negative energy balance is a key feature. Pregnant or lactating mares are most likely to develop the disease, particularly if they are older, excessively fat, and have recently suffered reduced feed intake because of onset of parturition, conditions such as laminitis or parasitism, or other causes of stress. The clinical course is marked by somnolence, complete anorexia, and colic, progressing to mania in some cases, although most simply become progressively more depressed. Some ponies develop ventral subcutaneous edema and most develop moderate diarrhea. Metabolic acidosis is a consistent feature in animals that die. The liver at autopsy is severely fatty and may have ruptured; the steatosis also extends to heart and skeletal muscle, kidney, and adrenal cortex. The hepatic steatosis is remarkable only by its severity; there may be some focal hepatocellular necrosis, and there is consistent prolongation of bromosulfophthalein retention times and elevation of serum alkaline phosphatase levels. Evidence of disseminated intravascular coagulation is seen as serosal hemorrhages and microscopic thrombi in various organs, and even gross infarction of myocardium and kidney. Small lipid emboli may be detected in frozen sections of lung, myocardium, and brain in these animals; their relationship to the microthrombosis is uncertain. The pathogenesis of this disease is obscure. Because the excess lipid in liver and blood is in the form of triglyceride, the implication is that the liver is capable of esterifying fatty acid mobilized from depot fat. The triglyceride thus formed is presumably then exported to the plasma as VLDL. It is believed that the primary cause of hyperlipemia is increased production via an increased rate of adipocyte lipolysis, leading to fatty acids and glycerol being released into the bloodstream and the increased hepatic synthesis of triglyceride as VLDL, rather than reduced clearance of VLDL from the serum. Another possibility is that there is an inability on the part of all tissues other than the liver to use fatty acids from VLDL at the normal rate, whereas triglyceride synthesis from fatty acids continues in the liver. In any event, at this stage lipids begin to accumulate in hepatocytes. It has been proposed that an underlying cause of pony hyperlipemia is a comparative resistance to insulin in susceptible animals, and that this is compounded in stressful episodes by increased levels of circulating cortisol. Various steroid hormones, including glucocorticoids, have been shown to interfere with insulin action, and hyperlipemic ponies often have elevated plasma insulin levels, which suggests reduced function of insulin receptors. However, plasma ketones are much less consistently elevated, which appears to be characteristic of equids; this suggests that the increased ketogenesis that one might expect in insulin resistance is not a typical feature in horses. Hepatic steatosis is common in companion animals. Following periods of stress or fasting, toy-breed dogs can develop profound hypoglycemia and a striking microvesicular hepatic steatosis. This may be the result of poor homeostasis of blood glucose levels. Most often, affected pups die from cerebral complications of hypoglycemia, but significant liver dysfunction also occurs. Dogs eating diets deficient in vitamin E may develop severe hepatic steatosis, but there are many circumstances in companion animals where the exact cause for individual cases of steatosis cannot be determined. The syndrome of feline hepatic steatosis most commonly occurs in obese, nutritionally stressed female cats, which display vomiting, anorexia, weakness and weight loss, jaundice, and hepatomegaly (Fig. 2-25 ). Neurobehavioral signs indicative of hepatic encephalopathy, other than drooling and depression, are rarely reported. Affected cats generally have hyperbilirubinemia, and a significant increase in serum alkaline phosphatase activity, in the face of normal or modest increases in γ-glutamyltranspeptidase activity, a key diagnostic feature of this syndrome. Untreated, the mortality rate is high. The liver has diffuse, macrovesicular or microvesicular steatosis, by definition affecting >50% of hepatocytes. Focal or zonal lipid accumulation in <50% of the parenchyma is considered more likely to be physiologic, or associated with other systemic abnormalities, rather than idiopathic feline hepatic steatosis. Bile pigment accumulates in canaliculi or Kupffer cells and can be confused with lipofuscin and ceroid. Figure 2-25 Hepatomegaly resulting from hepatic steatosis in a cat (Courtesy A.P. Loretti.) The pathogenesis of hepatocellular triglyceride accumulation in this disease is obscure, and likely multifactorial, involving increased mobilization and uptake of nonesterified fatty acids by the liver, alterations in formation and release of VLDL, and impaired oxidation of fatty acids within hepatocytes. Ultrastructural studies have demonstrated decreased numbers and abnormal morphology of hepatic peroxisomes as well as mitochondria, both of which are important in the oxidization of fatty acids, but whether these changes are significant or simply adaptive responses is unknown. Starvation may reduce the availability of proteins, choline, and other precursors necessary for lipoprotein synthesis. Severe hepatic steatosis can also develop in cats, concurrent with or secondary to other major medical problems such as diabetes mellitus, which alter metabolism of fat. Acute pancreatitis appears to be an important predisposing disease to secondary hepatic steatosis in cats—an association with clinical significance as it has a poorer prognosis than uncomplicated idiopathic hepatic steatosis. Secondary hepatic steatosis has also been reported with concurrent inflammatory liver disease, such as cholangitis, renal disease, small intestinal disease, neoplasia, and hyperthyroidism. Familial hyperlipoproteinemia has been described in cats, associated with congenital lipoprotein lipase deficiency. The condition is characterized by lipid vacuoles and ceroid accumulation in liver, spleen, lymph nodes, kidney and adrenal glands, multifocal xanthomas, and focal arterial degenerative changes. An autosomal recessive mode of inheritance is suspected. Primary idiopathic hyperlipidemia has also been reported in Miniature Schnauzer dogs and Beagles, although the metabolic defect has not been identified. Affected dogs have fasting lipemia, elevated plasma VLDL, and may have hyperchylomicronemia. Affected animals may develop severe vacuolar hepatopathy associated with both glycogen and triglyceride accumulation, with eventual stromal collapse and regenerative nodule formation. There is also an association between hyperlipidemia and gallbladder mucocele. An incompletely characterized condition known as hepatic lipodystrophy has been recognized in pedigree Galloway calves since 1965. Calves initially appear normal but develop lethargy, tremors, and opisthotonos, and die by 5 months of age. On postmortem examination, affected calves have an enlarged, pale, mottled liver. Histologically, there is marked hepatic steatosis with portal fibrosis and ductular reaction (Fig. 2-26 ). Vacuolar changes in the white matter of the brain are consistent with hepatic encephalopathy. A metabolic defect has been proposed. Figure 2-26 Hepatic steatosis, portal fibrosis, and bile duct hyperplasia resulting from hepatic lipodystrophy in a Galloway calf. (Courtesy M.J. Hazlett.) Lysosomal storage diseases In common with other tissues in animals with a heritable deficiency of specific lysosomal enzymes, liver cells may accumulate substrates normally catabolized by the missing enzyme. These lysosomal stores can be less obvious in the liver than in other tissues such as the central nervous system, and although they are unlikely to affect hepatic function, they can sometimes be recognized in liver biopsies (eFig. 2-7). However, hydropic and fatty changes in hepatocytes can obscure or lead to misidentification of lysosomal storage vacuoles. Kupffer cells and bile duct epithelium may be more severely affected than hepatocytes, which have additional catabolic and excretory pathways, including the ability for lysosomal exocytosis into the bile canaliculi. In animals with ceroid lipofuscinosis, lysosomal storage is minimal in hepatocytes compared to that in the brain. Examples of storage disorders associated with hepatomegaly and abnormal hepatic or Kupffer cell lysosomal inclusions include GM1 gangliosidosis (β-1-galactosidase deficiency) in cats, mucopolysaccharidosis type I (α-L-iduronidase deficiency) in dogs, and α-mannosidosis in cats. These and others are discussed in more detail in Vol. 1, Nervous system. eFigure 2-7 Vacuolated hepatocytes in a cat with a ganglioside lysosomal storage disease (GM2), causing abnormal lysosomal accumulation. Hepatic phospholipidosis is an excessive accumulation of phospholipids within the cytoplasm of hepatocytes. This condition is an acquired storage disorder caused by some therapeutic drugs and chemicals, including amiodarone and chlorphentermine. Despite the diversity of agents capable of causing phospholipidosis, most of these are cationic amphipathic molecules. Mechanistically, phospholipidosis is caused by cationic amphipathic molecules binding to cellular phospholipids and inhibiting complete digestion by lysosomal phospholipase A1, A2, or C within lysosomes; although less often, direct inhibition of phospholipase can occur, as in the case of gentamicin. This leads to an accumulation of lysosomal phospholipids that can develop acutely or only following long-term drug administration. The histologic appearance of phospholipidosis can be quite variable, but typically, hepatocellular and Kupffer cell cytoplasm contains numerous round clear vacuoles that are smaller than the diameter of the nucleus or larger, imparting a foamy appearance to the cytoplasm. Fine vacuoles are most often apparent adjacent to canaliculi. Zonal distribution can vary depending on the type of drug or chemical. Biliary epithelium can also be affected, with or without hepatocellular involvement. The clear vacuoles can be confused with microvesicular steatosis, and in most cases, ultrastructural examination is needed to identify phospholipidosis with certainty. At the ultrastructural level, there is a characteristic multilaminated whorl of material with a “fingerprint” pattern, termed myeloid bodies, within affected lysosomes. Amyloidosis In most species, hepatic amyloidosis is usually part of generalized amyloidosis. Systemic amyloidosis of domestic animals is typically associated with overproduction of amyloid A (AA), an amino-terminal fragment of serum amyloid A, a highly inducible acute-phase protein in most species. In humans, AA amyloidosis occurs either as a familial trait (familial Mediterranean fever), or secondary to a sustained acute-phase reaction in chronic inflammatory or neoplastic diseases. Amyloid infiltration of the liver occurs sporadically in cattle, horses, dogs, and cats as a secondary response to chronic disease or tissue-destructive process. In horses, hepatic amyloidosis occurs chiefly as a result of chronic inflammation and has been well recognized in horses used for the production of hyperimmune serum. Horses may develop icterus and other signs of hepatic failure, but cattle die first of the primary disease or from uremia resulting from concurrent renal amyloidosis. Dogs and cats typically develop signs of renal dysfunction, although cats may be presented with spontaneous hepatic rupture. Familial AA amyloidosis is recognized in Chinese Shar-Pei dogs. The condition resembles familial Mediterranean fever in humans, and is characterized by febrile episodes, swollen hock syndrome, and the development of renal and sometimes hepatic amyloidosis. Familial AA amyloidosis is also recognized in Abyssinian cats and suspected in Siamese and Oriental cats. Affected livers in horses are pale, enlarged with rounded edges, friable, and prone to fracture; in cattle, affected livers may be firm. Affected livers in all species are predisposed to rupture and bleeding (eFig. 2-8). Amyloid is deposited first in the parenchyma about the portal tracts and appears gray and waxy. Amyloid is deposited in the perisinusoidal space between the sinusoidal lining and hepatocytes and is sometimes found in the walls of the afferent vessels. The surrounded hepatocellular cords atrophy. On H&E staining, amyloid appears as homogeneous eosinophilic amorphous extracellular material (Fig. 2-27 ). Staining with Congo red results in apple-green birefringence when viewed with polarized light. Thioflavine T stain produces yellow-green fluorescent staining of amyloid when viewed under ultraviolet light. Figure 2-27 Amyloid in spaces of Disse compressing hepatocytes in a cat. eFigure 2-8 Amyloidosis. A large, pale liver with capsular rupture and hemorrhage in a cat with amyloidosis. (Courtesy J.L. Caswell.) Further reading Armstrong PJ, Blanchard G. Hepatic lipidosis in cats. Vet Clin North Am Small Anim Pract 2009;39:599-616. Armstrong PJ, Williams DA. Pancreatitis in cats. Top Companion Anim Med 2012;27:140-147. Badylak SF, Van Vleet JF. Tissue gamma-glutamyl transpeptidase activity and hepatic ultrastructural alterations in dogs with experimentally induced glucocorticoid hepatopathy. Am J Vet Res 1982;43:649-655. Beatty JA, et al. Spontaneous hepatic rupture in six cats with systemic amyloidosis. J Small Anim Pract 2002;43:355-363. Bobe G, et al. Invited review: pathology, etiology, prevention, and treatment of fatty liver in dairy cows. J Dairy Sci 2004;87:3105-3124. Brix AE, et al. Glycogen storage disease type Ia in two littermate Maltese puppies. Vet Pathol 1995;32:460-465. Cebra CK, et al. Hepatic lipidosis in anorectic, lactating Holstein cattle: a retrospective study of serum biochemical abnormalities. J Vet Intern Med 1997;11:231-237. Center SA, et al. Ultrastructural hepatocellular features associated with severe hepatic lipidosis in cats. Am J Vet Res 1993;54:724-731. Center SA, et al. A retrospective study of 77 cats with severe hepatic lipidosis: 1975-1990. J Vet Intern Med 1993;7:349-359. Dannatt L, Porter TA. An outbreak of ovine white liver disease in south west England. Vet Rec 1996;139:371-373. Downs-Kelly E, et al. Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy. J Mol Neurosci 2000;15:251-262. Gregory BL, et al. Glycogen storage disease type IIIa in curly-coated retrievers. J Vet Intern Med 2007;21:40-46. Haskins ME, et al. Hepatic storage of glycosaminoglycans in feline and canine models of mucopolysaccharidoses I, VI, and VII. Vet Pathol 1992;29:112-119. Helman RG, et al. The lesions of hepatic fatty cirrhosis in sheep. Vet Pathol 1995;32:635-640. House JK, et al. Hemochromatosis in Salers cattle. J Vet Intern Med 1994;8:105-111. Jakowski RM. Right hepatic lobe atrophy in horses: 17 cases (1983-1993). J Am Vet Med Assoc 1994;204:1057-1061. Johnstone AC, et al. The pathology of an inherited hyperlipoproteinaemia of cats. J Comp Pathol 1990;102:125-137. Kennedy S, et al. Histopathologic and ultrastructural alterations of white liver disease in sheep experimentally depleted of cobalt. Vet Pathol 1997;34:575-584. Kishnani PS, et al. Canine model and genomic structural organization of glycogen storage disease type Ia (GSD Ia). Vet Pathol 2001;38:83-91. Lee FY, et al. Activation of the farnesoid X receptor provides protection against acetaminophen-induced hepatic toxicity. Mol Endocrinol 2010;24:1626-1636. Loeven KO. Hepatic amyloidosis in two Chinese shar pei dogs. J Am Vet Med Assoc 1994;204:1212-1216. Macleod NS, Allison CJ. Hepatic lipodystrophy of pedigree Galloway calves. Vet Rec 1999;144:143-145. McCaskey PC, et al. Accumulation of 2,8 dihydroxyadenine in bovine liver, kidneys, and lymph nodes. Vet Pathol 1991;28:99-109. Mogg TD, Palmer JE. Hyperlipidemia, hyperlipemia, and hepatic lipidosis in American miniature horses: 23 cases (1990-1994). J Am Vet Med Assoc 1995;207:604-607. Munson L, et al. Diseases of captive cheetahs (Acinonyx jubatus jubatus) in South Africa: a 20-year retrospective survey. J Zoo Wildl Med 1999;30:342-347. Niewold TA, et al. Familial amyloidosis in cats: Siamese and Abyssinian AA proteins differ in primary sequence and pattern of deposition. Amyloid 1999;6:205-209. Ohlenbusch PD. A possible relationship between climate, vegetation, management and occurrence of hard yellow liver disease. Vet Hum Toxicol 1990;32:583. O'Toole D, et al. Hepatic failure and hemochromatosis of Salers and Salers-cross cattle. Vet Pathol 2001;38:372-389. Pearson EG, et al. Hepatic cirrhosis and hemochromatosis in three horses. J Am Vet Med Assoc 1994;204:1053-1056. Sanchez CR, et al. Use of desferoxamine and S-adenosylmethionine to treat hemochromatosis in a red ruffed lemur (Varecia variegata ruber). Comp Med 2004;54:100-103. van der Linde-Sipman JS, et al. Fatty liver syndrome in puppies. J Am Anim Hosp Assoc 1990;26:9-12. van der Linde-Sipman JS, et al. Generalized AA-amyloidosis in Siamese and Oriental cats. Vet Immunol Immunopathol 1997;56:1-10. Whitney MS, et al. Ultracentrifugal and electrophoretic characteristics of the plasma lipoproteins of miniature schnauzer dogs with idiopathic hyperlipoproteinemia. J Vet Intern Med 1993;7:253-260. Wilkerson MJ, et al. Clinical and morphologic features of mucopolysaccharidosis type II in a dog: naturally occurring model of Hunter syndrome. Vet Pathol 1998;35:230-233. Xenoulis PG, Steiner JM. Lipid metabolism and hyperlipidemia in dogs. Vet J 2010;183:12-21. Yamazaki Y, et al. Role of nuclear receptor CAR in carbon tetrachloride-induced hepatotoxicity. World J Gastroenterol 2005;11:5966-5972. Types and Patterns of Cell Death in the Liver Irreversible damage leads to cell death that is evident histologically as long as the affected animal survives for a sufficient period of time, usually hours. Although all cells in the liver are susceptible, most interest and evaluation has been directed toward hepatocytes, biliary epithelium, and sinusoidal endothelium. The 2 predominant types of cell death are necrosis, also termed oncotic necrosis, and apoptosis, although there are other less common forms of cell death. These types of cell death can be separated mechanistically and histologically to a certain extent. Types of cell death In routine diagnostic settings, the terms apoptosis and necrosis can be based on descriptive criteria recognized in routine H&E-stained sections of liver. There have been attempts to refine the terminology and criteria for cell death in the liver, but this is still hindered by our incomplete understanding of different cell death pathways and cellular responses in various diseases. Apoptosis Apoptosis is a form of programmed cell death that permits removal of cell debris without much leakage of cell contents or inflammation. Key features of apoptosis include retention of plasma membrane integrity until the later stages of the process; proteolysis of intracellular cytoskeletal proteins by aspartate-specific proteases, leading to collapse of subcellular components; chromatin condensation and marginalization; nuclear fragmentation; plasma membrane bleb formation; and eventual cell fragmentation into smaller apoptotic bodies bound by intact plasma membrane (Fig. 2-28 ). These bodies are rapidly phagocytosed and degraded by neighboring hepatocytes or Kupffer cells. The rapid disappearance of these fragments means that very few apoptotic bodies in a section can indicate a rapid rate of hepatocellular death and turnover. Biochemically, apoptotic cells are characterized by phosphatidylserine on the outer surface of the cell membrane, increased mitochondrial permeability with release of internal proteins and activation of caspases. Figure 2-28 Three apoptotic hepatocytes (arrows) in a region of injury in the liver of a dog. Apoptosis is the main means of physiologic removal of damaged or aged cells and for remodeling of tissue. Liver mass is maintained by a balance of mitosis and apoptosis. Experimental support for the importance of this balance comes from studies of mutant mice deficient in the principal mediator of apoptosis, the receptor Fas (Fas/CD95), which develop prominent hepatocellular hyperplasia over time. Apoptosis can be initiated by extrinsic or intrinsic events involving the death receptors or mitochondria, respectively. Extrinsic mechanisms can be activated via cell surface receptors termed death receptors when they bind their cognate ligands. Death receptors include Fas, tumor necrosis factor-α (TNF-α) receptor and death receptor 4 (DR4) and 5 (DR5). The main signaling molecules in the liver are the Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), and TNF-α. When the membrane-bound death receptors bind their ligand, they trigger formation of a multiprotein complex, the death-inducing signaling complex (DISC). Conformational changes in this complex trigger the activation of caspase 8, the key mediator of apoptosis and subsequent activation of the effector caspases: caspases 3, 6, and 7. More details on hepatocellular apoptosis are available in recent reviews. Intrinsic mechanisms involve mitochondrial injury with release of proapoptotic factors. Activators of the latter pathway include oxidative stress, DNA damage, toxins, endoplasmic reticulum (ER) stress, including the unfolded protein response, lipid peroxidation, ultraviolet and γ-irradiation, and deprivation of growth factors. All of the pathways initiated by these agents converge on the mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP). Mitochondrial membrane permeability is effected either through direct membrane damage, or more commonly by opening of regulated transmembrane pores by bax and other proapoptotic members of the Bcl-2 family. Cytochrome C and other intermembrane mitochondrial factors released from the mitochondrion form an apoptosomal complex in the cytoplasm that activates the distal caspase pathways, including caspase 9, that execute many of the cell fragmentation events. Alternatively, MOMP can be triggered by another mechanism involving multiprotein channels within the mitochondria, leading to swelling and release of proapoptotic molecules. Functional classification of cell death has been developed on the basis of chemical rather than the histologic-morphologic criteria because different pathways can lead to similar morphologies. For example, intrinsic pathways can be divided into caspase-dependent and caspase-independent pathways. Injury to the ER via oxidative stress, hypoxia, calcium depletion, inflammation, and altered glycosylation, among other mechanisms, leads to the “unfolded protein response (UPR)” that can also activate the distal caspase pathway by a separate mechanism. Hepatocytes are also susceptible to an intrinsic mechanism of apoptosis that is active after removal of hepatotrophic influences, including those that function via the constitutive androstane receptor, and other nuclear receptors. In addition, lysosomes can undergo selective membrane permeabilization, leading to a partial release of their contents in response to death signaling mediated by oxidative stress, some lipid mediators, and by Bcl-2 family members. Release of lysosomal proteases can cooperate with the caspase cascade or act in a caspase-independent fashion. Both extrinsic and intrinsic pathways lead, in turn, to disruption of the cytoskeleton and nucleus primarily through the activation and direct action of caspases 3, 6, and 7. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. If the degree of mitochondrial damage is sufficient to exhaust ATP stores, membrane ion homeostasis deteriorates and leads to a mechanism of cell death more consistent with necrosis. For example, although mild to moderate oxidative stress may initiate intrinsic pathways of apoptotic cell death, processes leading to marked oxidative stress typically cause cell death by necrosis, not only resulting from the severity of mitochondrial damage, but also by direct inhibition of the proapoptotic caspase cascade. Loss of cell anchorage to the extracellular matrix can activate a form of programmed cell death related to apoptosis that is termed anoikis. Detached cells can die intact, as in exfoliation, or undergo typical fragmentation into apoptotic bodies. Hepatocyte survival in vitro depends on their integrins that adhere to the extracellular matrix, but it is still unknown how loss of these contributes to cell death of hepatocytes in the intact liver. Phagocytosis of apoptotic fragments follows flipping of phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaf, where it can then be recognized by the phosphatidylserine scavenger receptor. Various assays can be used to detect portions of these pathways in hepatocytes; caspase-cleaved cytokeratin 18 can be recognized by antibodies to an internal domain, whereas chromatin fragmented by a calcium-activated endonuclease generates double-strand breaks that can be tagged by the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) technique. However, there are still few diagnostic situations in which these indicators have been shown to be useful. Increased numbers of apoptotic hepatocytes can be more readily assessed with a suitable cytologic marker. In routine sections, a previous increase in apoptosis can be suggested by increased amounts of lysosomal debris and pigment in Kupffer cells, but this is not specific for apoptosis. Rapid phagocytic removal of apoptotic hepatocyte fragments can minimize secondary inflammation in cases of minor injury. However, phagocytosis of apoptotic bodies by Kupffer cells and hepatic stellate cells is not innocuous and can engender inflammation and fibrosis. Kupffer cells and resident macrophages express several ligands (TNF-α, TRAIL, and Fas ligand) that lead to increased hepatocyte apoptosis. Similarly, hepatic stellate cells are stimulated to release profibrotic cytokines and type 1 collagen following phagocytosis of apoptotic bodies. In addition, apoptotic cells release nucleotides ATP and uridine triphosphate (UTP), which can bind to purinergic receptors on macrophages and hepatic stellate cells and which may provide additional stimulus for fibrosis and inflammation. Excessive apoptosis is currently viewed as a driver of hepatic inflammation and fibrosis. Necrosis Necrosis is morphologically recognizable by initial cell swelling and subsequent loss of plasma membrane integrity, leading to large organelle-free blebs and cell lysis. Some authors prefer the term “oncosis” to emphasize the cell swelling response in necrosis to contrast with cell shrinkage in apoptosis (Fig. 2-29 ). Necrosis occurs when cells are depleted of ATP and lack sufficient energy to maintain membrane associated ionic pumps, leading to swelling and gross calcium influxes that result in disruption of the mitochondrial and plasma membranes, including release of lysosomal enzymes. Oncosis is generally regarded as a severe injury to cell membrane integrity or other vital functions, leading to enzyme leakage, inflammation, and tissue repair. Release of cellular contents typically elicits a secondary inflammatory response and provides the serum enzymes that are useful clinically to detect liver necrosis. However, these sequelae can also occur after hepatocyte apoptosis, either because the capacity for phagocytic removal of dead cells is exceeded, or because the insult worsens such that necrosis may supervene. Figure 2-29 Necrosis of hepatocytes is initiated by cell swelling evident in this toluidine blue–stained section of liver. (Courtesy V. Meador) Necrosis is often a consequence of profound loss of mitochondrial function involving the opening of the membrane permeability transition (MPT) pore, a megachannel composed of inner- and outer-membrane proteins. This results in mitochondrial depolarization and ATP depletion because of inhibited oxidative phosphorylation. Poly-ADP-ribose polymerase (PARP) is a DNA repair enzyme that can deplete injured cells of available ATP when it is activated to repair the multitude of DNA strand breaks induced by cell damage. Conversely, PARP is swiftly cleaved in apoptosis to maintain ATP levels. Much of the ATP in hepatocytes is used for membrane homeostasis of Na+, K+, and Ca2+ ions. ATP depletion or physical damage to membranes of the cell surface, mitochondria, and ER leads to loss of ion homeostasis, altered cellular volume regulation, and increased intracellular calcium ion concentrations. Activation of calcium-dependent endonucleases, proteases (e.g., calpains), and phospholipases is responsible for the terminal events in necrosis. The source of calcium may influence the type of cell death, as calcium influx from the plasma membrane is associated with necrosis, but calcium released from the ER is more likely to trigger apoptosis. Newer evidence suggests that necrosis is not entirely a passive process. For example, necrosis can be initiated via cell surface receptor–driven processes when TNF is present in high concentrations. The term coagulative necrosis may be applied to groups or zones of intact, but dead hepatocytes that have shrunken slightly, stain intensely with eosin, and may have visible but distorted nuclei (Fig. 2-30 ). These cells may also be dehydrated but, unlike apoptosis, the removal of water is not an active process, and the affected cells do not undergo spontaneous fragmentation. It seems that coagulative necrosis, which is often seen in acute hepatotoxicity, is the result of sudden and catastrophic denaturation of cytosolic protein, which imparts a rather dense, rigid texture to the dead cells, somewhat preserving their shape. The term lytic necrosis has been used for areas of necrosis in which the hepatocytes are disintegrating, usually in the presence of infiltrating phagocytes, especially neutrophils. This is consistent with the later stages of postnecrotic inflammation that follow necrosis rather than apoptosis. Figure 2-30 Coagulative necrosis of infarcted hepatocytes in a cat resulting from compression by an adjacent mass. One might expect that the different types of cell death might be informative in relation to causes and pathogenesis. However, apoptosis, necrosis, and mixed responses are common in liver injury in vivo. Many insults can initiate either necrosis or apoptosis of hepatocytes; these include hypoxia, reactive oxygen metabolites, hepatotoxic chemicals, viral infections, bacterial toxins, and inflammation. Some of the molecular events are common to both. Mitochondrial damage and several other activation responses are common to apoptosis and necrosis, and programmed cell death responses can be activated before they are overwhelmed by more intense injury responsible for necrosis. Susceptibility therefore depends on many factors, including the level and duration of insults, replication status, and integrity of the various homeostatic and cytoprotective functions. Indeed, review of caspase-cleaved substrates reveals that different toxic drugs do not cause a uniform pattern of caspase activation and that it is likely that there are distinct pathophysiologic pathways of apoptosis. Clearly, apoptosis is not a consistent and stereotypic response. There are important misconceptions regarding differences between apoptosis and necrosis and their effects on the liver. It is often assumed that apoptosis, unlike necrosis, leads to a “clean” death that does not provoke inflammation or elevation of transaminases and other markers of inflammation. However, there is evidence that phagocytosis of apoptotic bodies, termed efferocytosis, particularly by Kupffer cells can, in fact, activate the Kupffer cells and stimulate additional cell death. Various inflammatory markers are elevated in both acute and chronic apoptosis. The view that apoptosis, because it involves release of intact membrane bodies, does not lead to transaminase elevation has been shown to be incorrect in several studies. Injection of Fas ligands has been shown to produce significant increases in serum transaminases within hours and that this response is blocked in Bcl-2 transgenic mice. In addition, deletion of anti-apoptotic proteins results in elevated transaminase levels. Other forms of cell death There are a number of other forms of cell death. These include autophagy, pyroptosis, necroptosis, entosis, netosis, and parthanatos, which can be identified by biochemical means and specific forms of inhibition. A complete description is beyond the scope of this text. Tissue patterns of cell death Focal necrosis Focal necrosis is very common in autopsy material. The lesions are microscopic or barely visible to the naked eye and are usually numerous. Their designation as focal depends on their size and on a random distribution relative to the lobules. There may be a tendency for focal necrosis to occur nearer to the portal vessels than to the periphery of the circulatory fields, and to be concentrated in some lobular agglomerates rather than others. Focal necrosis occurs in many infections, parasitic migrations, and instances of biliary obstruction, and in these, the designation focal hepatitis will often be more appropriate, because most are attended by some degree of focal inflammation. The infectious causes may be viral (Fig. 2-31 ) or bacterial. Many septicemic bacterial infections consistently produce focal hepatic lesions; examples are salmonellosis, tularemia, pseudotuberculosis, listeriosis in the fetus and newborn, and Mannheimia haemolytica septicemia in lambs. The focal necrosis may be the outcome of a Kupffer cell reaction, as in salmonellosis, or of bacterial embolism. The cause can usually be determined by histologic examination. Figure 2-31 Focal necrosis caused by canid herpesvirus 1 in a dog. In cattle, focal necrosis in a few or many visible foci is common at autopsy and is common enough to be important at slaughter; it is responsible for the descriptive appellation “sawdust liver” (Fig. 2-32 ). The pathogenesis is not known and probably varies, but it may be caused by organisms from the gut that reach the liver in the portal blood. The lesion is not specific and consists of focal parenchymal necrosis with disruption of reticulin fibers and infiltration of neutrophils and lymphocytes; frank suppuration does not occur. This lesion is said to be more frequent in livers from feedlot-fattened cattle. Figure 2-32 Focal hepatitis—the so-called “sawdust liver” of cattle. Focal necrosis in biliary obstruction follows rupture of distended canaliculi or smaller cholangioles, with the formation of small bile lakes. Necrosis of larger bile ducts caused by neutrophilic cholangitis or destructive cholangitis can also release bile. The yellow pigment is readily visible microscopically and provokes small granulomas with giant cells. Focal necrosis is of very little functional significance for the liver, even when numerous. The lesions heal with some scarring, but this too probably disappears in time. They are of diagnostic importance in some diseases such as salmonellosis, and as indicators of possible bacteremia for meat inspectors. Lobular necrosis Although various terms are used, the simplest way to visualize these patterns of necrosis is by using the hepatic lobule concept. The time-honored designation “centrilobular necrosis” is a common lesion in response to intoxication or hypoxia. Centrilobular (zone 3) necrosis is the most common form of zonal necrosis in domestic animals. The hepatocytes in the centrilobular zone are particularly vulnerable to necrosis, in part because they are farthest from incoming arterial and portal venous blood bearing oxygen and essential nutrients. They also contain the greatest concentration of cytochromes P450 that activate various exogenous compounds into reactive metabolites capable of injuring or killing hepatocytes (see later section on Toxic hepatic disease). Severe viral infections, such as canine adenovirus 1 and Rift Valley fever virus, can produce centrilobular necrosis, and the reasons for the increased susceptibility of the hepatocytes of this zone in these diseases are not established. Plausible explanations include zonal expression of entry receptors used by viruses to infect hepatocytes or ischemia-related hepatocellular swelling and sinusoidal damage because of virus-induced endothelial injury that reduce effective perfusion of the centrilobular hepatocytes. Centrilobular degeneration and necrosis are seen commonly in animals that have died rather slowly. It is assumed that, in the agonal period, the hepatocytes in this zone are disproportionately affected by tissue hypoxia as a result of the failing circulation. This necrosis is more extensive if the animal is anemic. Centrilobular necrosis is also seen in passive venous congestion of the liver, most often in cases of acute and dramatic decompensation of cardiac function, as atrophy of hepatocytes is a more common outcome. In the liver with centrilobular necrosis, there is usually a prominent zonal pattern, which takes the form of a fine, regular, pallid network of surviving, often fatty, hepatocytes in the periportal zone, which stands up above the red, collapsed areas adjacent to the hepatic venules (Fig. 2-33 ). The zonal necrotic insult frequently affects the sinusoidal endothelium, allowing erythrocytes to enter the perisinusoidal space and contribute to the redness of the necrotic zones. Histologically, necrosis is confined to the centrilobular region (Fig. 2-34 ), although bridging can occur in more severe cases (Fig. 2-35 ). Necrotic cells that are removed can be replaced by stagnant blood, at least in the acute phase. However, this red-on-yellow zonal pattern is difficult to interpret grossly because hepatic steatosis may also have a zonal distribution without having significant necrosis. Figure 2-33 Enhanced zonal pattern of hepatocellular necrosis in a horse. (Courtesy R. Panciera.) Figure 2-34 Acute centrilobular necrosis in an ox. Figure 2-35 Bridging centrilobular hemorrhagic necrosis caused by Xamia sp. poisoning in a sheep. However, vascular influences on the susceptibility to necrosis mean that segments of the lobule can be differentially affected. Frequently, the areas of necrosis are joined to one another, thus cutting the conventional lobules into segments, and at the same time, outlining the periphery of the circulatory fields of the hepatic lobules. Some of these areas of necrosis extend up to larger portal triads, because the periphery of some lobules may lie against the larger portal tracts. Often, the hepatocytes between the necrotic and more normal zones show hydropic degeneration or fatty change. If the insult is of short duration, quite extensive centrilobular necrosis may be followed by phagocytic infiltration and hepatocellular proliferation and complete restoration of normal structure and function within a few days. Severe centrilobular necrosis may be followed shortly by proliferation of bipotential progenitor cells found near cholangioles, termed the ductular reaction, and mature bile duct epithelia that also respond to the regenerative stimulus. With restitution of the normal complement of hepatocytes, the proliferative response in the progenitor cells and biliary tract subsides unless the original insult is continuous or repeated. Some intoxications can produce selective midzonal (zone 2) necrosis, affecting only a narrow, sharply defined band of hepatocytes (Fig. 2-36 ). It may be more diffuse within the lobule, so that periportal or centrilobular degeneration may be superimposed on the more severe midzonal lesion. Figure 2-36 Acute midzonal necrosis in a foal. Periportal (zone 1) necrosis is also an uncommon lesion, perhaps more often seen than midzonal necrosis, and can be caused by direct-acting hepatotoxins that do not require metabolism by the cytochromes P450 to produce toxic moieties. More than one pattern of zonal necrosis can be found in the same liver. The various forms of zonal necrosis cannot reliably be distinguished from one another grossly, but one may expect to see in periportal necrosis a reversal of the pattern seen in centrilobular necrosis; that is, in periportal necrosis, the surviving hepatocytes about the hepatic venules may appear as pale, raised islands within a regular network of red, collapsed periportal tissue. Careful scrutiny may reveal the smallest hepatic venules at the center of the pale islands. Paracentral necrosis, a form of coagulative necrosis, occurs when an isolated portion of the centrilobular region of the lobule (or complete hepatic acinus of Rappaport—perhaps visualized more easily as an entire acinus) dies and is viewed in transverse section (Fig. 2-37 ). It is possibly an ischemic lesion or infarct produced by an occlusion of a terminal portal venule, such as may occur in disseminated intravascular coagulation. Its appearance in certain of the acute hepatotoxicities probably represents the death of a single complete acinus as a result of local vascular insufficiency, although, theoretically, high local microsomal enzyme activity or local deficiency of hepatocellular protective factors may play a part. Occlusion and rupture of a bile ductule or cholangiole are other potential causes of paracentral necrosis. Figure 2-37 Paracentral necrosis. Massive necrosis Massive necrosis refers to necrosis of an entire hepatic lobule, not necessarily necrosis of the liver as a whole. By accepted definition, every cell in the affected lobule is dead, including the hepatocytes of the limiting plate (Fig. 2-38 ). Without surviving parenchyma to support regeneration, affected lobules collapse, so that portal areas and hepatic venules are approximated and the intervening stroma is condensed. Such a liver must regenerate from surviving hepatocytes in other, less severely affected lobules or by proliferation of progenitor cells that mature into hepatocytes. The distribution of massive hepatic necrosis often relates to the distribution of larger vessels. Collapse, condensation, and subsequent scarring are characteristic, the end result being known as postnecrotic scarring. The liver is not uniformly involved typically. Large areas of parenchyma remain intact, and these enlarge amid scarring areas during compensatory regeneration. Figure 2-38 Massive necrosis with destruction of the periportal limiting plate and ductular reaction caused by Amanita poisoning in a cat. A liver that is the seat of massive necrosis may be of normal size or smaller. Fine red threads of fibrin may be present on the surface, especially in the grooves between the lobes. There is a surface mosaic appearance of red, gray, or yellow areas intermingled with areas of dark red. The gray or yellow areas of parenchyma, representing surviving tissue, form irregular, coalescing patches that may be <1.0 cm in diameter. The intermingled red areas represent areas of necrosis, hemorrhage, and collapse, and these are depressed a few millimeters below the surface. In the healing stage, the depressed areas of hemorrhage and necrosis are condensed, shrunken, and scarified so that the surface of the liver is traversed by fine or heavy scars that separate large nodules of regenerative hyperplasia. Further acute episodes may be superimposed so that the presented lesion may be a mixture of acute massive necrosis and postnecrotic scarring. Hepatosis dietetica of swine is a now uncommon syndrome of massive hepatic necrosis in association with its immediate or late effects, namely “yellow-fat disease,” degeneration of skeletal and cardiac muscle, serous effusions, ulceration of the squamous mucosa of the stomach, and fibrinoid necrosis of arterioles. These lesions may occur alone or in any combination, although all seldom occur in one animal. They are known to be of nutritional origin, and the fact that the various lesions can occur separately indicates the complexity of the pathogenesis. Experimental observations have revealed the need for concurrent deficiencies of sulfur-containing amino acids, tocopherols, and trace amounts of selenium if hepatic necrosis is to develop. Selenium protects efficiently against the hepatic necrosis and massive effusions, and tocopherols are probably protective against other lesions that occur as part of the syndrome. The pathogenesis is incompletely understood, but is in part related to the generation of free radicals, exacerbated by deficiency of free-radical scavengers such as vitamin E, and selenium, protective against reactive oxygen radicals through its role in glutathione peroxidase and some other selenoproteins. Hepatosis dietetica occurs in rapidly growing pigs fed diets largely of grain and containing protein supplements lacking in either quality or quantity. There is some evidence that in pigs that are nutritionally predisposed, a cold and damp environment or some other stress may precipitate the disease. Death usually occurs without signs of illness or after a short period of dullness. Melena, dyspnea, weakness, and trembling may be observed in some cases. Jaundice is indicative of a relapsing course. Affected pigs are usually in good condition. The carcass may be anemic if ulceration of the gastric mucosa has occurred, and, in these cases, free and digested blood may be found in the stomach and intestine. Jaundice is not common, but yellow staining of adipose tissues (yellow-fat disease) is. In relapsing cases, hemorrhagic diathesis may occur, manifested mainly by hemorrhage into and about joints. Protein-rich fluid collects in the serous cavities in small volume. Fine strands of fibrin are present in the peritoneal cavity. Pulmonary edema accompanies myocardial lesions that consist of intramural and subendocardial hemorrhages with focal areas of hyaline degeneration. The changes in the liver dominate the autopsy findings. The massive hepatic necrosis is of the typical appearance described earlier, and in a number of cases, both acute and chronic lesions are found (Fig. 2-39 ). The sites of severest injury are the dorsal parts on the diaphragmatic surface. The right lobe may escape and later undergo marked hypertrophy. The gallbladder is often edematous. Figure 2-39 Hepatosis dietetica in a pig. (Courtesy University of Guelph.) The histologic changes that occur in this syndrome are described elsewhere with the particular organs involved. Briefly, centrilobular necrosis of the liver is typical (eFig. 2-9). Additionally, fibrinoid degeneration of small arteries occurs in some cases. The arterial degeneration may occur in any organ or in most organs but is relatively common in only the small vessels of the mesentery, gut, and heart (see Mulberry heart disease, in Vol. 3, Cardiovascular system). eFigure 2-9 Centrilobular necrosis in hepatosis dietetica. Piecemeal necrosis Models of immune-mediated hepatocyte necrosis have emerged from studies of human viral hepatitis and some forms of drug-induced chronic hepatitis. This pattern of necrosis amid sites of more active inflammation at the limiting plate of hepatocytes immediately adjacent to the edge of the portal tract is referred to as piecemeal necrosis. The mechanisms may involve either direct damage to hepatocytes by the uptake of antigen-antibody complexes, or cooperation between macrophages and T lymphocytes. These may cause cell-mediated destruction of hepatocytes that have taken up these complexes or, perhaps, native antigen or virus. Whatever the agency, the mode of cell removal in this sort of injury often takes the form of single-cell necrosis or apoptosis, which may be directly triggered by immunologically competent cells. This type of liver injury, in which inflammation characteristically disrupts the limiting plate, giving an irregular appearance to the periportal zone, is discussed further in the section on Chronic hepatitis. Necrosis of sinusoidal lining cells Injury to the endothelium of hepatic sinusoids can develop in a variety of forms. The sinusoidal endothelial cells may lose their typical fenestrae or pores, limiting contact between plasma and hepatocellular microvilli, and if there is an increased deposition of extracellular matrix (ECM) beneath the altered endothelium, the alteration is termed “capillarization of sinusoids,” leading to reduced hepatic function. Sinusoidal endothelial cells may relax their connections to the fine meshwork of the hepatic “reticulin” or detach completely forming microemboli in the sinusoid downstream. In addition, the residual, exposed space of Disse has an increased risk of sinusoidal thrombosis. Acute endothelial injury can occur as a result of ischemia-reperfusion injury, acetaminophen or pyrrolizidine toxicity, and effects of endotoxin, all of which can lead to localized ischemia and necrosis. Other endothelial toxins include ngaione poisoning and microcystin-LR, a highly toxic cyclic heptapeptide produced by Microcystis aeruginosa, an aquatic cyanobacterium (blue-green alga). Microcystin-LR is selectively injurious to hepatic sinusoidal endothelial cells by inhibiting protein phosphatases. When the cytoskeleton becomes hyperphosphorylated, endothelial cells undergo apoptosis, leading to hemorrhagic necrosis. Some forms of peliosis hepatis may develop following endothelial necrosis. More chronic injury can produce fibrosis, leading to diminished or disrupted sinusoidal blood flow. Alteration of the sinusoidal blood flow can be a primary event leading to hepatocyte hypoxia with liver dysfunction and disruption of the portal circulation. Secondary endothelial cell injury can result when hepatocytes are being destroyed by the elaboration of toxic molecules within their cytoplasm, it is to be expected that the sinusoidal lining cells may also suffer should the products of these biotransformations spill into the space of Disse. Erythrocytes increase in the perisinusoidal space and, when endothelial damage is severe, the regions of necrosis are hemorrhagic. Necrosis of sinusoidal endothelial cells is seen very early in the course of many hepatotoxicities. Sinusoidal phagocytes are sometimes vulnerable to necrosis by virtue of their role in clearing the portal blood of particulate or colloidal material; should these particles be toxic or infectious, Kupffer cell necrosis may occur alone, but more usually, there is damage to surrounding hepatocytes as well. Necrosis of bile duct epithelium It is unusual for the bile duct epithelium to be singled out by specific lethal insults, but this is seen in intoxication by sporidesmin (see section on Toxic hepatic disease). Usually, there is accompanying portal inflammation. Some experimental toxicants, such as α-naphthylisothiocyanate, are lethal to bile duct epithelia and elicit a local inflammatory cholangitis. Idiosyncratic drug-induced destructive cholangitis can lead to acute cholangiolar injury as well as chronic cholestasis with damage and loss of bile ducts (destructive cholangitis). Treatment with sulfonamides, other drugs, and viral infection have been linked to this response (Fig. 2-40 ). Figure 2-40 Acute necrosis of biliary epithelium in a dog following exposure to trimethoprim-sulfa. Further reading Copple BL, et al. Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure. Toxicol Sci 2002;65:309-318. Guicciardi ME, et al. Apoptosis and necrosis in the liver. Compr Physiol 2013;3:977-1010. Luedde T, et al. Cell death and cell death responses in liver disease: mechanisms and clinical relevance. Gastroenterol 2014;147:765-783. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011;147:728-741. Responses of the Liver to Injury The liver is a remarkably versatile organ. Given its central position in the body, supplied by blood draining the gastrointestinal tract, and the fact that it is the key organ involved in detoxification of exogenous (xenobiotics) and endogenous compounds (endobiotics), the liver must respond to injury, including metabolic, infectious, and hemodynamic insults. This section describes the 3 main categories of liver response to injury: regeneration, fibrosis, and ductular reaction (biliary hyperplasia). Hepatic regeneration Mild injury involving the hepatocytes, biliary epithelium, endothelial cells, and mesenchymal elements typically is completely resolved quickly through regeneration. This is particularly true in younger animals, as the vigor of regeneration diminishes with age. Even in cases of massive hepatocellular necrosis, complete recovery is possible if the reticulin framework of the liver persists and surviving hepatocytes have sufficient time to regenerate and replace the lost hepatic mass. In adulthood, the liver is a stable organ with limited replication of mature hepatocytes, biliary epithelium, and other cells in the liver. However, loss of hepatic mass through injury, such as infections, toxic insult, or surgical removal, will transform the mature hepatocytes, biliary epithelium, and/or bipotential progenitor cells, as well as hepatic stellate cells and endothelial cells, into actively proliferating cells until normal functional mass has been replaced. As much as 70% of the normal liver can be removed surgically without clinical insufficiency, and in the course of a few weeks, it is back to its normal mass. Depending on the nature of the injury, regeneration occurs via one of 2 pathways. In cases of moderate injury, cell loss is replaced by proliferation of mature hepatocytes or biliary epithelium, providing swift replacement of parenchymal loss. In cases of more severe injury, or conditions that inhibit replication of mature cells, there is proliferation of bipotential progenitor cells, called a ductular reaction. These cells reside in the periportal region of the lobule at the level of the canals of Hering, the site where bile canaliculi and biliary epithelial cells first join and form the beginning of the epithelium-lined biliary tree. They are recognized histologically as small, basophilic cells that form single-cell arrays or ductules with narrow lumens. Depending on the differentiation pathway that is followed, they can mature into hepatocytes or bile duct epithelium. Regeneration of mature hepatocytes following surgical removal is well known. An example from Greek mythology is Prometheus, punished for giving fire to the mortals, daily had his liver partially eaten by an eagle (a symbol of Zeus), only to regrow and be eaten again. Although the mythical daily regrowth of hepatic mass is more prodigious than the real-world response, hepatic regeneration is nonetheless remarkable. In rodents, partial hepatectomies can be repeated more than 10 times, followed by restoration of normal hepatic mass. Individual hepatocytes are able to replicate up to 70 times. Simple replacement of missing hepatocytes alone does not truly constitute regeneration because normal liver microscopic architecture is essential to maintain normal hepatic function. Although new lobes are not regenerated following hepatectomy, the remaining lobes enlarge, and the proliferated hepatocytes and other cellular elements progress through a series of steps that involve interaction of hepatocytes and angiogenic processes to restore the normal relationships between the hepatocytes and the sinusoids. Regeneration of mature hepatocytes is stimulated by several factors. These include polypeptide growth factors, such as hepatocyte growth factor, epidermal growth factor, transforming growth factor-α (TGF-α), and insulin-like growth factors. Hepatocyte growth factor is the most potent mitogen and is released by hepatic stellate cells to stimulate hepatocytes in a paracrine fashion. This growth factor can also be embedded in the ECM to be released by proteases. Hepatic regeneration is also enhanced by nutrition, with fasting slowing and dietary protein promoting regeneration. In addition, catecholamines and a broad variety of hormones support regeneration, with insulin, glucagon being the most significant. Cytokines, such as IL-6, produced by TNF-α–stimulated Kupffer cells, are other important effectors. When IL-6 binds its receptor, it stimulates mitogenic intracellular signaling and potentiates signaling by other growth factors. These effectors are examples of the important role of the innate immune system in hepatic regeneration. The autonomic nervous system also plays a role in hepatic regeneration, as denervation of the liver can significantly limit hepatic regeneration. Following hepatocyte proliferation, normal liver architecture must be preserved to maintain hepatic function. Several cell types are involved in this process. Following partial hepatectomy, the initial proliferation creates aggregates of hepatocytes 10-14 cells thick. These aggregates are penetrated by endothelial buds that eventually establish sinusoidal channels. Ingrowth of the endothelial cells is driven by regulators of angiogenesis, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Hepatic stellate cells replicate and synthesize ECM within ~4 days of partial hepatectomy and help establish normal hepatocyte-matrix interactions. Once the needed hepatic mass has been replaced, the regenerative process must be regulated to terminate cell proliferation. TGF-β and related family members, including activin, are the best known mediators responsible for curtailing hepatocyte proliferation. These mediators are produced primarily by the hepatic stellate cells. They can increase ECM production as well as suppress hepatocellular replication, are downregulated during proliferation, and increase once appropriate hepatic mass has been achieved. In severe, often toxic, liver injury, mature hepatocytes cannot replace damaged hepatic mass, and bipotential progenitor cells are engaged to restore the hepatic mass. At the interface of the portal tract connective tissue and the hepatic parenchyma, and in the vicinity of the canals of Hering, there is a compartment that serves as the site of quiescent progenitor cells. Proliferation of these cells gives rise to the “ductular reaction,” characterized by ductular structures lined by cuboidal, basophilic cells (Fig. 2-41A ). The relationship between the “oval cells” described in rodents and the ductular reaction still requires some clarification, but it is likely that the same cell type is involved. In addition, there may be progenitor cells among the mature biliary epithelial cells, and others may be marrow derived and blood-borne to the liver. Immunohistochemical staining of these cells in humans and rodents reveals that they contain cytokeratin markers of biliary epithelium and, simultaneously, hepatocyte markers such as albumin (Fig. 2-41B). Similar cells are present in animals as well. There are several circumstances that can trigger the ductular reaction other than massive necrosis, although this is the best recognized form. Both cholestasis and hypoxia can drive the ductular reaction causing proliferation in the periportal region for the former, and in the centrilobular region in the latter instance. The ability of these cells in the ductular reaction to differentiate into mature biliary or hepatocellular phenotypes is well established. It should be appreciated that in most clinical circumstances, hepatic regeneration is a less orderly process than that which occurs in healthy experimental animals. Regeneration requires normal arterial and venous inflow as well as venous and biliary drainage. Without a supportive environment, hepatic regeneration of hepatocytes, biliary epithelium, and nonparenchymal cells may be quite variable within different regions of the liver. Following massive necrosis, all remaining hepatocytes are likely to initiate a replicative response, whereas random focal necrosis will engender a local proliferation of hepatocytes. Scattered individual cell necrosis is repaired almost imperceptibly. In the more common pattern of centrilobular necrosis, seen in many toxic insults, complete repair is usually evident with a 24-48 hour period. Chronic or repetitive injury often results in nodule formation as discussed in the section on Fibrosis. Figure 2-41 A. Ductular reaction in an injured liver. B. Cytokeratin 7 stain. Ductular reaction (bile duct hyperplasia) Ductular reaction (biliary hyperplasia) is a characteristic reaction of the liver to various types of insult involving proliferation of bile duct epithelium, hepatic progenitor cells, and possibly metaplasia of mature hepatocytes. The histologic hallmark of the ductular reaction is the formation of new, irregular, and tortuous ductules or chains of cells formed from flattened or cuboidal basophilic epithelium. There are 3 main types of ductular reactions. (1) A common association with acute bile duct obstruction is proliferation of preformed bile duct epithelium, creating a rapid increase in bile duct surface area. This can protect hepatocytes from bile acid buildup through a recirculation process termed cholehepatic recirculation. (2) In cases of serious injury to hepatocytes, particularly when mature hepatocytes are not able to replicate, there is a prominent ductular reaction originating from hepatic bipotential progenitor cells (or oval cells). The bipotential nature of these cells is supported by the detection of biliary epithelial cytokeratins 7 and 19 in cells with an otherwise hepatocellular phenotype. A dramatic presentation of ductular proliferation is often evident in animals that succumb several days after a fatal hepatic intoxication or in horses with Theiler's disease. (3) Ductular reaction may also result from metaplasia of mature hepatocytes in regions of hypoxia. Ductular reaction may also develop secondary to local portal inflammation and fibrosis. Ductular formation can also occur in areas of hypoxia. Such ducts formed by metaplasia of mature hepatocytes can be found in centrilobular areas in animals with chronic heart failure and passive congestion, for example (Fig. 2-42 ). Figure 2-42 Ductule formation in the centrilobular region of a dog with chronic right-sided heart failure. However, reabsorption of bile constituents by biliary epithelium leads to a neutrophilic inflammatory reaction and local edema formation, which can be confused with a bacterial infection. With persistence, the ductular reaction can contribute to portal and periportal fibrosis as the ductular cells secrete profibrogenic growth factors, cytokines, and chemokines. Illustrative examples from natural disease are provided by the toxicoses of phomopsin, pyrrolizidine alkaloids, and aflatoxin, and by equine serum hepatitis and ovine white-liver disease. Fibrosis Hepatic fibrosis is a potentially reversible form of wound healing in which there is an accumulation of various ECM components. In cases of acute or self-limited injury, fibrosis may resolve completely and normal liver architecture can be restored, but when damage is too extensive to be repaired, a scar may be permanent, and if the injury is persistent, there is progressive deposition of ECM, leading to the “final common pathway” and ending in fibrosis and cirrhosis. In normal liver, the ECM exists in a state of dynamic balance, with synthesis and degradation occurring in balance to maintain normal levels of all constituents. It is found in the portal tracts, along the sinusoids in the space of Disse, around the central vein and in Glisson's capsule. Normally, the hepatic ECM constitutes only 3% of the relative areas on a tissue section and only 0.5% of the total liver wet weight. The main constituents of the hepatic ECM are collagen, proteoglycans, laminin, fibronectin, and matricellular proteins. In the space of Disse, the matrix is composed primarily of collagens IV and VI. Following injury, however, fibrillar collagens I and III as well as fibronectin are deposited. These deposits disrupt the normal structure and function of the sinusoids, leading to capillarization of sinusoids, discussed later. The ECM is not merely scaffolding for the liver. It also functions as a repository for various growth factors and metalloproteinases in latent form, and it can bind a variety of survival factors, such as hepatocyte growth factor and TNF-α, that may protect the growth factors and inhibit hepatocyte apoptosis. In addition, interactions between cells and the ECM can affect the phenotype of the hepatocytes and stellate cells and the composition of the ECM. Thus the process of hepatic fibrosis is distinct from the condensation of the hepatic reticular framework that can be seen following hepatocyte loss. The process of ECM production in the injured liver is initiated by a process that activates hepatic mesenchymal cells into myofibroblasts. These mesenchymal cells constitute a prototypical mesenchymal cell type that mediates injury and repair in a number of tissues, including the kidney, skin, lung, and bone marrow. All of these cell types have the ability to produce ECM, and have contractile ability. The best recognized group in this family is the hepatic stellate cells, but there are other important groups in this family, including portal fibroblasts, bone marrow–derived cells, and, via epithelial-mesenchymal transition, hepatocytes that can acquire a mesenchymal phenotype. Portal fibroblasts, particularly those surrounding the bile ducts, are an important source of ECM formation in the portal tracts. This is particularly evident in cases of biliary tree injury. Portal fibroblasts/myofibroblasts are distinct from stellate cells based on distinct protein markers. Hepatic stellate cells are found in the space of Disse, often between hepatocytes. In normal circumstances, these cells store lipid, primarily vitamin A (retinyl esters), in characteristic large round vacuoles. In injured liver, hepatic stellate cells become activated. A principal morphologic change involves the loss of the lipid vacuole and transformation into a spindle-shaped myofibroblast. In some species, stellate cells contain immunohistochemically detectable α–smooth muscle actin before activation, and in other species, activation is required for expression of this protein. When activated, they replicate robustly at sites of injury and are the main source of collagen and other ECM components, such as proteoglycans, fibronectin, and hyaluronan, lining the space of Disse. Activated stellate cells also release proinflammatory, profibrogenic, and promitotic cytokines. Other features of stellate cells include the ability to present antigen and their consistent contact with individual fibers of autonomic nerve endings. Hepatic blood flow can be significantly modified by activated stellate cells. Blood flow through sinusoids can be restricted by the contraction of the stellate cells that extend processes around the sinusoidal endothelial cells. In health, there is a balance between the nitric oxide–stimulated vasodilation and endothelin-1–driven vasoconstriction. During active fibrosis and cirrhosis, there is an increase of endothelin-1, produced by sinusoidal endothelial cells causing contraction of the stellate cell–derived myofibroblasts. Fibrosis can form within portal tracts expanding their area, or as septa extending into the parenchyma and eventually bridging between portal tracts or central veins. Fibrosis in the portal tracts is primarily generated by the activity of portal fibroblasts, rather than hepatic stellate cells. Biliary injury leads to a rapid proliferation of periductular myofibroblasts derived from portal fibroblasts and ECM production. The volume of the portal tracts is increased by the generation of the ECM, ductular proliferation, myofibroblast proliferation, and edema. Fibrosis can be less florid and more insidious when it lines the space of Disse, disrupting the normal exchange between the plasma and hepatocyte microvilli. The essential microvascular unit of the liver is the sinusoid, lined by fenestrated endothelial cells, and separated from the hepatocytes by the space of Disse. A porous basal membrane–like matrix in the space of Disse helps to maintain the differentiated states of the hepatocytes and hepatic stellate cells that inhabit the space of Disse. When there is excess ECM lining the space of Disse, the adjacent sinusoidal endothelial cells lose their characteristic fenestrations, and the sinusoids are transformed functionally into capillaries. This process is known as capillarization and leads to significantly impaired exchange between the hepatocytes and the plasma. This causes diminished hepatic function when the lesion is extensive. The cells responsible for the ECM deposition are primarily in the myofibroblast family, including hepatic stellate cells. Because collagen has a finite half-life, and as hepatic fibrosis exists in a balance of synthesis and removal, it is potentially reversible. Elimination of the source of injury or successful therapy can lead to stellate cell apoptosis and a reduction in the synthesis of inhibitors of the enzymes responsible for the degradation of the collagen and other ECM elements, primarily matrix metalloproteinases. This has been demonstrated in rodent models of hepatic fibrosis produced by bile duct ligation, and models using carbon tetrachloride. However, over time, collagen can mature and fibrils can cross-link and become resistant to enzymatic degradation, leading to permanent fibrosis. Long-term or permanent fibrotic changes can also be caused by severe acute injury that leads to an initial focus of fibrosis and is so severe that it affects local vascular supply. The distribution of fibrosis in the liver can reflect the pathogenesis of the injury responsible. • Inflammatory disorders that produce piecemeal necrosis of hepatocytes, typically viral hepatitis in humans and often an idiopathic disorder in veterinary species, can evolve to form portal-to-portal, or portal-to-central, fibrous septa. • In the event of inflammation or obstruction involving the bile ducts, proliferation of periductular myofibroblasts and prominent ductular reaction can lead to biliary fibrosis or a pattern of portal-to-portal bridging termed biliary cirrhosis when accompanied by nodular regenerative proliferation of hepatocytes (Fig. 2-43 ). Figure 2-43 Bridging portal fibrosis and bile duct hyperplasia secondary to bile duct obstruction in a horse. • Postnecrotic scarring occurs after massive necrosis, where large areas of parenchyma are destroyed. The reticulin network collapses and condenses and portal areas converge, resulting in broad, irregular bands of scar tissue with variable irregular areas of parenchymal regeneration interspersed (Fig. 2-44 ). Figure 2-44 Postnecrotic scarring in the liver of a sheep. (Courtesy P. Stromberg) • Diffuse hepatic fibrosis is the outcome of chronic parenchymal injury, such as prolonged inflammation or multiple episodes of zonal necrosis. The fibrosis throughout the lobules bridges connective-tissue tracts in portal areas and hepatic venules to produce pseudolobulation, in which small areas of parenchyma are separated by a pattern of fibrosis on the scale of true lobules. This pattern of fibrosis can lead to capillarization of sinusoids, or intrahepatic bypass of portal and arterial blood, both of which can reduce the influences of growth factors and nutrients on liver regeneration. The accompanying hypoxia is important in the genesis of the hepatocellular atrophy that is almost always concomitant with diffuse hepatic fibrosis. • Centrilobular (periacinar) fibrosis is the most common pattern following zonal necrosis in hypoxic and toxic injury. Good examples are seen in animals with prolonged passive venous congestion of the liver, especially when this is caused by extracardiac sources of increased venous pressure, rather than to congestive heart failure (Fig. 2-45 ). Otherwise, this pattern of fibrosis is a response to toxic injury; poisoning by pyrrolizidine alkaloids may cause it in ruminants, and extraordinary development of periacinar fibrosis may follow accidental exposure to nitrosamines in several species. Figure 2-45 Centrilobular fibrosis (cardiac fibrosis), most severe at the center of the lobe in chronic passive congestion in a dog. Cirrhosis Cirrhosis has several definitions, and consensus regarding the essential features is difficult to obtain. In general, it is agreed that cirrhosis is not merely an end stage of ECM accumulation in the liver, but, in fact, is a multifaceted distortion of hepatic parenchymal architecture and hepatic vascular anatomy. By one definition, cirrhosis is “a diffuse process characterized by fibrosis and the conversion of normal liver architecture into structurally abnormal nodules.” The key features are the formation of regenerative nodules of hepatocytes surrounded by fibrous septa and vascular disorders that often integrate both central veins and portal tracts. Other than dogs, most veterinary species develop diffuse hepatic fibrosis following chronic injury, but do not develop regenerative nodules. Chronically injured canine livers often have a robust formation of regenerative nodules separated by fibrous septa typical of cirrhosis (Fig. 2-46A-C ). Portosystemic shunts and venous occlusion may result, interfering with hepatic function and driving portal hypertension. Note that not all nodular livers are cirrhotic. Clinically, cirrhosis results in hepatic insufficiency, mainly in the form of ascites and hypoproteinemia rather than excretory dysfunction and icterus, although, in advanced cases, all manifestations of fatal liver failure can occur. The hallmarks of cirrhosis are: 1. Bridging fibrous septa, ranging from delicate bands to broad scars that replace multiple adjacent lobules. These fibrous septa contain vascular channels, originating from sinusoids in collapsed parenchyma or angiogenesis that allow a considerable portion of blood to bypass hepatocytes. These microvascular channels in fibrous septa resist flow more than normal sinusoids. This factor, as well as the relative increase in flow in these vessels shunting around the nodules, contributes to portal hypertension. 2. Impaired exchange between hepatocytes and sinusoidal blood, because of the increase in perisinusoidal ECM. 3. Parenchymal nodules, created by the regenerative attempts of entrapped hepatocytes, and varying in size from <3 mm in diameter (micronodular) to several centimeters in diameter (macronodular). These nodules are composed of trabeculae that are typically 2 or more cells thick, with relative reduction of sinusoidal space. Some expanding nodules may compress the vessels within fibrous septa, contributing to portal hypertension. Nodule formation is prominent in dogs, but significantly less so in most other domestic species. 4. Ongoing damage and reorganization of the hepatic connective tissue, sometimes with areas of portal and arterial thrombosis and segmental ischemia is typical, but not always present. Figure 2-46 A. Regenerative nodules in the liver of a dog. B. Cut surface of the liver. C. Microscopic appearance of the regenerative nodule and adjacent septum. Cirrhosis is usually the end result of several pathogenic processes, namely, cell death (necrosis or apoptosis) and active inflammation with chronic fibrosis. Cirrhosis is not a synonym for chronic hepatic fibrosis, although some insults that cause chronic diffuse fibrosis in the liver can lead to cirrhosis. The nodular pattern of regeneration in cirrhosis is caused by expanding islands of surviving parenchyma entrapped between bands of scar tissue. At the point of liver failure, these regenerative attempts are insufficient to restore function. Despite the increased hepatocellular mass, the inability to restore normal function is attributed to altered circulation through the bridging scar tissue, portal hypertension and shunting, deposition of ECM in the perisinusoidal space, reduced supply of portal growth factors, inhibition of proliferation of ECM, and toxic inhibition of hepatocellular proliferation. The characteristic fibrovascular septa that bridge portal and central vascular tracts are readily observed in liver sections, but the portal-hepatic shunting therein can be difficult to identify. Clinical evidence for portosystemic shunting, such as impaired ammonia or bile acid clearance, can reflect acquired portocaval shunts that often develop as a result of portal hypertension from chronic hepatic fibrosis. Acquired portosystemic shunting Any chronic liver disease that causes sufficient fibrosis or atrophy to restrict portal blood flow significantly and produce portal hypertension has the potential to cause the development of collateral portosystemic shunts. These are described later in the section on Vascular factors in hepatic injury and circulatory disorders. Further reading Anthony PP, et al. The morphology of cirrhosis. Recommendations on definition, nomenclature, and classification by a working group sponsored by the World Health Organization. J Clin Pathol 1978;31:395-414. Desmet VJ. Ductal plates in hepatic ductular reactions. Hypothesis and implications. I. Types of ductular reaction reconsidered. Virchows Arch 2011;458:251-259. Desmet VJ. Ductal plates in hepatic ductular reactions. Hypothesis and implications. III. Implications for liver pathology. Virchows Arch 2011;458:271-279. Dranoff JA, Wells RG. Portal fibroblasts: underappreciated mediators of biliary fibrosis. Hepatol 2010;51:1438-1444. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011;6:425-456. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60-66. Overturf K, et al. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151:1273-1280. Hepatic Dysfunction Hepatic failure is a syndrome that results from inadequate hepatic function. The liver provides numerous essential processes, and liver failure can affect metabolic, synthetic, catabolic (detoxification), and immune functions. There is no specific point at which diminished function can clearly be defined as “failure,” given the various functions of the liver, but it is best understood as the point at which liver function is not capable of sustaining life. The liver is an organ with very large functional capacity and regenerative potential. Signs of insufficiency do not develop until the reserves in production, supply, and recovery are exhausted. The central role of the liver in many systemic functions means that liver disease can be manifested as lesions or dysfunctions elsewhere, for example, in the brain, skin, blood, and gut. Failure may develop suddenly, frequently following exposure to toxic compounds, or failure may develop in a slow inexorable fashion as a chronic disease processes. Hepatic failure, particularly chronic liver failure, may not affect all processes equally, and one or more features of liver failure may predominate in individual cases. Acute liver failure is uncommon, and is clinically evidenced as a severe and rapid liver injury. There are a number of causes. The consequences are also multiple and varied, including abrupt loss of normal metabolic and immunologic functions, as well as hepatic encephalopathy, coagulopathy, jaundice, photosensitization (in herbivores), and possibly dysfunction of other organs. In humans, there are recognized viral infections that can cause acute liver failure, but there are currently no known animal viruses capable of causing this syndrome, although recent studies suggest that equine serum hepatitis (Theiler's disease) may be caused by an equine pegivirus. Toxin-induced acute liver failure is likely the most common form in veterinary medicine. The toxic injury may come from natural sources, such as those found in plants and fungi, or from overdose of or idiosyncratic reactions to therapeutic drugs. Hyperthermia from environmental heating or prolonged seizure activity can also cause acute hepatic failure. Acute ischemic injury from profound hypotension secondary to sepsis or cardiac failure may also occur. Chronic liver failure results from progressive destructive processes, such as fibrosis or inflammation, or a combination of the two. The hallmarks of chronic liver failure in dogs are fibrosis and nodular regeneration, but other species are more prone to a fibrotic response alone. The progressive loss of critical functional hepatic mass caused by ongoing hepatocyte injury, shunting of portal blood within fibrous septa bypassing hepatocytes, and abnormal bile drainage, lead to a terminal stage of hepatic insufficiency. Chronic liver failure can be subdivided into 2 forms: end-stage liver, the typical fibrotic liver with progressive loss of functional hepatic mass, and “acute on chronic failure,” a circumstance in which a patient with compensated chronic liver disease acutely develops failure. The causes for the acute transition to failure are not certain, but the buildup of various toxins, such as aromatic amino acids, benzodiazepines, and ammonia, among others, may be involved. Other theories include increased bacterial or fungal translocation from the gut, leading to hepatic infection and sepsis. Liver failure, whether acute or chronic, affects a number of critical processes. These disturbances include hepatic encephalopathy, cholestasis and jaundice, hepatogenous photosensitization, hemorrhagic diathesis, hepatorenal syndrome, ascites, and hepatocutaneous syndrome. Hepatic encephalopathy (HE) The neurologic manifestations of hepatic failure are variable and nonspecific; they range from dullness, through complete unawareness and compulsive aimless movement, to mania and generalized convulsions. There is considerable variation in the clinical signs of hepatic encephalopathy between different species. Sheep rarely show more than dullness and central blindness, with perhaps some compulsive chewing movements and tremor. The picture in cattle is similar, but mania and aggression may also be seen, whereas frenzy is more often recorded in horses. The closer clinical observation of cats and dogs may reveal more subtle behavioral changes, and inappetence and vomiting are commonly reported in carnivores with portosystemic shunts. Ptyalism is common in cats with HE. The clinical context in which HE occurs can be important. In animals with acute hepatic disease, HE is a serious sign, usually indicating imminent death. However, in animals with more chronic conditions, such as portosystemic shunting or deficiency of a urea cycle enzyme, neurologic signs can be intermittent for many months, and may disappear after appropriate dietary modification. The pathogenesis of HE involves more than just neurons, as astrocyte and glial cell responses are now evident. Older literature, particularly in vitro studies, which did not take all of these features into account, will likely need reinterpretation. Ammonia toxicity is regarded as the major part of the clinical signs and the brain lesions; however, there are a number of other factors that may play a role. Blood ammonia is largely of dietary origin, derived from protein and urea by microflora in the large bowel. Ammonia is also derived from hepatic deamination of amino acids and from metabolism of glutamine in peripheral tissues. Ammonia is normally removed in the first pass of portal blood through the liver, wherein it is incorporated with carbon dioxide into carbamoyl phosphate that enters the urea cycle. In shunting or hepatic failure, ammonia that bypasses hepatic clearance accumulates in the circulation. Ammonia is able to cross the blood-brain barrier, and initially, this influx leads to astrocyte injury. Astrocytes are the site of ammonia detoxification in the brain, and they eliminate ammonia by the synthesis of glutamine through amidation of glutamate by the enzyme glutamine synthetase. Elevated blood ammonia increases the accumulation of glutamine in astrocytes, resulting in osmotic stress and astrocyte swelling caused by cytotoxic edema. Affected astrocytes that are adjacent to endothelium cannot maintain the blood-brain barrier, and this permits increased ammonia entry into the brain. Direct ammonia-induced effects on brain endothelial cells are also possible. Briefly, the consequences of increased ammonia in the brain include a net decrease in energy metabolism within the brain, increased edema formation because of astrocyte dysfunction, and injury to neurons, leading to an imbalance in neurotransmitters that emphasizes neural inhibition. The pathogenesis of HE may vary depending on the type of liver failure—acute or chronic—that produces the cerebral injury. Also, not all parts of the brain are affected in a similar fashion. In acute hepatic failure, cerebral edema may be the leading cause of death because of increased intracerebral pressure with possible herniation. The increase in intracranial pressure is, however, linked to brain ammonia levels. Currently, it is thought that swollen astrocytes release vasogenic factors, such as nitric oxide, causing intracerebral hyperemia, and that this leads to edema formation. Systemic inflammatory responses can enhance the severity of HE. Neuroinflammation, mediated by microglia, is evident in acute liver failure and tends to increase with increasing duration of HE. Synergetic interactions between increased ammonia and systemic or neuroinflammation can increase the severity of HE. However, the specific mechanisms by which systemic inflammation triggers neuroinflammation are not currently known. In chronic hepatic failure, ammonia and neuroinflammation are likely to contribute synergistically to HE. Ammonia plays a role as a directly neurotoxic agent, altering neurotransmission, and potentially contributing to cerebral energy failure through inhibition of α-ketoglutarate dehydrogenase, a rate-limiting enzyme in the tricarboxylic acid cycle. Chronic increase in brain ammonia is associated with disrupted neural transmission involving all of the different neurotransmitter systems of the brain. The main neurotransmitter systems affected involve neuropeptides, with an evolution toward an increase in the inhibitory γ-aminobutyric acid (GABA)ergic system, and downregulation of the excitatory glutaminergic system. Increased intracerebral ammonia leads to an increase in extracellular glutamate, which will initially drive upregulation of N-methyl-D-aspartate (NMDA) receptors, causing neuronal damage, but eventually, there is a reduction in NMDA receptors and glutaminergic signaling, leading to neuroinhibition. In addition, increased ammonia alone has been linked to neuroinflammation, possibly through direct activation of microglia, which also interferes with normal neural transmission. Newer studies in animal models have demonstrated that agents acting on specific targets in the brain, including phosphodiesterase 5, type A GABA receptors, and mitogen-activated kinase (p38), can improve cognitive function in mild forms of experimental hepatic encephalopathy. Survival can be increased by treatment with NMDA receptor agonists. Studies have also implicated alterations in inhibitory and excitatory neurotransmitters, including endogenous benzodiazepines, and serotonin, as well as their receptors. Although infusion of ammonia or hyperammonemia caused by deficiency of urea cycle enzymes reproduces similar neurologic signs and vacuolar lesions, other noxious substances in the alimentary tract are also believed to contribute to hepatic encephalopathy resulting from liver failure. These include variably toxic amines, captans, thiols, and short-chain fatty acids, which are normally removed from the portal blood in one passage through the liver after production in the large bowel. Thus there are many factors to consider in the pathogenesis of HE. The microscopic lesions of HE are subtle and variable, likely because postmortem examinations of the brain are performed at various time points of chronicity and clinical severity. There are also significant differences in the microscopic lesions between species. In humans, the hallmark of HE is Alzheimer type II astrocytosis, in which astrocytes are enlarged with swollen nuclei, margination of chromatin, and prominent nucleoli. These can be found in large animals, particularly horses. In dogs, Alzheimer type II changes are rare if they occur at all, but vacuoles in gray matter, particularly in brain stem nuclei, predominate. Lesions in cats have features of both the dog and the horse (see Vol. 1, Nervous system). These changes can be observed best in animals with chronic liver disease and portosystemic shunts; the brain lesions can be minimal in animals with acute liver failure. Cholestasis and jaundice Cholestasis is the term for impaired bile secretion and flow as well as a failure to secrete organic and inorganic components of bile, with accumulation of these elements in blood. Normal bile formation and flow is dependent on the activity of a series of membrane transporters found in enterocytes, hepatocytes, and biliary epithelium. In rare cases, hereditary mutations can lead to cholestasis, but more often, cholestasis is caused by exposure to injurious drugs, hormones, proinflammatory cytokines, or obstruction. Clinically, cholestasis leads to increased levels of bilirubin and bile acids in the blood because of retention. Injury to the biliary tree leads to increases in serum alkaline phosphatase. Disturbance of bile flow can originate from altered function of hepatocytes, termed hepatocellular cholestasis, or because of obstruction of the biliary tree, termed obstructive cholestasis. Hepatocellular cholestasis can be attributed to impaired uptake, metabolism, secretion, or transport of bile constituents. Obstructive cholestasis is related to obstruction of bile flow at the level of the major bile ducts or gallbladder. Jaundice (icterus) occurs when the tissues, particularly the sclerae, are pigmented yellow because of an excess of bile pigments, primarily bilirubin, in the plasma. Jaundice can arise from hepatocellular cholestasis or obstructive cholestasis and, in addition, from prehepatic causes, such as an overproduction of bilirubin from heme catabolism in hemolytic diseases (Fig. 2-47 ). The associated hypoxia caused by anemia may facilitate cholestasis in hemolytic diseases. Figure 2-47 Deeply bile-stained liver from a jaundiced dog with immune hemolytic anemia and cholecystitis. (Courtesy A.P. Loretti.) Histologically, both forms of cholestasis share common features, but there are additional lesions associated with duct obstruction in the portal tracts and bile ducts. The main histologic manifestation of cholestasis is accumulation of homogeneous waxy brown bile pigment in bile canaliculi. Bile regurgitated from hepatocytes can also be found in Kupffer cells following phagocytosis. Bilirubin can be found in the hepatocellular cytoplasm in most species, especially if frozen sections are examined, but it is quite rare for bile to be evident in canine hepatocytes. Bile accumulation is more severe in the centrilobular regions of the lobules, but can extend to the periportal regions in severe cases. With time, canalicular plugs are cleared, and they may not be evident in more chronic cases of cholestasis. Hepatocyte rosettes, collections of 2 or more hepatocytes surrounding a dilated canaliculus are common. Hepatocellular degeneration may also be evident, sometimes with bile staining of the cytoplasm of injured hepatocytes. Scattered apoptotic cells may be present, although significant necrosis is not usually a feature of cholestasis. In more severe cases of obstructive cholestasis, confluent areas of bile-stained hepatocellular necrosis, termed bile infarcts, can develop. Bile infarcts are rare in dogs and cats, but can be found in other species. Cholate stasis refers to hepatocellular degeneration in the periportal region, often accompanied by a proliferation of small-caliber bile ducts (ductular reaction). Affected hepatocytes have swollen pale cytoplasm, and they may contain copper granules. This lesion is most common in horses with obstruction and rare in dogs. In hepatocellular cholestasis, there are no distinctive lesions in the portal tracts. However, in obstructive cholestasis, there are a number of distinctive changes. Initially, there is edema in the portal tract and an inflammatory infiltrate that is most often predominantly neutrophilic. There is an accompanying proliferation of small-caliber bile ducts arranged in an apparently haphazard fashion (ductular reaction). There may be degeneration or proliferative changes in pre-existing interlobular bile ducts, often associated with inflammation, depending on the cause of the obstruction. Ductal ectasia may also be present. With time, there is an increase of fibrosis that expands the portal tract outline and can bridge between portal tracts in cases of severe obstruction. In addition, there is concentric fibrosis surrounding the bile ducts. Proliferation of bile ducts, termed ductular reaction, is a constant feature. Mixed inflammatory cells, usually with fewer neutrophils than that seen in acute cholestasis, as well as macrophages containing pigment found in the portal tract. Hepatocellular cholestasis arises when there is a disturbance in 1 or more of the 3 main steps involved in bile excretion. They include failure (1) to take up bilirubin, bile acids, or other bile constituents; (2) to conjugate bilirubin, bile acids, or other constituents; and (3) to transport and excrete conjugated bilirubin, bile acids, or other bile constituents into canaliculi. Most of these bile metabolism issues are mediated by a series of hepatobiliary transporters, molecular pumps involved in the transport of bilirubin, bile acids, and other organic ions in hepatocytes, canaliculi, and biliary epithelium, although some pumps serve to export substances as well. Hepatocellular uptake of unconjugated and conjugated bilirubin, like other organic ions, is mediated by members of the organic anion-transporting polypeptide (OATP) family found on the basolateral aspects of hepatocytes. Bilirubin is conjugated by uridine diphosphate–glucuronyl transferase in the endoplasmic reticulum of hepatocytes. From there it is transported into the bile. Bile acids are imported from the plasma by the Na+-taurocholate cotransporting polypeptide (NTCP). Transport of bile acids, the main osmotic driving force for bile formation (bile acid–dependent flow), into the canaliculus is performed by the bile salt export pump (BSEP). Aquaporin channels facilitate the movement of water into the canaliculus in response to the osmotic forces produced by the bile acids. Movement of bilirubin into the canaliculus for excretion is accomplished by the multidrug-resistance–associated protein-2 (MRP-2). Reduced glutathione is also a substrate for MRP-2, and it is reduced glutathione and bicarbonate that are the main elements of the bile acid–independent portion of bile flow, the second most significant force in bile flow. Bile is continually modified by transporter-driven processes of secretion and absorption during its transit to the duodenum. In hepatocellular cholestasis, drugs or endotoxin-induced proinflammatory cytokines interfere with the activities of molecular pumps or inhibit their synthesis. Endotoxin exposure both reduces activity of OATP and NCTP, but also interferes with the activity of BSEP and MRP-2. Hepatocellular cholestasis can also occur through disruption of the structural integrity of the canaliculi, as discussed later for Lantana camara toxicity. Poisons such as phalloidin and cytochalasin, which disrupt the polymerization cycle of pericanalicular actin microfilaments, are cholestatic because these contractile filaments are required for propelling bile along the canaliculi. In horses, fasting can cause plasma bilirubin increases and jaundice. The mechanism may be the impaired uptake of bilirubin from the plasma or possibly impaired conjugation of bilirubin when energy supplies are low. Rarely, cholestasis and its indicators can occur in the absence of significant hepatocellular damage and other signs of liver failure. This can occur in toxicity by L. camara in ruminants and as an idiosyncratic reaction to some drugs. In obstructive cholestasis, the activities of NTCP and OATP transporters are both reduced, causing bile acids and bilirubin to accumulate in the blood. Transport of bile acids out of the hepatocyte and into the canaliculus is maintained by BSEP. The regulation of transcription of the various transporters in hepatocellular or obstructive cholestasis is controlled by the interaction of bilirubin or bile acids with several nuclear receptors, primarily PXR (pregnane X receptor) and, to a lesser extent, others, such as CAR (constitutive androstane receptor) and FXR (farnesoid X receptor). During cholestasis, nuclear receptors orchestrate protection of hepatocytes from injury by reducing the expression of basolateral uptake transporters and increasing the expression of basolateral export pumps, as well as reducing new bile acid synthesis and increasing the rate of intrahepatic metabolism of bile acids. However, these adaptive responses are not always sufficient to prevent cholate-induced hepatotoxicity. Severe diffuse liver necrosis obviously impairs bile excretion at various levels, but the severity of cholestasis or of jaundice depends on the amount of liver affected, the chronicity, the supply of heme for catabolism, and nonhepatic routes of bilirubin excretion. Focal liver injury can have substantial local cholestasis, but clinically detectable cholestasis does not occur because bilirubin can be cleared by unaffected parts of the liver. Similarly, segmental duct obstructions that spare some parts of the liver can lead to cholestasis but not jaundice. This can occur in anorexic or dehydrated cats, in which bile may become dehydrated and viscous, causing ductal obstruction. Some of the various transporters and conjugating enzymes involved in bilirubin excretion can be genetically defective. Congenital hyperbilirubinemia in mutant Southdown sheep is the result of impaired hepatic uptake of unconjugated bilirubin. These animals have few liver lesions, but eventually develop chronic renal disease, the reason for which is not clear. Unconjugated bilirubin levels in the plasma are consistently elevated, but sufficient excretion takes place to prevent them from becoming icteric. They become photosensitized, indicating that excretion of phylloerythrin (phytoporphyrin) is less efficient than that of bilirubin. Hyperbilirubinemia in mutant Corriedale sheep is a defect in excretion of conjugated bilirubin similar to Dubin-Johnson syndrome in humans and mutant rat strains. In humans and rats, there is a mutation in Mrp-2 causing hypofunction and a similar mutation is likely in affected sheep. Affected sheep have an elevation of plasma bilirubin (just over half of which is conjugated), but there is no obvious jaundice. Nevertheless, phylloerythrin (phytoporphyrin) excretion in these Corriedale sheep is also sufficiently impaired to produce photosensitization. There is impaired excretion of other conjugated metabolites, and there is dark pigmentation of the liver by polymerized residues of retained catecholamine metabolites. This pigment, resembling lipofuscin, accumulates in lysosomes in the pericanalicular cytoplasm. The recognition of jaundice at postmortem sometimes involves differentiation of bile staining of tissues from the yellow staining caused by accumulation of carotenoid pigments. These latter are limited to fat depots and are to be expected in certain species such as horses; sometimes there are breed influences, as seen in the yellow fat of Channel Island breeds of dairy cattle. The yellow discoloration of fat depots of older cats is less well understood. In animals fed ox liver, carotenoids may again be responsible, and in others, there may be some contribution by ceroid-type pigments. Distinction of the fatty pigments from bile depends on the absence of the former from pale, nonfatty tissues, such as periosteum and dermal collagen. Photosensitization Photosensitization is the term applied to inflammation of skin (usually unpigmented) because of the action of ultraviolet light of wavelengths 290-400 nm on photodynamic compounds that have become bound to dermal cells. In primary photosensitization, these compounds may have been deposited unchanged in the skin after ingestion, before the normal liver is capable of excreting the native compound. This is seen, for example, after ingestion of hypericin in St. John's wort (Hypericum perforatum). Photodynamic agents may also be produced by aberrant endogenous metabolism. This can occur, for example, in congenital erythropoietic protoporphyria caused by ferrochelatase deficiency in Limousin and Blonde d'Aquitaine calves. Hepatogenous photosensitization almost always accompanies cholestasis of more than a few days' duration in herbivores that are kept in sunlight and that have been eating green feed. Phytoporphyrins (formerly termed phylloerythrins) are green photoactive catabolites of plant porphyrins (mainly chlorophyll) that are generated by the alimentary microflora of herbivores. Some phytoporphyrin is absorbed and normally excreted in the bile by the transporters that eliminate bilirubin. Cholestasis in herbivores can increase retention of phytoporphyrin in the blood, and it can result in photosensitive dermatitis of unpigmented areas of skin exposed to sunlight for several days (Fig. 2-48 ). It is possible, however, for mild photosensitization to appear in the absence of gross or microscopic evidence of cholestasis in animals grazing alfalfa, Paspalum, pangola, or Panicum grasses; however, it is unclear if these are related to phytoporphyrin or other photoactive products of these forages. Absence of serum biochemical evidence for cholestasis or liver damage is used to differentiate primary from secondary photosensitization in these circumstances. Figure 2-48 Hepatogenous photosensitization caused by Panicum miliaceum (French millet) in a sheep. (Courtesy P. Hoskin.) If no hepatic changes can be discerned in photosensitized animals, the possibility of primary photosensitization must be considered, but hepatogenous photosensitization cannot be excluded unless adequate liver function tests are performed. Hemorrhage and liver failure Hemorrhagic diathesis characterized by widespread ecchymoses and petechiae can occur when the liver is injured and becomes the site of significant clotting factor consumption. However, recent investigations suggest that clinically significant hemorrhage is not common in acute liver failure. The liver is the source of plasma proteins involved in the clotting cascade, but these are normally supplied in substantial excess, and can be induced as part of the acute-phase response to inflammation. Thus coagulopathy with hemorrhage is most likely to occur when there is acute liver necrosis, for example, in acute canine infectious hepatitis or xylitol toxicity. Under these conditions, there is significant intrahepatic consumption of clotting factors at sites of endothelial necrosis in the damaged liver. Although the damaged liver also fails to resupply the clotting factors, consumption is probably a minor contributing influence. In more chronic liver diseases with hypoproteinemia, coagulation tests may be prolonged, but hemorrhagic diathesis is unlikely, unless there is an additional demand for hemostasis, for example, during the trauma of surgery. Although production of clotting factors is often reduced in liver failure, there is a commensurate reduction in anticoagulant synthesis as well. Portal hypertension and endothelial cell dysfunction may also be a contributing cause for bleeding in chronic liver disease. Overall, the pathogenesis of hemorrhagic tendencies in chronic liver disease is complex, multifactorial, and incompletely understood. Consumption of clotting factors can also occur in septic diseases that affect the liver and other tissues. Nephropathy Acute liver failure may be accompanied by oliguria and biochemical indications of renal failure. Hepatorenal syndrome is a complication of advanced cirrhosis in humans, characterized by renal failure in which there are no intrinsic renal morphologic or functional causes. The pathogenesis of hepatorenal syndrome is related to deterioration in effective arterial blood volume because of splanchnic arterial vasodilation and reduced venous return and cardiac output. Intense compensatory vasoconstriction of the renal circulation results in decreased glomerular filtration and resultant renal failure. In addition, some hepatic toxins such as acetaminophen may produce injury in the nephrons as well. Edema and ascites Ascites (retention of excess low-protein peritoneal fluid) is a feature of chronic liver failure, but is more frequently associated with systemic venous congestion (e.g., right-sided heart failure) or hypoproteinemia secondary to protein-losing renal or alimentary tract conditions. It is likely that ascites in advanced liver disease is influenced by both mechanical and dynamic influences on blood flow through a damaged liver. Reduced synthesis of albumin and globulins by the failing liver can reduce vascular oncotic pressure, but edema resulting from this mechanism is generalized. Experimentally, restriction of blood flow from the liver leads to a swift increase in sinusoidal plasma within the space of Disse, leading to increased lymph flow into the thoracic duct and through the hepatic capsule. This explains rapid ascites formation in cases of hepatic outflow obstruction or veno-occlusive diseases. There are several theories that explain the complicated process of ascites formation in chronic liver disease. The theory that best encompasses the known circulatory changes is known as the peripheral arteriolar vasodilation hypothesis. In end-stage livers, there is progressive sinusoidal and portal vein hypertension with collateral vein formation and acquisition of shunting vessels to the systemic vasculature. Sinusoidal hypertension appears to be an important feature as ascites rarely develops with prehepatic portal hypertension. The main factor leading to ascites formation is splanchnic vasodilation. In chronic liver failure, there is increasing portal vein hypertension and a local release of vasodilators, such as nitric oxide, leading to splanchnic arterial dilation. With progression, the extent of the arterial vasodilation increases to the point that effective arterial volume and pressure drops, leading to activation of vasoconstrictor and antinatriuretic factors, including activation of the renin-angiotensin-aldosterone system and the sympathetic nervous system, promoting retention of sodium and fluid. Intestinal capillary pressure and permeability are increased as a result of the increased portal vein hypertension, promoting excess fluid transit to the abdominal cavity. Over time, sodium retention by the kidneys is insufficient to compensate for the progressive arteriolar vasodilation as well as the movement of sodium and fluid to the extracellular space of the abdomen. This movement is enhanced by hypoproteinemia caused by reduced synthesis by the liver and dilution secondary to fluid retention. Consequently, there is persistent activation of antinatriuretic systems, and fluid continues to accumulate. There is evidence that increased venous return caused by reduced peripheral arterial vasodilation stimulates endogenous natriuretic substances, but this response is insufficient to counteract the more significant release of antinatriuretic signals. Retention of peritoneal fluid can also sometimes result from mechanical obstructions to mesenteric and peritoneal lymphatics by inflammatory or neoplastic lesions. Peritoneal fluids with a higher protein and cell content that accumulate in various abdominal inflammatory conditions are considered exudates (see section on Peritonitis in Vol. 2, Alimentary system and peritoneum). Hepatocutaneous syndrome An idiopathic vacuolar hepatopathy with parenchymal collapse and nodular regeneration has been reported in dogs and cats that have been presented clinically with hepatocutaneous syndrome (necrolytic migratory erythema, superficial necrolytic dermatitis). The skin disease resembles necrolytic migratory erythema in humans, a well-defined paraneoplastic syndrome typically associated with hyperglucagonemia secondary to glucagon-secreting pancreatic neoplasia, but also reported in individuals with hepatitis, cirrhosis, celiac disease, chronic malabsorption, and inflammatory bowel disease. In dogs, hepatocutaneous syndrome is most commonly associated with liver disease, including severe vacuolar hepatopathy, idiopathic hepatocellular collapse, and hepatopathy secondary to anticonvulsant drug administration, and more rarely with glucagonoma and gastric carcinoma. Clinical presentation is typically because of the dermatitis; skin lesions include erythema, crusting, exudation, ulceration, and alopecia, affecting footpads, periocular, perioral, anogenital regions, and pressure points. The dermatologic lesions are described more fully in Vol. 1, Integumentary system. Affected dogs have depressed plasma amino acid concentrations, inconsistent elevations of plasma glucagon levels, and may also become diabetic. The liver of affected dogs is usually grossly nodular, resembling cirrhosis (eFig. 2-10A); however, histologically, there is typically moderate to severe vacuolation of hepatocytes, with parenchymal collapse accompanied by nodular regeneration (eFig. 2-10B). Inflammation, necrosis, and fibrosis are not usually prominent. Although some studies report fibrosis typical of cirrhosis, more characteristically, there is a network of reticulin and fine collagen fibers representing the remnants of collapsed hepatic lobules, with proliferation of bile ductules. The vacuolated hepatocytes stain with oil red O for lipids. The hepatic lesions have been suggested to support an underlying metabolic, hormonal, or toxic etiology. In humans, persistent hyperglucagonemia stimulates prolonged gluconeogenesis, resulting in secondary hypoaminoacidemia. Although hepatic insufficiency can increase glucagon levels in animal models, in dogs, the link between liver disease and hypoaminoacidemia remains unclear. A hypermetabolic state with exaggerated amino acid catabolism has, however, been suggested. Other biologically active molecules that are normally cleared by the normal liver or generated by a damaged liver should also be considered. eFigure 2-10 A. Nodular liver from a dog with hepatocutaneous syndrome. (Courtesy J.L. Caswell.) B. Hepatic parenchymal collapse with nodular regeneration, hepatocutaneous syndrome. Further reading Asakawa MG, et al. Necrolytic migratory erythema associated with a glucagon-producing primary hepatic neuroendocrine carcinoma in a cat. Vet Dermatol 2013;24:466-469. Bernal W, et al. Acute liver failure. Lancet 2010;376:190-201. Gines P, et al. Management of cirrhosis and ascites. N Engl J Med 2004;350:1646-1654. Gross TL, et al. Superficial necrolytic dermatitis (necrolytic migratory erythema) in dogs. Vet Pathol 1993;30:75-81. Kimmel SE, et al. Clinicopathological, ultrasonographic, and histopathological findings of superficial necrolytic dermatitis with hepatopathy in a cat. J Am Anim Hosp Assoc 2003;39:23-27. March PA, et al. Superficial necrolytic dermatitis in 11 dogs with a history of phenobarbital administration (1995-2002). J Vet Intern Med 2004;18:65-74. Mia AS, et al. Unconjugated bilirubin transport in normal and mutant corriedale sheep with Dubin-Johnson syndrome. Proc Soc Exp Biol Med 1970;135:33-37. Mullen KD, Prakash RK. Hepatic Encephalopathy. New York: Humana Press; 2012. Nyland TG, et al. Hepatic ultrasonographic and pathological findings in dogs with canine superficial necrolytic dermatitis. Vet Radiol Ultrasound 1996;37:200-205. Quinn JC, et al. Secondary plant products causing photosensitization in grazing herbivores: their structure, activity and regulation. Int J Mol Sci 2014;15:1441-1465. Schrier RW, et al. Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis. Hepatol 1988;8:1151-1157. Stravitz RT, et al. Minimal effects of acute liver injury/acute liver failure on hemostasis as assessed by thromboelastography. J Hepatol 2012;56:129-136. Tripodi A, Mannucci PM. The coagulopathy of chronic liver disease. N Engl J Med 2011;365:147-156. Wagner M, et al. New molecular insights into the mechanisms of cholestasis. J Hepatol 2009;51:565-580. Wagner M, et al. Nuclear receptors in liver disease. Hepatol 2011;53:1023-1034. Witte ST, Curry SL. Hepatogenous photosensitization in cattle fed a grass hay. J Vet Diagn Invest 1993;5:133-136. Postmortem and Agonal Changes in Liver The liver, rich in nutrients for bacteria and freely exposed to agonal invaders from the intestine, undergoes postmortem decomposition very rapidly. Gas bubbles generated by anaerobic saprophytes form first in the hepatic blood vessels, but soon suffuse large portions of the organ. The vessels and adjacent parenchyma are stained by hemoglobin. The substance of the organ becomes soft and clay-like, and the formation of putrefactive gases may make it foamy. On the capsular surface, irregular, pale foci are visible; they superficially may resemble infarcts or fatty areas but can be observed to increase in size during the postmortem interval, and microscopically are without cellular reaction. Bacilli are present in large numbers in such foci. Green-black pigmentation of the capsule and superficial parenchyma occurs where the liver is in contact with gut, and the lobes surrounding the gallbladder become stained by bile. Microscopic structural changes occur in the liver, approaching and immediately following death. Shrinkage of liver cells, possibly because of a period of anaerobic catabolism of glycogen, and widening of centrilobular (periacinar) sinusoids because of hepatic congestion, are seen after death. Dissociation of liver cells may be complete, with every cell in every cord separated and free from adjacent cells, so architectural patterns are lost. The early expression of this change affects centrilobular cells, which become detached, rounded in contour, condensed, and hyperchromatic. The dissociation is particularly evident in feline panleukopenia and leptospirosis, related in part to antemortem changes. Vascular Factors in Hepatic Injury and Circulatory Disorders Hepatic artery The hepatic artery delivers approximately of the afferent hepatic blood supply, but ~40-50% of the oxygen. Hepatic artery flow responds to alterations in portal vein flow to sustain overall perfusion of the liver at a nearly consistent level via what is termed the hepatic arterial buffer response. Hepatic arterial flow is believed to be regulated via adenosine levels in the space of Mall. Adenosine is produced at a constant level, but with decreased portal flow less adenosine is washed out, raising local concentrations and causing the hepatic artery to dilate and increase arterial flow. Other mediators of arterial flow are recognized, and they include hydrogen sulfide, nitric oxide, and the autonomic nervous system. Complete loss of arterial flow can be, after a brief period of injury, compensated for via the portal flow because the liver normally only extracts ~40% of the oxygen supplied by the portal vein. Within the liver, branches of the hepatic artery form several patterns, including (1) a peribiliary plexus, (2) a vasa vasorum for the portal vein, or (3) an array of terminal hepatic arterioles that drain directly into the sinusoids. In addition, the hepatic artery provides branches to supply the liver capsule. The peribiliary plexus surrounds intrahepatic bile ducts and likely plays a role in an exchange of bile constituents and vasoactive factors. Hepatic arterial occlusions occur rather commonly in animals but usually involve small intrahepatic branches and are of little consequence. Large segments of the liver may be necrotic in cats as a result of thrombosis of the aorta and hepatic artery. Verminous arteritis may occlude the hepatic artery in horses. The extent of necrosis depends on how completely the obstruction excludes collateral circulation and also on the oxygen tension of the portal blood. Ischemic areas of liver can be sequestered, but in some instances, bacteria such as clostridia and other anaerobes can flourish and release potent toxins with systemic effects. Portal vein The portal vein drains the large and small intestine, stomach, pancreas, gallbladder, and spleen and normally contributes of hepatic blood flow. Because the portal vein collects blood from the abdominal viscera, the rate of flow is variable, depending on physiologic factors such as eating, which increases, or stress, which decreases, portal flow. Portal venous blood has considerably more oxygen than most systemic venous blood and contributes ~60% of the liver's oxygen needs. The portal vein branches successively until the finest branches, the terminal portal venules, drain into the sinusoids via side branches, the inlet venules. Entry of blood into the sinusoids is regulated via sphincters in the terminal inlet venules. Portal blood flow is likely streamlined, rather than turbulent, but there is little agreement regarding existence of specific patterns of flow. Streamlining may account for the different regional distributions sometimes observed with metastatic tumors and infections. The umbilical vein usually drains to the left lobe, so hematogenous umbilical infections tend to localize in the left lobe. The liver cannot regulate portal venous flow, so hepatic blood flow is largely balanced by arterial supply that varies with the portal venous supply to maintain a relatively consistent hepatic perfusion. Hepatic microcirculation is well regulated at the level of inflow by sphincters in the finest branches of the portal venules, the inlet venules, as well as the terminal branches of the hepatic arterioles and at the level of outflow by sphincters that regulate passage of blood from the sinusoids to the terminal hepatic venules. In dogs, the spiral smooth muscle that invests the wall of sublobular hepatic veins can contract and alter hepatic venous outflow in various conditions, particularly in shock, where contraction leads to acute congestion and pooling of blood in the liver and the organs that drain into the portal vein. Hepatic blood thus flows evenly through the sinusoids under a very-low-pressure gradient. The liver receives ~25% of the cardiac output, even though it represents only ~2.5% of body mass. Approximately 25% of the weight of the liver in situ is blood. Obstruction of the portal vein, if sudden and complete, can produce a condition akin to strangulation of the gut, and death occurs quickly without significant hepatic change; however, surgical diversion of the portal vein into the vena cava produces only transient injury to the liver. Obstruction of a large branch of the portal vein in cattle, sheep, and cats leads to acute ischemia of a wedge of tissue in which necrosis may be zonal or massive. Obstruction of many small portal radicles is common, with necrosis of many lobules. It is evident that if a collateral supply develops and oxygenation remains adequate, obstruction of portal radicles will have no immediate effect on the hepatic parenchyma, save perhaps to make it more sensitive to toxic injury. The parenchyma in the affected lobe, deprived of hepatotrophic factors, loses much of its regenerative power and atrophies fairly rapidly, allowing condensation and scarification of the stromal tissues. Loss of portal venous inflow and obstruction of hepatic venous outflow, a double-hit insult, can lead to complete infarction of the liver as seen in hepatic lobe torsion. Arterial supply can be obstructed as well in this situation. Acute increase in pressure in the portal vein may occur in any severe episode of widespread acute hepatic necrosis; the cause appears to be simple obstruction of the sinusoidal flow by thrombosis and actual sinusoidal disruption. In such animals, there is severe acute congestion of the liver, slight ascites, free fibrin accumulations in the abdomen (not the firm capsular adhesions seen in passive congestion), and distended portal lymphatics. Obstruction of the extrahepatic portal vein is quite uncommon. External compression may occur because of adjacent abscesses or neoplasms. Portal vein thrombosis may be caused by damage to the portal vein by local inflammatory processes or be associated with states of hypercoagulability or retrograde intravascular growth of hepatic neoplasms. In dogs, thrombosis of the portal vein has been associated with distant neoplasia, immune-mediated hemolytic anemia, protein-losing nephropathy and enteropathy, pancreatitis, peritonitis, and corticosteroid administration (Fig. 2-49 ). Portal vein obstructions of slow development are expected to lead to portal hypertension and its consequences. Atresia or hypoplasia of the extrahepatic portal vein can be demonstrated in some cases of primary portal vein hypoplasia and is discussed previously in the section on Developmental disorders. Figure 2-49 Portal vein thrombosis in a dog. Obstruction of intrahepatic portal vessels is a consequence of progressive fibrosing lesions of primary hepatic disease centered on the portal triads. The small portal vessels may be obliterated in the proliferative portal lesions, and new connections may be established, including small functional arteriovenous communications. Regenerative hepatic nodules and neoplasms may deform and compress portal vessels in some locations. Portal hypertension is defined as increased blood pressure within the portal vein caused by resistance to normal flow rates. Portal hypertension can arise from disturbances of venous blood flow in any of the following 3 sites: prehepatic, intrahepatic, and posthepatic. Prehepatic portal hypertension is relatively uncommon and occurs when blood flow through the portal vein is impaired before it enters the liver. The most common cause is portal vein thrombosis. Intrahepatic portal hypertension arises from increased resistance to blood flow at the level of the hepatic parenchyma. Intrahepatic portal hypertension can be subdivided into 3 forms: presinusoidal (including intrahepatic portal vein hypoplasia, intrahepatic arterioportal fistulae, periportal neoplastic or inflammatory infiltrates, and periportal fibrosis), sinusoidal (cirrhosis or long-term inflammatory disease with fibrosis and capillarization of sinusoids, amyloidosis, neoplastic infiltrations), and postsinusoidal (perivenular fibrosis, veno-occlusive disease). Posthepatic causes of portal hypertension are uncommon, but can result from increased resistance to blood flow in the major hepatic veins, caudal vena cava, or right heart. Causes include partial or complete thrombosis of the hepatic veins, which is very rare; obstruction of the hepatic veins by neoplastic occlusion (e.g., pheochromocytoma); and kinking of the caudal vena cava, associated with abdominal trauma. Congestive heart failure can also lead to portal hypertension. Regardless of cause, persistent portal hypertension can lead to acquired portosystemic shunts (discussed later), with the exception of passive congestion, which rarely, if ever, results in the development of shunt vessels. These shunts are usually numerous and composed of distended thin-walled veins, which may connect the mesenteric veins and the caudal vena cava. Ascites is common in conditions that develop acquired shunts because of the associated portal hypertension. Efferent hepatic vessels Efferent flow begins in the sinusoids and passes through terminal hepatic venules and larger hepatic veins to the vena cava. In the sinusoidal network, flow is interconnected in many directions. However, in normal conditions, lobule outflow is closely matched to inflow. In dogs, hepatic veins have substantial spiral smooth-muscle sphincters that regulate outflow, but these are not evident in other domestic species. Splanchnic engorgement during anesthesia and anaphylaxis is an indication that these smooth muscles control canine hepatic outflow in a dynamic manner. Obstruction at the level of the large hepatic veins can occasionally occur because of suppurative phlebitis, hepatic abscesses, neoplasms, or other physical obstacles that impinge on the hepatic veins. Similar space-occupying lesions in or around the vena cava in the diaphragm or mediastinum can also affect hepatic outflow, along with systemic venous return. The amount and arrangement of conventional connective tissue around the terminal hepatic veins and larger hepatic veins influence the patterns of fibrosis observed in various patterns of centrilobular injury. Hypoxia from anemia, heart failure, or shock can cause centrilobular necrosis, as can many toxic agents that are preferentially injurious to zone 3 hepatocytes. Necrosis at this level elicits a tissue repair response involving the fibrous connective tissue along the terminal hepatic venules. Tissue fibrosis can increase the resistance of the hepatic parenchyma and slow local outflow. This can redirect sinusoidal blood to alternative less-affected venules, unless the necrosis is extensive. Hepatic veno-occlusive disease is the obliteration of small intrahepatic veins that may begin with damage to the sinusoidal endothelium, accumulation of red cells and fibrin in the subintimal space, and subsequent subendothelial fibrosis. This pattern of postnecrotic fibrosis can contribute to the development of portal hypertension. Veno-occlusive disease is a feature of pyrrolizidine toxicosis in humans ingesting these alkaloids in so-called bush tea. In domestic animals, occlusive changes in terminal venules are notable in poisoning by ragwort (Senecio jacobea) in cattle and also in dogs treated experimentally with the pyrrolizidine alkaloid monocrotaline. Veno-occlusive disease has also been reported as a consequence of prolonged chemotherapy, radiotherapy, and bone marrow transplantation in humans, and similarly in dogs treated with irradiation or busulfan. Idiopathic veno-occlusive disease with perivenular fibrosis around central and sublobular veins, causing Budd-Chiari–like syndrome (see later), has also been reported as a rare occurrence in the dog and cat. Total or subtotal obliteration of central hepatic or sublobular veins by subintimal accumulation of collagen and fibrous tissue has also been reported in captive snow leopards and cheetahs. Passive congestion of the liver develops when the blood pressure in the hepatic veins increases relative to that of the hepatic portal veins and can occur in any species. It is almost always the consequence of cardiac dysfunction, including acute and chronic heart failure, pericardial disease, and processes that obstruct the flow of blood into or out of the heart, such as abscesses, heartworm disease, and local neoplasia. Right-sided heart failure, in particular, produces elevated pressure within the caudal vena cava that later involves the hepatic vein and its tributaries. Acute passive congestion of the liver occurs when there is sudden cardiac decompensation, particularly on the right side of the heart, or shock. Grossly, there is slight enlargement of the liver, which is typically dark red, and blood flows freely from any cut surface (Fig. 2-50 ). The intrinsic lobular pattern of the liver may be slightly more pronounced, particularly on the cut surface, because centrilobular areas are congested (dark red) in contrast to the more normal color of the remainder of the lobule. The microscopic picture in acute passive congestion is initially characterized by distention of central veins and adjacent sinusoids, with accompanying distension of lymphatics in the stroma of hepatic veins, portal triads, and the capsule. The appearance of the liver differs with the duration and severity of the congestion. Fatty change, trabecular atrophy, and necrosis of centrilobular hepatocytes with retention of the perisinusoidal reticulum framework develop quickly. Erythrocytes tend to move into the perisinusoidal spaces left by the lost hepatocytes, and may be seen trapped there when blood drains from sinusoids and veins in freshly fixed sections. The intrahepatic network of lymphatics at this stage becomes very distended and may form extensive cavernous channels about hepatic veins and venules, in portal triads, and just beneath the capsule. With increased venous pressure, transudation of red blood cells and high-protein-content edema can develop, leading to polymerization of released fibrinogen on the capsular surface that yields a fibrin coating of the capsule and blood-tinged abdominal fluid. Distension of the space of Disse may be seen, but rarely, if ever, in tissue well fixed soon after death, and is best regarded as a postmortem artifact. By comparison, perisinusoidal edema is more obvious in hepatic congestion associated with shock or inflammatory conditions. Figure 2-50 Acute passive congestion of the liver in a cria with a congenital heart defect. In chronic passive congestion, the amounts of fibrous connective tissue in the liver increase in various patterns that differ among species. The capsular surface becomes thicker and more opaque, and can develop a finely nodular texture with the formation of capsular plaques (eFig. 2-11A). In dogs and cats, the edges of the central lobes become rounded, whereas the margins of lateral and caudate lobes are sharpened by peripheral atrophy and fibrosis. If the cause of the congestion is still present, there is usually copious ascites at this stage. In species in which fibrosis is more pronounced, a chronically congested liver has a distinct reticulated acinar pattern, often more obvious beneath the capsule than in deeper parenchyma. This pattern is known as “nutmeg liver” and is the result of the contrast of red centrilobular zones of congestion with loss of hepatocytes, among pale swollen periportal parenchyma composed of viable hepatocytes that are fatty (eFig. 2-11B). This classic pattern of chronic passive congestion is most obvious in ruminants and horses. The “nutmeg” pattern can be mimicked by some forms of toxic periacinar necrosis or fatty change but should always be distinguishable from them by the presence of fibrous plaques in Glisson's capsule in the passively congested liver. The pale lobular pattern is less obvious in carnivores and should not be expected in animals that have insufficient adipose reserves for mobilization. Over longer periods, centrilobular fibrosis links terminal hepatic venules with one another and with the larger portal triads in a pattern known as cardiac fibrosis. eFigure 2-11 A. Chronic passive congestion in the liver of a cat. B. Cut surface of a bovine liver with chronic passive congestion associated with traumatic reticulopericarditis. (Courtesy K.G. Thompson.) Inflammatory changes in the outflow veins are sometimes observed. Acute inflammation and thrombosis of sublobular veins are typical of acute salmonellosis in many species, but these changes are terminal and not associated with hepatic dysfunction. Inflammatory cell infiltrates are occasionally observed in the larger hepatic veins, and have been described in dogs with parvoviral myocarditis. Muscular hypertrophy of walls of hepatic veins has been described in dogs with arteriovenous fistulae. Fibrous remodeling of these veins has been associated with nitrosamine intoxication in domestic animals. The term Budd - Chiari syndrome is used to describe the clinical features associated with hepatic venous outflow obstruction caused by thrombosis of the main hepatic veins in humans. Hepatic vein thrombosis is a rare occurrence in dogs and cats. In veterinary medicine, it is more appropriate to use specific morphologic diagnostic terms to describe pathologic changes that develop in response to various mechanical causes of postsinusoidal obstruction of hepatic venous flow, resulting in development of hepatomegaly, postsinusoidal portal hypertension, high-protein ascites, and acquired portosystemic collateral shunting. Causes include obstruction of flow caused by tumors (e.g., intraluminal leiomyosarcoma or other sarcoma, adrenal pheochromocytoma with extensive invasion into the caudal vena cava in the dog) or abscesses in the liver or caudal vena cava, hepatic venous or vena caval thrombosis, congenital malformation (fibrous web), kink or acquired stricture occluding the lumen of the vena cava or hepatic veins (intrahepatic postsinusoidal venous obstruction), or cardiac abnormalities impairing right atrial function (cor triatrium dexter, neoplasia). Histologic changes include distension of the hepatic veins and perivenous sinusoidal congestion, which, with chronicity, leads to perivenous fibrosis typical of chronic passive congestion. Thrombosis of the caudal vena cava has been described in detail in cattle, where rupture of a hepatic abscess into the caudal vena cava is the most common etiology (Fig. 2-51 ). Sequelae include pulmonary emboli, endoarteritis, multifocal pulmonary abscessation, and chronic suppurative bronchopneumonia. Figure 2-51 Thromboembolism of the caudal vena cava in an ox. (Courtesy K.G. Thompson.) Acquired portosystemic shunts Congenital shunts have been described previously in the section on Developmental disorders. Acquired portosystemic shunts within and external to the liver can develop in association with various chronic liver diseases that lead to portal hypertension. These shunts tend to be multiple, small, and tortuous (Fig. 2-52 ). They may be difficult to identify postmortem when they are collapsed. They are easily destroyed by routine dissection, so they must be noted before removal of the abdominal viscera. Acquired shunts can be associated with evidence of portal hypertension, such as distension of the portal veins and ascites, but when shunts are multiple and well established, portal hypertension is somewhat relieved. It is important to distinguish these varicose dilations that develop in response to portal hypertension from true congenital portosystemic shunts. Acquired shunts arise in vestigial nonfunctional portosystemic communications that dilate and become functional in response to portal hypertension. They tend to develop between mesenteric veins and the caudal vena cava, right renal vein, or gonadal veins, and are multiple, taking the form of a plexus of tortuous, thin-walled vessels. Esophageal shunts and varicosities are common in humans with cirrhosis, but are much less important in domestic animals. This may be related to postural differences such that shunts are more likely to enter the vena cava, where central venous pressure is least. In dogs, acquired shunts are most numerous along the caudal mesentery. Figure 2-52 Acquired shunt vessels in a dog. Peliosis hepatis/telangiectasis Peliosis hepatis is a term used to designate a hepatic vascular disorder characterized by cystic, blood-filled spaces in the liver. The distinction between telangiectasis and peliosis hepatis is subtle at best, and both conditions can be considered together. There are 2 forms of peliosis hepatis, although both can appear in the same animal. Intrinsic weakness of the local reticulin framework leading to sinusoidal dilation is the hallmark of the phlebectatic form of peliosis hepatis, and the other form is characterized by death of hepatocytes and sinusoidal distention (parenchymal type). Peliosis hepatis lesions occur throughout the liver as dark red areas, irregular in shape but well circumscribed, and ranging from pinpoints to many centimeters in size (Fig. 2-53 , eFig 2-12). Sectioned or capsular surfaces are depressed after death, and on cutting, they appear as cavities from which the blood drains to reveal a delicate network of residual stroma. The histologic lesion is characterized by multiple, dilated, blood-filled spaces sometimes surrounded by fibromyxoid stroma. The blood-filled cystic spaces may be lined by endothelium. In humans, these lesions have an idiosyncratic association with administration of various drugs, including anabolic and contraceptive steroids. Infectious peliosis hepatis caused by Bartonella henselae or B. quintana infection has also been described in immunosuppressed human patients. Figure 2-53 Peliosis hepatis (telangiectasis) in the liver of an ox. (Courtesy K.G. Thompson.) eFigure 2-12 Peliosis hepatis in the liver of a cat. (Courtesy K.G. Thompson.) Peliosis hepatis has been reported in cattle, dogs, and cats, but the pathogenesis is unknown. B. henselae DNA has been identified by PCR from the liver of a single canine case, but does not seem to be involved in the feline disorder. Bovine hepatic peliosis hepatis (telangiectasis) and human peliosis hepatis have been suggested to share a similar pathogenesis. Primary alteration of the sinusoidal barrier, with rupture of the reticulin fibers and increased deposition of basement membrane components in the perisinusoidal region and fibrosis, may alter oxygen and substrate exchange between hepatocytes and the blood, leading to hemodynamic imbalance, hepatocyte atrophy, and eventually to sinusoidal disruption. Peliosis hepatis in livers is quite common in older cats and cattle (Fig. 2-54 ). There is no evidence clinically of related liver dysfunction. In cats, the cavities are rather more frequent in the subcapsular zone and rarely exceed 2-3 mm in size. There are often other senile changes in these livers, such as chronic fatty change, nodular hyperplasia, and chronic hepatitis. Figure 2-54 Histologic appearance of peliosis hepatis in a dog. A specific condition termed “peliosis” develops in cattle poisoned by plants of Pimelea spp. This form begins as diffuse periportal sinusoidal dilation. Because these changes are also found in these animals in the spleen and in other organs with sinusoidal microcirculation, it seems that the lesions may be adaptive to progressive and dramatic increases in total blood volume. In the late stages of the intoxication by Pimelea, the liver may resemble a huge, blood-filled sponge (Fig. 2-55 ). The animals eventually die of a combination of hemodilutional anemia and circulatory failure. Figure 2-55 Periportal sinusoidal dilation resulting from chronic Pimelea poisoning in an ox. (Courtesy R. Kelly.) Further reading Arey LB. Throttling veins in the livers of certain mammals. Anat Rec 1941;81:21-33. Brown PJ, et al. Peliosis hepatis and telangiectasis in 18 cats. J Small Anim Pract 1994;35:73-77. Buchmann AU, et al. Peliosis hepatis in cats is not associated with Bartonella henselae infections. Vet Pathol 2010;47:163-166. Cave TA, et al. Idiopathic hepatic veno-occlusive disease causing Budd-Chiari-like syndrome in a cat. J Small Anim Pract 2002;43:411-415. Crawford JM, Burt AD. Anatomy, pathophysiology and basic mechanisms of disease. In: Burt AD, et al., editors. Macsween's Pathology of the Liver. 6th ed. New York: Churchill Livingstone; 2012. p. 2-77. Eipel C, et al. Regulation of hepatic blood flow: the hepatic arterial buffer response revisited. World J Gastroenterol 2010;16:6046-6057. Epstein RB, et al. A canine model for hepatic venoocclusive disease. Transplantation 1992;54:12-16. Fine DM, et al. Surgical correction of late-onset Budd-Chiari-like syndrome in a dog. J Am Vet Med Assoc 1998;212:835-837. Gunn C, et al. Hepatic dearterialization in the dog. Am J Vet Res 1986;47:170-175. Kalt DJ, Stump JE. Gross anatomy of the canine portal vein. Anat Histol Embryol 1993;22:191-197. Kelly WR. The pathology and haematological changes in experimental Pimelea spp. poisoning in cattle (“St. George disease”). Aust Vet J 1975;51:233-243. Kelly WR, Seawright AA. Pimelea poisoning of cattle. In: Keeler RF, et al., editors. Effects of Poisonous Plants on Livestock. New York: Academic Press; 1978. p. 293-300. Kitchell BE, et al. Peliosis hepatis in a dog infected with Bartonella henselae. J Am Vet Med Assoc 2000;216:519-523. Marcato PS, et al. Pretelangiectasis and telangiectasis of the bovine liver: a morphological, immunohistochemical and ultrastructural study. J Comp Pathol 1998;119:95-110. Seawright AA. Phlebectatic peliosis hepatis in Australian cattle. Vet Hum Toxicol 1984;26:208-213. Schoeman JP, Stidworthy MF. Budd-Chiari-like syndrome associated with an adrenal phaeochromocytoma in a dog. J Small Anim Pract 2001;42:191-194. Shulman HM, et al. Induction of hepatic veno-occlusive disease in dogs. Am J Pathol 1987;126:114-125. Thompson JS, et al. Adequate diet prevents hepatic coma in dogs with Eck fistulas. Surg Gynecol Obstet 1986;162:126-130. Van Winkle TJ, Bruce E. Thrombosis of the portal vein in eleven dogs. Vet Pathol 1993;30:28-35. Yamamoto K. Ultrastructural study on the venous sphincter in the sublobular vein of the canine liver. Microvasc Res 1998;55:215-222. Inflammatory Diseases of the Liver and Biliary Tract The inflammatory response in the liver is unusual for 3 main reasons. First, hepatic microvasculature is structurally and functionally different from tissues with capillary vasculature. Microvascular permeability to plasma proteins, a hallmark of acute inflammation in most tissues, is a normal property of the fenestrated sinusoidal endothelium of the liver. Thus sinusoidal edema is not a prominent feature of acute parenchymal inflammation, although edema can be observed in the capsule, portal tracts, connective tissue of the terminal and sublobular hepatic veins (particularly in dogs), and in the wall of the gallbladder. Microvascular blood flow in hepatic sinusoids is also less responsive to the actions of various vasoactive mediators that alter blood flow in most other acutely inflamed tissues. Second, resident Kupffer cells play an important and complex role in liver inflammation, injury, and repair. Kupffer cells function in the innate immune response, acting as the final component of the gut barrier by phagocytosing pathogens, immunoreactive material, and endotoxin entering the liver via the portal circulation. However, Kupffer cells also exhibit a range of different activated phenotypes, depending on the local metabolic and immune environment. Classically activated macrophages secrete proinflammatory cytokines, including tumor necrosis factor-α, interleukin-1 (IL-1), IL-6, IL-12, and inducible nitric oxide synthase (iNOS), influencing cell populations in the liver and elsewhere, whereas alternatively, activated macrophages express anti-inflammatory mediators, including IL-10 and contribute to resolution of inflammation and promote repair. Dysregulation of the complex control of inflammatory responses in Kupffer cells can contribute to chronic inflammation in the liver. Third, the liver has central regulatory influences on many proinflammatory insults and inflammatory mediators. As the major source of acute-phase proteins, including secreted pathogen recognition receptors (PRRs), short pentraxins, components of the complement system, and regulators of iron metabolism, hepatocytes are essential constituents of innate immunity and largely contribute to the control of a systemic inflammatory response. The liver is also the site of degradation of most soluble plasma proteins, and Kupffer cells are the main site of clearance of immune complexes from the circulation, playing an important role in the development of immunotolerance to potential antigenic substances absorbed from the intestine. A full discussion of immune surveillance, and the complex innate and adaptive immune responses involved in initiation and regulation of liver inflammation, is beyond the scope of this chapter, and readers are directed to current review articles on the subject. These unusual aspects of the inflammatory response in the liver can make it more difficult to differentiate certain degenerative and inflammatory conditions in this organ. Ongoing cell death and repair can appear inflammatory, and acute leukocyte responses can cause necrosis and apoptosis in the liver. The term “necroinflammatory” is convenient when the underlying pathogenetic mechanisms of necrosis and inflammation are unknown. Increased numbers of leukocytes (sinusoidal leukocytosis) are observed in hepatic sinusoids in many acute or subacute bacteremias, as well as in conditions of steroid excess in dogs. This change can be diagnostically useful but does not constitute evidence of hepatitis unless there is obvious infiltration of granulocytes, monocytes, or lymphocytes into the perisinusoidal space. Extramedullary hematopoiesis can also appear as focal aggregates of myeloid cells in the perisinusoidal compartment. This change can be distinguished from inflammatory infiltrates by the presence of immature myeloid cells. Occasionally, hematopoietic cells from the splenic red pulp can be artifactually extruded into the portal vasculature and appear in the liver, usually as nucleated cell aggregates in the larger portal veins. Infectious agents capable of causing hepatitis include viruses, bacteria, fungi, protozoa, and helminths. Autoimmune and idiosyncratic drug responses also occur, but often the etiology of acute or chronic hepatitis cannot be determined. Hepatic inflammation The liver is subject to infectious and degenerative insults that elicit inflammatory responses in various patterns, for which the general term hepatitis is appropriate. The term hepatitis is used for focal or diffuse hepatic conditions that are either caused by infectious agents or characterized by a leukocytic infiltrative inflammatory response, irrespective of the cause. This definition allows inclusion of viral infections that are hepatotropic, even though the lesions are mainly characterized by hepatocellular necrosis or apoptosis rather than by the inflammatory response to the agent. The term hepatitis is also used for responses to some hepatic toxicants, metals, or drug metabolic idiosyncrasies in which there is a prominent leukocytic response to damaged cells. However, in responses in which single necrotic hepatocytes elicit a mild neutrophilic or histiocytic response, the term hepatitis is less appropriate because the pattern of injury is primarily degenerative. Cholangitis refers to inflammation of the biliary tree . More specifically, choledochitis refers to inflammation of the bile ducts, and cholecystitis refers to inflammation of the gallbladder. Cholangiohepatitis applies to hepatic inflammation centered on the biliary tract and extending into adjacent hepatic parenchyma. Patterns and character of inflammation in hepatitis vary according to causative agent, severity and stage of disease, the route of entry into the liver, and pathogenesis of liver injury. Some viral pathogens, such as canine adenovirus 1, can cause acute and diffuse hepatitis, with centrilobular to widespread hepatocellular necrosis, mixed leukocyte infiltrates, sinusoidal congestion, and edema. By comparison, most infectious causes of hepatitis, for example, toxoplasmosis, various herpesviruses, and various bacteria, produce a more patchy pattern of inflammation with focally intense leukocyte and Kupffer cell responses in the vicinity of areas of necrosis. The distribution, character, and chronicity of these focal lesions are important diagnostically, but they typically do not damage enough functional hepatic parenchyma, ducts, or vasculature to produce systemic signs of liver failure. Foci of hepatitis with necrosis are common incidental findings, and these are assumed to reflect localized responses to bacteria that arrive via the portal system. Occasionally, such focal necroinflammatory lesions are large and numerous, for example, in cattle with rumenitis. Acute hepatitis Hallmarks of acute hepatitis typically include a combination of inflammation, typically a granulocytic inflammatory cell infiltrate, foci of hepatocellular apoptosis and necrosis, and in some instances, evidence of regeneration. Leukocyte infiltrates in acute diffuse hepatitis tend to accumulate mainly in the vicinity of the portal tracts, around major bile ducts, or sometimes in the capsule and around the central veins. Small but important numbers of neutrophils and mononuclear cells, including lymphocytes, are usually seen in the perisinusoidal space and among hepatocytes, and are often focally concentrated in sites of necrosis, or adjacent to infectious organisms. Edema is an unusual feature of acute hepatitis, but is seen in severe injury, for example, in infectious canine hepatitis. Grossly, hepatic edema is most obvious in the gallbladder, large extrahepatic bile ducts, hepatic lymph nodes, and sometimes in the capsule. Microscopically, edema is also evident in the portal triads, in the connective tissue of the terminal and sublobular veins, and sometimes by an increase in the perisinusoidal space. Kupffer cells are key participants in the acute inflammatory responses in the liver. They can enlarge and accumulate vacuoles and lysosomal debris during regular phagocytic removal of microorganisms, cell debris, and extravascular erythrocytes. They can also be activated to secretory histiocytes that release various cytokines and other mediators that induce hypertrophic or proliferative responses of hepatocytes, stellate cells, and endothelium. Activated Kupffer cells are larger and more prominent or numerous in sections, their nuclei are larger and vesicular, and their cytoplasm is basophilic and may contain vacuoles or ingested particulate matter. In overwhelming infections, many of the Kupffer cells and adjacent sinusoidal endothelial cells and hepatocytes undergo necrosis. Chronic hepatitis Chronic liver disease in domestic animals has historically been classified into several different entities based on morphologic criteria, but fibrosis is a consistent feature. In human medicine, classification of chronic hepatitis has been simplified, and morphologic divisions such as chronic active hepatitis and chronic persistent hepatitis, originally defined in specific clinical contexts, have been abandoned because of problems of evolving definitions and application, and lack of correspondence with prognosis. The single designation “chronic hepatitis” is now used for chronic necroinflammatory disease lasting more than 6 months, further modified by specifying the etiology, type and severity of inflammation, and degree and distribution of apoptosis and/or necrosis (disease activity or grade), and the degree of fibrosis (disease chronicity or stage). Numerous systems for grading and staging chronic hepatitis have been developed. In human medicine, chronic hepatitis is usually the result of chronic infection with hepatotropic viruses, and, less commonly, autoimmune, drug-induced, or associated with inherited metabolic diseases, such as Wilson disease. In veterinary medicine, chronic liver disease may develop following chronic bile duct obstruction, infection with hepatotropic infectious agents, familial or hereditary metabolic diseases, or may be toxic, drug-induced, or possibly autoimmune in origin. However, the majority of chronic liver disease is idiopathic, reflecting deficiencies in our current level of understanding of the etiologic, pathophysiologic, and clinical implications of the patterns of inflammation and necrosis seen in domestic animals. Regardless of the etiology, initial acute liver damage will not progress to fibrosis or cirrhosis unless the inflammation and damage are protracted, for example, by ongoing hepatocellular injury mediated by immunologic mechanisms, including antibody- and lymphocyte-mediated cytotoxicity, or ongoing oxidative damage. Clinical signs are nonspecific in the early stages, but as the disease progresses to involve more of the liver and impair regeneration, icterus, ascites, and hepatic encephalopathy may develop as typical correlates with hepatic insufficiency. Chronic hepatitis of humans has several characteristic lesions. These are also evident in animals, although not always to the same degree or frequency as described in the various forms of viral, immune-mediated, and idiosyncratic hepatitis in humans. The first is periportal interface hepatitis, sometimes referred to as “piecemeal necrosis” (Fig. 2-56 ). This necroinflammatory change initially destroys the limiting plate of periportal hepatocytes, and may continue to erode into the hepatic parenchyma, expanding the portal areas. Portal inflammation is variable in intensity, and includes infiltration by lymphocytes and plasma cells. Bridging necrosis, with tracts of necrosis dissecting across the hepatic lobule between portal triads or between portal areas and central veins, may also develop. Degenerative changes affecting hepatocytes in areas of interface hepatitis include cell swelling and apoptosis. Bile duct degeneration, multifocal necrosis, and hepatocellular regeneration, in the form of 2-cell–thick hepatic plates and mitotic figures, may also be seen. Deposition of collagen and basement membrane material in the space of Disse leads to capillarization of hepatic sinusoids. Single or small groups of hepatocytes may be isolated and entrapped in expanded portal areas. Fibrosis may progress to bridge portal tracts and central veins, disrupting hepatic lobular architecture and culminating in the development of cirrhosis. Figure 2-56 Periportal interface hepatitis in chronic hepatitis in a dog. Further reading Batts KP, Ludwig J. Chronic hepatitis: an update on terminology and reporting. Am J Surg Pathol 1995;19:1409-1417. Beighs V, Trautwein C. The innate immune response during liver inflammation and metabolic disease. Trends Immunol 2013;34:446-452. Bode JG, et al. Hepatic acute phase proteins—regulation by IL-6 and IL-1 cytokines involving STAT3 and its crosstalk with NF-kB-dependent signaling. Eur J Cell Biol 2012;91:496-505. Dixon LJ, et al. Kupffer cells in the liver. Compr Physiol 2013;3:785-797. Goodman ZG. Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J Hepatol 2007;47:598-607. Ishak K, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22:696-699. Ishak KG. Pathologic features of chronic hepatitis. Am J Clin Pathol 2000;113:40-55. Knodel RG, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatol 1981;1:431-435. Knolle PC, Thimme R. Hepatic immune regulation and its involvement in viral hepatitis infection. Gastroenterol 2014;146:1193-1207. Ludwig J. The nomenclature of chronic active hepatitis: an obituary. Gastroenterol 1993;105:274-278. Sterczer A, et al. Chronic hepatitis in the dog—a review. Vet Q 2001;23:148-152. Szabo G, Csak T. Inflammasomes in liver diseases. J Hepatol 2012;57:642-654. Chronic hepatitis in dogs Chronic hepatitis is a relatively common diagnosis in dogs, characterized by hepatocellular apoptosis or necrosis, a variable mononuclear or mixed inflammatory cell infiltrate, evidence of regenerative attempts, and fibrosis of variable extent and pattern. Although various infectious etiologies and drug- and toxin-induced chronic hepatic disease have been described, the majority remains idiopathic, although excess hepatic copper accumulation is the best characterized cause of chronic hepatitis in the dog. Breeds of dogs reported to be at increased risk for chronic hepatitis include the Bedlington Terrier, Doberman Pinscher, West Highland White Terrier, Labrador Retriever, American and English Cocker Spaniel, Skye Terrier, Standard Poodle, Dalmatian, and English Springer Spaniel. Hepatic copper accumulation in association with chronic hepatitis has been documented in most of these breeds, but apart from the recognized genetic mutation in the COMMD1 gene in Bedlington Terriers, leading to a primary copper storage disease with impaired copper excretion, the pathogenesis of the breed-related copper accumulation remains unclear. In the dog, disease grade is correlated with increasing hepatocellular apoptosis, proliferation, expression of nitric oxide synthase isoforms, and total hepatic iron, whereas disease stage is correlated with increasing α-smooth muscle actin–labeled periductular myofibroblasts and perisinusoidal stellate cells, CK7-positive ductular cells, the extracellular matrix protein tenascin-C, and expression of genes important in the production and regulation of hepatic fibrosis, including platelet-derived growth factors PDGFB and PDGFD, thrombospondin 1, transforming growth factors β1 and β2 (TGFB1 and TGFB2), matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinase 1, and the collagen genes COL1A1 and COL3A1. Given the importance of autoimmune hepatitis in human medicine, several studies have evaluated the possible contributions of immune mechanisms in dogs. Upregulation of major histocompatibility complex class II antigen expression in hepatocytes has been demonstrated in Doberman hepatitis in association with lymphocyte infiltration, and it has been proposed that hepatocytes presenting a putative major histocompatibility complex class II molecule–associated autoantigen could be targets for T-cell–mediated immune attack. An association with specific major histocompatibility complex DLA class II haplotypes has been described in Doberman Pinschers and English Springer Spaniels with subclinical or clinical hepatitis, and it has been suggested that the highly polymorphic DLA genes may be involved in altered susceptibility to chronic hepatitis. However, a primary autoimmune pathogenesis has yet to be demonstrated. The presence, mechanism, and role of hepatic copper accumulation in dogs continues to generate ongoing interest. Although copper is an essential metal cofactor for cuproenzymes, free copper ions can catalyze the formation of reactive hydroxyl radicals capable of causing oxidative injury (see the Copper section later in Toxic hepatic disease). The liver is central to copper homeostasis, and biliary excretion is the major route for regulating levels of copper in the body. Hepatic copper accumulation can arise (1) as the result of a primary metabolic defect in hepatic copper metabolism, (2) secondary to abnormal hepatic function with cholestasis and altered biliary copper excretion, or (3) as a consequence of excess dietary copper intake. Secondary copper accumulation occurs in the majority of human patients with primary biliary cirrhosis, prolonged extrahepatic bile duct obstruction, or chronic liver disease; however, dogs appear to be relatively resistant to hepatocellular copper accumulation as a result of cholestasis, at least in experimental studies following ligation of the common bile duct. In the dog, the reference range for hepatic copper is generally considered to be ≤400 µg/g dry weight (DW);.concentrations >1,800-2,000 µg/g DW are considered pathogenic. In primary genetic copper storage disorders and excess copper intake, copper accumulation appears at least initially to be primarily centrilobular, extending throughout the lobule as the condition progresses, with hepatic copper concentrations usually >2,000 µg/g DW. In copper storage secondary to abnormal hepatic function, copper usually accumulates in smaller amounts in the periportal parenchyma or without a consistent pattern, in concentrations typically <2,000 µg/g DW. When the hepatic concentration of copper surpasses 400 µg/g DW, the excess begins to accumulate within lysosomes. These copper-laden lysosomes become consistently demonstrable by the histochemical stains rubeanic acid and rhodanine, at copper concentrations exceeding 400 µg/g DW (Fig. 2-57 ), although some studies note visible copper granules at hepatic copper concentrations as low as 200 µg/g DW. A reasonable estimate of copper burden can be made from stained sections; however, biochemical determination of copper is more reliable and can be useful to follow the effectiveness of copper chelation therapy. Care should be taken when collecting liver for copper levels, as once the normal lobular architecture is lost because of injury and regenerative nodule formation, copper distribution within the liver becomes heterogeneous. Hepatocytes in regenerative nodules often have relatively low levels of copper, possibly as an adaptation that facilitates their ability to proliferate and form nodules, or as a function of inadequate time for significant copper accumulation in the hepatocytes forming nodules. The intervening areas of parenchymal collapse may contain an abundance of copper. Because of this regional variation, small samples, such as those taken from needle biopsies, are often inaccurate, and larger samples are required. Figure 2-57 Copper staining (rhodanine) in the liver of a West Highland White Terrier. Regardless of the reasons for copper accumulation, lysosomal copper can exceed a threshold or be released when hepatocytes die, and thereby contribute to the development of hepatitis. Hepatic pathology does not typically occur at concentrations <2,000 µg/g DW, although higher values can still be found in some dogs with normal liver histology, and other factors, such as antioxidant levels and other oxidative stress from drugs or other factors, may influence the relationship between copper concentration and injury. A hereditary, autosomal recessive copper-associated hepatopathy associated with impaired biliary copper excretion and progressive accumulation of copper within hepatocytes has been well documented in Bedlington Terriers. The majority of affected Bedlington Terriers have been found deficient in a protein, COMMD1 (copper metabolism MURR1 domain protein 1), caused by deletion of exon 2 in the COMMD1 gene, although there are other less common mutations that can also occur. Although its function is not fully understood, the COMMD1 protein has been shown to interact with the copper transporter ATP7B, important in Wilson disease, a copper storage disorder in humans, and its absence in affected dogs may impair ATP7B-mediated copper export from hepatocytes into the canaliculus. Homozygous affected dogs have the highest copper levels. Bedlington Terriers are the only breed to date shown to accumulate copper continuously throughout life, and hepatic copper concentrations in these animals may be very high; as much as 12,000 µg/g DW has been recorded, and levels >5,000 µg/g are common. Affected dogs are usually presented with signs of progressive liver failure, including ill-thrift, wasting, ascites, and signs of encephalopathy. An acute form may occur in some dogs, with acute hepatic necrosis and release of copper into the systemic circulation, where it provokes a hemolytic crisis and rapidly developing anemia and icterus (eFig. 2-13). The initial histologic lesion is multifocal centrilobular hepatitis, with foci of macrophages, lymphocytes, plasma cells, and neutrophils among the copper-laden hepatocytes in zone 3. Apoptotic hepatocytes, some containing copper granules, appear at the periphery of some foci. Copper levels >3,000 µg/g DW result in widespread massive necrosis in some dogs. Survivors may progress to develop postnecrotic cirrhosis. Grossly, the livers in later stages are fibrotic, pale, and finely nodular. eFigure 2-13 Icteric Bedlington Terrier with an acute hemolytic crisis concurrent with chronic copper-associated hepatitis. (Courtesy University of Guelph.) Chronic hepatitis is well documented in Doberman Pinschers. The disease is more common in middle-aged female dogs. Histologic changes in the livers of dogs exhibiting clinical signs of advanced hepatic disease include piecemeal necrosis of periportal zone 1 hepatocytes, with a mixed inflammatory cell infiltrate, as well as necrosis of zone 3 hepatocytes with bridging necrosis crossing the lobule (Fig. 2-58 ). Copper accumulation is evident in centrilobular hepatocytes, with various degrees of portal fibrosis, bile duct proliferation, bridging fibrosis, and development of cirrhosis in the most severely affected dogs. Intrahepatocyte bile pigment accumulation and intracanalicular bile stasis are present in periportal zones, along with iron accumulation in Kupffer cells and macrophages. Figure 2-58 Periportal interface hepatitis with mixed inflammatory infiltrates and bridging portal fibrosis in a Doberman Pinscher dog. Elevated concentrations of hepatic copper have been reported in many, but not all, Doberman Pinschers with chronic hepatitis, and the significance of increased hepatic copper concentration in this breed remains controversial. A recent investigation into the possibility of impaired copper excretion in affected Doberman Pinschers reported that 5 dogs with elevated liver copper and persistent subclinical hepatitis but without demonstrable cholestasis had comparable rates of plasma copper clearance to control dogs, but reduced rates of biliary excretion of 64Cu, suggesting that impaired copper excretion may play a role in disease in this breed. Significantly reduced levels of mRNA of various proteins involved in copper binding, transport, and excretion, including ATP7A, ATP7B, ceruloplasmin, and metallothionein, have been documented in Dobermans with clinical hepatitis with high hepatic copper concentrations, along with a reduction in gene expression of components of the antioxidant defense system, including SOD1, catalase, as well as reduced levels of glutathione, so continued investigations are needed to further clarify the pathogenesis of chronic hepatitis in Dobermans. West Highland White Terriers are at increased risk of developing chronic hepatitis and cirrhosis. There is evidence to support familial hepatic copper accumulation in some West Highland White Terriers, although the mode of inheritance is not completely understood. Hepatic copper accumulates in zone 3 hepatocytes, up to ~8 months of age, with concentrations rarely >2,000 µg/g DW. Clinical illness directly attributable to copper hepatotoxicity (concentrations >2,000 µg/g DW) in West Highland White Terriers is, however, apparently uncommon. Idiopathic chronic hepatitis progressing to cirrhosis does also occur in this breed, and may be distinguished on the basis of a different zonal location and morphology of the inflammatory lesions. In the idiopathic disease, inflammatory foci are smaller, composed of a single apoptotic hepatocyte or fragments of cells accompanied by a few lymphocytes and plasma cells, and are commonly localized to zone 1, or may be random in distribution. In dogs with copper toxicosis, foci of inflammation and necrosis were larger, always found around the central vein among copper-laden hepatocytes, and were composed of debris-filled macrophages, lymphocytes, plasma cells, and scattered neutrophils, with occasional apoptotic hepatocytes around the periphery. Distinguishing between copper toxicosis and idiopathic chronic hepatic disease may be difficult in cirrhotic livers, which, irrespective of the underlying cause, may have reduced copper burdens because of connective-tissue displacement of hepatic parenchyma, and typically lower concentrations of copper in regenerative nodules. Chronic hepatitis been reported in genetically related Skye Terriers, accompanied by modest and somewhat inconsistent hepatic copper accumulation. Lesions ranged from hepatocellular degeneration and necrosis with mild inflammation in zone 3, to chronic hepatitis and cirrhosis with marked intracanalicular cholestasis. Hepatic copper concentrations ranged from 800-2,200 µg/g DW, and copper-containing hepatocytes were found predominantly in zone 3. Copper-associated chronic hepatitis has been described in Labrador Retrievers, characterized by centrilobular infiltrates of macrophages containing intracytoplasmic copper and hemosiderin, fewer neutrophils, mononuclear inflammatory cells, as well as scattered foci of hepatocellular necrosis, lobular collapse, periacinar to bridging fibrosis, nodular regeneration, and in some cases cirrhosis. Hepatic copper concentrations in one study were typically >2,000 µg/g, whereas mean hepatic copper concentrations in a separate study were also found to be significantly higher in affected dogs (614 µg/g, range 104-4,234 µg/g) compared to age- and sex-matched control dogs (299 µg/g, 93-3,810 µg/g). A concurrent general increase in hepatic copper concentrations in a study population of Labrador Retrievers spanning 30 years was postulated to be associated with increased dietary copper availability, the result of a change in the form and bioavailability of supplemental copper added to commercially produced dog foods. A positive association between dietary copper levels and hepatic copper concentrations has been demonstrated, and high dietary copper has been suggested as a risk factor for development of copper-associated hepatitis in susceptible animals within the breed. Concurrent renal proximal tubular dysfunction with glucosuria, and increased renal copper has been described in some Labrador Retrievers with copper-associated hepatitis, and sporadically in other breeds. Chronic liver disease associated with elevated hepatic copper concentrations has also been reported in Dalmatians. A range of necroinflammatory changes has been reported, including multifocal, piecemeal, centrilobular to massive hepatic necrosis, and cirrhosis, although, in one study of 10 dogs, various degrees of piecemeal necrosis and bridging fibrosis were the most common histologic change, with either primarily lymphocytic or neutrophilic inflammatory infiltrates. Morphologic or biochemical evidence of cholestatic liver disease was not prominent. Hepatic copper concentrations ranged from 745-8,390 µg/g DW (mean 3,197 µg/g) in one report of 10 dogs, aged 2-10 years, with a variable zonal distribution. Three previous cases in young Dalmatians reported hepatic copper concentrations of 7,940 µg/g DW, 1,916 µg/g wet weight, and 2,356 µg/g wet weight, and 2 of these reports describe diffuse positive staining for copper in all hepatocytes, with the strongest staining observed in centrilobular hepatocytes. Chronic hepatitis has been reported in American and English Cocker Spaniels and English Springer Spaniels, characterized clinically in the later stages by ascites, weight loss, and icterus. Affected dogs develop chronic hepatitis and cirrhosis, and, at postmortem, livers are typically small and firm, with multiple small regenerative nodules (Fig. 2-59 ). Histologically, there is moderate to severe portal hepatitis, with inflammatory infiltrates of predominantly lymphocytes, plasma cells, and fewer neutrophils, and variable degrees of portal fibrosis and bridging fibrosis. One study of American Cocker Spaniels reported diffuse fibrosis typical of lobular dissecting hepatitis in 7 of 13 affected dogs, in addition to patterns of fibrosis more typical of cirrhosis. Piecemeal necrosis and limiting plate destruction have been reported in Cocker Spaniels, whereas in English Springer Spaniels, hepatocyte necrosis and apoptosis in areas of inflammatory infiltrates in both portal areas and scattered throughout the hepatic parenchyma was more typical. Biliary hyperplasia or marked ductular reaction was noted in affected Cocker Spaniels. Hepatic copper staining is variable, and hepatic copper accumulation is not a consistent feature. Figure 2-59 Chronic hepatitis in an American Cocker Spaniel. α1-Antitrypsin (α1-AT) deficiency has been suggested to play a role in chronic hepatitis in some dog breeds, including English Cocker Spaniels, although whether this is an epiphenomenon or cause of chronic liver disease has not yet been proven. α1-AT, a plasma glycoprotein synthesized mainly by hepatocytes, is a member of the serine proteinase inhibitor (serpin) superfamily, and is a potent inhibitor of neutrophil elastase. Hereditary α1-AT deficiency in humans is associated with mutations that perturb the protein's tertiary structure and promote polymerization. These misfolded forms of α1-AT accumulate within the rough endoplasmic reticulum of hepatocytes, forming PAS-positive globules. α1-AT deficiency has been associated in humans with neonatal hepatitis, juvenile cirrhosis, and adult hepatocellular carcinoma. Lobular dissecting hepatitis, associated with predominantly sinusoidal inflammation and fibrosis, has been described in young dogs with ascites and acquired portosystemic shunts. The liver is usually small, pale with a predominantly smooth surface, and occasional hyperplastic nodules (eFig. 2-14). The histologic lesion is characterized by dissection of lobular parenchyma by reticulin and fine collagen fibers into individual and small groups of hepatocytes, accompanied by a variable, mixed inflammatory infiltrate of lymphocytes, plasma cells, and lesser numbers of neutrophils and macrophages (Fig. 2-60A, B ). Activated fibroblastic cells, likely hepatic stellate cells, may also be prominent along sinusoids. Hepatocytes form rosettes or pseudoductular structures, and regenerative nodules may be present. Portal inflammation and periportal fibrosis are not conspicuous features of this disease. The etiology of this disorder remains unknown. Figure 2-60 A. Lobular dissecting hepatitis with diffuse fine interstitial fibrosis isolating hepatocytes in a juvenile Golden Retriever dog. B. Reticulin stain demonstrating pattern of lobular dissection. eFigure 2-14 Lobular dissecting hepatitis in a dog. (Courtesy J.L. Caswell.) Chronic hepatitis in other species Copper-associated liver disease has also been described in domestic cats. A hepatopathy with excess hepatic copper (4,074 µg/g DW) was first reported in a Siamese cat, characterized by enlarged, finely vacuolated, and individual necrotic hepatocytes with centrilobular and midzonal copper accumulation. Chronic hepatitis and cirrhosis, with marked accumulation of stainable copper in macrophages in fibrous septa and with sparser staining in regenerative nodules, was reported in a European Shorthair cat with similarly elevated liver copper concentrations (4,170 µg/g DW; the upper reference range limit for hepatic copper concentration is cats is <180 µg/g DW). A recent retrospective study described an additional 11 cats with presumed primary copper-associated hepatopathy, characterized by elevated hepatic copper concentrations (>700 µg/g DW), diffuse or centrilobular and intermediate zone copper staining, without any other co-occurring cholestatic disorders. Affected cats had hepatocyte vacuolation consistent with glycogen accumulation, fibrillar collagen in the perivenular region, and variable mild parenchymal collapse, with bridging fibrosis in one cat. Cats also accumulate copper in the liver secondary to other cholestatic hepatobiliary disorders, such as chronic cholangitis/cholangiohepatitis, or extrahepatic bile duct obstruction, although copper staining in these cases is principally in hepatocytes in portal and intermediate zones. Chronic hepatitis seen as end-stage livers occurs in horses (eFig. 2-15) and ruminants, particularly cattle, and is often assumed to be associated with ingestion of hepatotoxins, such as pyrrolizidine alkaloids in forage, or a consequence of prolonged administration of hepatotoxic therapeutics, although in the absence of specific histologic lesions and/or a corroborative clinical history, the underlying causes are rarely discovered. eFigure 2-15 Chronic hepatitis with cirrhosis in a horse. Further reading Andersson M, Sevelius E. Breed, sex and age distribution in dogs with chronic liver disease: a demographic study. J Small Anim Pract 1991;32:1-5. Azumi N. Copper and liver injury: experimental studies on the dogs with biliary obstruction and copper loading. Hokkaido Igaku Zasshi 1982;57:331-349. Bexfield NH, et al. Chronic hepatitis in the English springer spaniel: clinical presentation, histological description and outcome. Vet Rec 2011;169:415-419. Bexfield NH, et al. DLA class II alleles and haplotypes are associated with risk for and protection from chronic hepatitis in the English Springer Spaniel. PLoS ONE 2012;7:e42854. Boisclair J, et al. Characterization of the inflammatory infiltrate in canine chronic hepatitis. Vet Pathol 2001;38:628-635. Boomkens SY, et al. Hepatitis with special reference to dogs. A review on the pathogenesis and infectious etiologies, including unpublished results of recent own studies. Vet Q 2004;26:107-114. Center SA. Chronic liver disease: current concepts of disease mechanisms. J Small Anim Pract 1999;40:106-114. Center SA, et al. Digital image analysis of rhodanine-stained liver biopsy specimens for calculation of hepatic copper concentrations in dogs. Am J Vet Res 2013;74:1474-1480. Dill-Macky E. Chronic hepatitis in dogs. Vet Clin N Am Small Anim Pract 1995;25:387-398. Dyggve H, et al. Association of Doberman hepatitis to canine major histocompatibility complex II. Tissue Antigens 2010;77:30-35. Favier RP, et al. Copper-induced hepatitis: the COMMD1deficient dog as a translation animal model for human chronic hepatitis. Vet Q 2011;31:49-60. Fieten H, et al. Association of dietary copper and zinc levels with hepatic copper and zinc concentration in Labrador Retrievers. J Vet Intern Med 2012;26:1274-1280. Fieten H, et al. Canine models of copper toxicosis for understanding mammalian copper metabolism. Mamm Genome 2012;23:62-75. Fuentealba C, Aburto EM. Animal models of copper-associated liver disease. Comp Hepatol 2003;2:5-16. Fuentealba C, et al. Chronic hepatitis: a retrospective study in 34 dogs. Can Vet J 1997;38:365-373. Haynes JS, Wade PR. Hepatopathy associated with excessive hepatic copper in a Siamese cat. Vet Pathol 1995;32:427-429. Haywood S, et al. Pathobiology of copper-induced injury in Bedlington terriers: ultrastructural and microanalytical studies. Anal Cell Pathol 1996;10:229-241. Hoffman G. Copper-associated liver diseases. Vet Clin Small Anim 2009;39:489-511. Hoffmann G, et al. Copper-associated chronic hepatitis in Labrador Retrievers. J Vet Intern Med 2006;20:856-861. Hurwitz BM, et al. Presumed primary and secondary hepatic copper accumulation in cats. J Am Vet Med Assoc 2014;244:68-77. Jensen AL, Nielson OL. Chronic hepatitis in three young standard poodles. J Vet Med A 1991;38:194-197. Johnston AN, et al. Hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis: 72 cases (1980-2010). J Am Vet Med Assoc 2013;242:372-380. Kanemoto H, et al. Expression of fibrosis-related genes in canine chronic hepatitis. Vet Pathol 2011;48:839-845. Kanemoto H, et al. American Cocker Spaniel chronic hepatitis in Japan. J Vet Intern Med 2013;27:1041-1048. Klomp AEM, et al. The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis. J Hepatol 2003;39:703. Langlois DK, et al. Acquired proximal renal tubular dysfunction in 9 Labrador Retrievers with copper-associated hepatitis (2006-2012). J Vet Intern Med 2013;27:491-499. Mandigers PJ, et al. Hepatic 64Cu excretion in Dobermanns with subclinical hepatitis. Res Vet Sci 2007;83:204-209. Mandigers PJ, et al. Chronic hepatitis in Doberman pinschers. A review. Vet Q 2004;26:98-106. Mandigers PJ, et al. Association between liver copper concentration and subclinical hepatitis in Doberman Pinschers. J Vet Intern Med 2004;18:647-650. Meertens NM, et al. Copper-associated chronic hepatitis and cirrhosis in a European Shorthair cat. Vet Pathol 2005;42:97-100. Mekonnen GA, et al. Tenascin-C in chronic canine hepatitis: immunohistochemical localization and correlation with necro-inflammatory activity, fibrotic stage, and expression of alpha-smooth muscle actin, cytokeratin 7, and CD3+ cells. Vet Pathol 2007;44:803-813. Noaker LJ, et al. Copper associated acute hepatic failure in a dog. J Am Vet Med Assoc 1999;214:1502-1505. Poitout F, et al. Cell-mediated immune responses to liver membrane protein in canine chronic hepatitis. Vet Immunol Immunopathol 1997;57:169-178. Poldervaart JH, et al. Primary hepatitis in dogs: a retrospective review (2002-2006). J Vet Intern Med 2009;23:72-80. Rolfe DS, Twedt DC. Copper-associated hepatopathies in dogs. Vet Clin North Am Small Anim Pract 1995;25:399-417. Rutgers HC, et al. Idiopathic hepatic fibrosis in 15 dogs. Vet Rec 1993;133:115-118. Sevelius E, et al. Hepatic accumulation of α1-antitrypsin in chronic liver disease in the dog. J Comp Pathol 1994;111:401-412. Smedley R, et al. Copper-associated hepatitis in Labrador Retrievers. Vet Pathol 2009;46:484-490. Spee B, et al. Differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in Doberman pinschers. Comp Hepatol 2005;4:3-14. Spee B, et al. Copper metabolism and oxidative stress in chronic inflammatory and cholestatic liver diseases in dogs. J Vet Intern Med 2006;20:1085-1092. Speeti M, et al. Lesions of subclinical Doberman hepatitis. Vet Pathol 1998;35:361-369. Speeti M, et al. Upregulation of major histocompatibility complex class II antigens in hepatocytes in Doberman hepatitis. Vet Immunol Immunopathol 2003;96:1-12. Sterczer A, et al. Chronic hepatitis in the dog—a review. Vet Q 2001;23:148-152. Stockhaus C, et al. A multistep approach in the cytologic evaluation of liver biopsy samples of dogs with hepatic diseases. Vet Pathol 2004;41:461-470. Thornburg LP. Histomorphological and immunohistochemical studies of chronic active hepatitis in Doberman Pinschers. Vet Pathol 1998;35:380-385. Thornburg LP. A perspective on copper and liver disease in the dog. J Vet Diagn Invest 2000;12:101-110. Thornburg LP, et al. Hepatic copper concentrations in purebred and mixed-breed dogs. Vet Pathol 1990;27:81-88. Thornburg LP, et al. The relationship between hepatic copper content and morphologic changes in the liver of West Highland white terriers. Vet Pathol 1996;33:656-661. Twedt DC, et al. Clinical, morphological and chemical studies on copper toxicosis of Bedlington terriers. J Am Vet Med Assoc 1979;175:269-275. van De Sluis BJ, et al. Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16. Hum Mol Genet 1999;8:501-507. van De Sluis B, et al. Identification of a new copper metabolism gene by positional cloning in a purebred dog population. Hum Mol Genet 2002;15:165-173. van den Ingh TSGAM, et al. Morphological classification of parenchymal disorders of the canine and feline liver. In: Rothuizen J, et al., editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Chapt. 7. Philadelphia: Saunders Elsevier; 2006. p. 85-101. van den Ingh TSGAM, Rothuizen J. Lobular dissecting hepatitis in juvenile and young adult dogs. J Vet Intern Med 1994;8:217-220. Vince AR, et al. Hepatic injury correlates with apoptosis, regeneration, and nitric oxide synthase expression in canine chronic liver disease. Vet Pathol 2013;51:932-945. Webb CB, et al. Copper-associated liver disease in Dalmatians: a review of 10 dogs (1998-2001). J Vet Intern Med 2002;16:665-668. Whittemore JC, et al. Hepatic copper and iron accumulation and histologic findings in 104 feline liver biopsies. J Vet Diagn Invest 2012;24:656-661. Miscellaneous inflammatory liver disease Nonspecific reactive hepatitis refers to hepatic inflammation characterized by light inflammatory infiltrates, primarily in the portal tracts, without any evidence of hepatocellular necrosis. It is often observed during inflammation of the splanchnic organs and is a reactive response, rather than primary inflammation of the liver. Giant cell hepatitis is an uncommon lesion in animals, but is recorded in cats, calves, and foals (Fig. 2-61 ). There is evidence for maternal leptospirosis in some cases reported in foals. Two cases are reported in young cats with concurrent thymic lymphomas. The liver is deeply bile stained. Histologically, the acinar structure is effaced, and the blood vessels engorged. Hepatocytes are large and syncytial and may contain 10 or more nuclei. The pale or ballooned cytoplasm contains bile pigments, and cytoplasmic invaginations into hepatocyte nuclei are common. Inflammatory cells are not conspicuous. Liver parenchymal giant cell transformation occurs in humans in a wide variety of congenital and neonatal liver disorders, including bile duct obstruction associated with biliary atresia, viral and bacterial infections, some metabolic disorders such as galactosemia, some cases of Down syndrome and other genetic disorders, as well as in idiopathic neonatal hepatitis, a cholestatic condition of undetermined cause. Although giant cells were originally suggested to be a marker of infantile obstructive cholangiopathy in humans, their association with a wide range of disorders supports an alternative conclusion that giant cell formation represents a nonspecific reaction of the infant's hepatocytes to various types of injury. Figure 2-61 Giant cell hepatitis in a cat. Hypertrophic hepatic cirrhosis in calves is described from Germany and occasionally observed elsewhere. Death may occur in liver failure within a few days to weeks of birth, or the disease may be discovered at slaughter for veal. The liver is moderately enlarged with rounded borders, very firm but smooth on the surface, and gray. Histologically, there is some biliary hyperplasia, but the lesion is dominated by diffuse fibrosis infiltrated in the early stages by mononuclear inflammatory cells. No cause has been identified. Similar lesions of congenital hepatic fibrosis may be found in unborn or aborted calves. Systemic granulomatous disease has been reported in cattle grazing hairy vetch (Vicia villosa). The disease is characterized clinically by dermatitis, pruritus, diarrhea, wasting, and high mortality. Histologic lesions include infiltration of skin and internal organs, including portal areas of the liver, by monocytes, lymphocytes, plasma cells, eosinophils, and multinucleated giant cells. The pathogenesis is unknown, although the inflammatory reaction has characteristics of a type IV hypersensitivity reaction. Alternatively, vetch lectin may act to activate T lymphocytes directly to initiate the cellular response. Further reading Messer NT, Johnson PJ. Idiopathic acute hepatic disease in horses: 12 cases (1982-1992). J Am Vet Med Assoc 1994;204:1934-1937. Panciera RJ, et al. Hairy vetch (Vicia villosa Roth) poisoning in cattle: update and experimental induction of the disease. J Vet Diagn Invest 1992;4:318-325. Poonacha KB, et al. Leptospirosis in equine fetuses, stillborn foals, and placentas. Vet Pathol 1993;30:362-369. Suzuki K, et al. Giant cell hepatitis in two cats. J Vet Med Sci 2001;63:199-201. Inflammatory diseases of the biliary tract Inflammation of the gallbladder is termed cholecystitis. Inflammation of the large bile ducts is cholangitis, whereas inflammation of the smallest intrahepatic bile ductules is termed cholangiolitis. Cholangiolitis is uncommon in animals, and occurs mainly in conjunction with inflammation of larger ducts. Destructive cholangiolitis has been observed in dogs, and is associated with adverse drug reactions in humans. Cholecystitis Cholecystitis is uncommon and is often associated with concurrent cholelithiasis, although acalculous cholecystitis has been reported in the dog, with a single case report in a pig. Cholecystitis is thought to be caused by reflux of intestinal bacteria into the gallbladder via the bile ducts, or to hematogenous entry of bacteria from the adjacent hepatic circulation. Aerobic gram-negative bacteria are the most frequent isolates from canine cases, although occasionally anaerobic bacteria such as clostridia have been cultured. Campylobacter jejuni has been isolated from 2 dogs with bacteremia and cholecystitis. Occasionally, parasites, such as flukes that colonize the bile ducts, can enter the gallbladder and cause cholecystitis. Canine cholecystitis has been associated with various systemic disorders, including diabetes mellitus, severe enteritis, biliary stasis, septicemia, as well as with the use of immunosuppressive drugs, each of which may promote bacterial colonization of the gallbladder. In the acute lesion, histologic changes include neutrophilic inflammatory infiltrates in the wall and lumen of the gallbladder, with focal erosion or ulceration, and edema. More chronic stages develop typical mixed inflammatory infiltrates, with fibrosis. Occasionally, the infiltrate may be predominantly lymphoplasmacytic, with formation of lymphoid follicles within the mucosa. Gallbladder infarction characterized by transmural coagulative necrosis of the gallbladder wall with intravascular fibrin thrombi has been reported in dogs (Fig. 2-62 ). The arterial blood supply to the gallbladder is the cystic artery, a branch of the hepatic artery, and occlusion may cause partial or complete infarction. Predisposing factors have not been identified; however, the lack of significant concurrent inflammatory response suggests that this is not simply a sequela to underlying cholecystitis. Figure 2-62 Infarcted gallbladder in a dog. Gallbladder mucocele is discussed further under Ectopic, hyperplastic, and metaplastic lesions. Cholangitis/cholangiohepatitis Although pure cholangitis does occur, extension of inflammation from the ducts into the adjacent hepatic parenchyma can occur, and such lesions can quite accurately be regarded as cholangiohepatitis. Bile contains various antimicrobial factors, including β-defensins and bile acids that are inhospitable to most bacteria, except for some species with particular capsule adaptations. Bacterial cholangiohepatitis is usually caused by common opportunists of enteric origin, such as coliforms and streptococci. Some bacteremic organisms, including some Salmonella, can be cultured from the bile, but the mechanism by which they get there is unknown. Salmonellosis is a distinctive cause of fibrinous cholecystitis in cattle, especially calves. The pathogenesis of bacterial cholangitis/cholangiohepatitis depends on various predisposing conditions, similar to those involved in the pathogenesis of pyelonephritis. These include infection by bacteria that reach the ducts hematogenously, facilitated by localization in the peribiliary plexus or by extension from sinusoidal Kupffer cells or foci of necrosis. Descending cholangitis is occasionally observed in cattle with suppurative hepatitis with extension from abscesses directly into the ducts or via the portal lymphatics. Cholangiohepatitis of this origin may be restricted in its distribution to biliary fields, but in some cases, it does become quite diffuse in the biliary system. Alternatively, bacteria can ascend the ducts from the intestine, facilitated by bile stasis caused by mechanical or functional obstructions. The inflammatory and proliferative responses in cholangitis further interfere with bile flow and exacerbate the ductular spread of bacterial infections once they are established. Again, these lesions can be quite regionally variable, and may be missed on hepatic biopsy if only small areas are sampled. The course and pathologic changes in cholangiohepatitis vary greatly, from fulminating suppurative infection to persistent mild inflammation that, over a period of months or years, leads to hepatic fibrosis of biliary distribution. Severe suppurative cholangiohepatitis may follow a short course to death, the effects being those of the infection itself, which may become septicemic, rather than of hepatic injury. At autopsy, the liver is swollen, soft, and pale, and its architecture is blurred. Few or many suppurative foci may be visible beneath the capsule and on the cut surface (eFig. 2-16). They are small, sometimes miliary in distribution, and not encapsulated. Lesions in other organs may be those of septicemia and jaundice. Microscopically, the larger ducts contain purulent exudate, and the smaller ones are disintegrated. Dense masses of neutrophils, liquefied or not, are present in the portal triads and infiltrate the degenerate parenchyma. eFigure 2-16 Cholangiohepatitis in a cat. (Courtesy J.L. Caswell.) In subacute and chronic cholangiohepatitis, the inflammation is more proliferative than exudative. The liver is enlarged and may be of normal shape, or distorted because of irregular areas of atrophy and regenerative hyperplasia. Its surface may be smooth or finely granular; the capsule is thickened and may bear fibrous tags or be adherent to adjacent viscera. Within areas of duct obstruction, retention of bile pigments can be found in the regions served by the occluded ducts, but systemic icterus (or photosensitization in herbivores) is unlikely unless a large portion of liver is affected. On cut surface, the enlarged portal tracts are easily visible and accentuate the architecture of the organ. Eventually, the new fibrous tissue replaces the parenchyma, and in chronic diffuse cases in which the original infection persists, continuous fibroplasia may produce hepatic enlargement, the organ becoming huge, gray, and gristly. This spectrum of liver changes is typical of alsike clover poisoning (“big-liver disease”) in horses (discussed in the later section on Toxic hepatic disease). Alternatively, the chronic fibrosis may occur in wedge-shaped areas oriented to a small bile duct. The enlarged interlobular ducts may be readily visible and frequently contain plugs of inspissated secretion and debris. Microscopically, the reaction remains centered on portal tracts. These are expanded in subacute cases by infiltration of leukocytes and macrophages and the proliferation of small ducts and, in chronic cases, chiefly by organizing fibrous tissue and proliferating bile ducts. Encroachment on the parenchyma is minimal but inevitable. Continued degeneration of the periportal parenchyma is probably an additional stimulus to local fibroplasia, which, as well as thickening the smallest portal triads, extends along their length and links up with neighboring triads, thus subdividing the acini into segments. The hepatic venules and sublobular veins are involved. Regenerative nodules are not a prominent feature of cholangiohepatitis unless large areas of parenchyma have been destroyed, in which case the least damaged lobes are expanded by coarse nodules. Neutrophilic cholangitis (suppurative or exudative cholangitis/cholangiohepatitis) is a relatively common disorder of cats and, much less commonly, dogs. This condition has been suggested to be the result of acute inflammation in the biliary tree associated with ascending bacterial infection, and in cats, the condition often occurs in conjunction with other disorders, including acute extrahepatic bile duct obstruction, pancreatitis, or inflammatory bowel disease. In the cat, the biliary and pancreatic ducts share a common entry to the duodenum, and simultaneous infectious inflammation of these systems is common. Escherichia coli is the most frequent bacterial isolate; however, Bacteroides, Klebsiella, hemolytic Streptococcus, and clostridia have also been reported. The acute stage is characterized by edema and neutrophilic portal infiltrates, inflammation and degeneration of bile ducts with neutrophils in ductular lumens and emigrating through the ductular epithelium. Some cases may progress to cholangiohepatitis, with infiltration of inflammatory cells into hepatic lobules and periportal hepatocellular necrosis. Areas of suppuration and hepatic abscessation may occur. Mixed cholangiohepatitis, with neutrophils, lymphocytes, and plasma cells infiltrating portal areas accompanied by bile duct proliferation, biliary epithelial degeneration, and various degrees of periportal to bridging fibrosis, may represent the subacute stage (Fig. 2-63 ), whereas concentric periportal fibrosis and pseudolobule formation may represent the most chronic stage of the disease. Figure 2-63 Neutrophilic bile duct inflammation with portal edema, early fibrosis, and a mixed infiltrate of neutrophils, lymphocytes and plasma cells extending across the limiting plate in a case of subacute cholangiohepatitis in a dog. An apparently distinct condition of cats, lymphocytic cholangitis (lymphocytic portal hepatitis, nonsuppurative cholangitis/cholangiohepatitis) is a slowly progressive disease characterized by lymphocytic infiltrates within the portal area, with variable degrees of bile duct or oval cell proliferation and peribiliary, portal-to-bridging fibrosis (Fig. 2-64 ). An immune-mediated pathogenesis has been proposed, and in one study, most cats with lymphocytic cholangitis had T-cell–predominant portal infiltrates, often with accompanying portal B-cell aggregates. The presence of various degrees of lymphocytic targeting and infiltration of bile ductules with concurrent degenerative changes in ductular epithelium, destructive cholangitis leading to ductopenia, and portal lipogranulomas may be useful in distinguishing this condition from hepatic lymphoma in the cat, along with T-cell receptor clonality assays. Figure 2-64 Lymphocytic cholangitis in a cat. Destructive cholangitis has also been described in dogs, and is characterized by destruction and loss of bile ducts in the smaller portal tracts, with subsequent inflammation composed of pigment-laden macrophages, neutrophils, and/or eosinophils, and in some instances, fibrosis. The extent of injury may be sufficient to cause marked icterus and acholic feces because of marked intrahepatic cholestasis. The pathogenesis of this lesion is unclear; however, toxic injury, idiosyncratic drug reactions (see Fig. 2-40) and infection by canine distemper virus have been implicated in some cases. Biliary tract obstruction Cholelithiasis (gallstone formation) is seldom observed in animals. The choleliths usually form in the gallbladder and are composed of a mixture of cholesterols, bile pigments, salts of bile acids, calcium salts, and a proteinaceous matrix. Choleliths of mixed composition are yellow-black or green-black and are friable. Pigment stones, composed of calcium bilirubinate, and cholesterol stones have also been reported in dogs. There may be hundreds of small stones or a few large ones. The large stones are usually faceted. The origin of these mixed gallstones is uncertain, but their development is probably secondary to chronic mild cholecystitis and related to disturbances of the resorptive activities of the gallbladder, whereby the bile salts are removed faster than the stone-forming compounds. Gallstones are usually asymptomatic. Occasionally, they lodge in and obstruct bile ducts and cause jaundice. The larger stones may cause pressure necrosis and ulceration of the mucosa, local dilations of the bile ducts, and saccular diverticula of the gallbladder. Calculi seldom form in the ducts, although calcareous deposits often do so in fascioliasis of cattle. Calcium bilirubinate calculi have been reported in the bile ducts of horses (Fig. 2-65 ), associated with intermittent jaundice. Figure 2-65 Cholelith obstructing the bile duct in a horse. Occasionally, particles of solid ingesta may find their way into the gallbladder; sand has been seen in sheep, and seeds in pigs. Biliary obstruction is rarely caused by impacted gallstones. Usually, it is the result of cholangitis or cholecystitis, the obstruction being produced by masses of detritus and biliary constituents, parasites, or cicatricial stenosis of the ducts. Adult ascarids may cause mechanical obstruction. Inspissated bile-stained friable plugs are occasionally responsible for obstructions in segments of the liver in horses. Tumors of the pancreas and duodenum, and tumors and abscesses of the hilus of the liver and portal nodes, may cause compression stenosis of the ducts. Edematous swelling of the papilla in enteritis may also be of significance. Biliary obstruction by abnormal intraluminal mucoid secretion (gallbladder mucocele) has been reported in dogs (see later section on Ectopic, metaplastic, and hyperplastic lesions). The consequences of biliary obstruction depend on the site and duration of the obstruction. When the main duct is involved, there is jaundice. When one of the hepatic ducts is involved, there is no jaundice, and depending on the efficiency of biliary collaterals, there may be no pigmentation of the obstructed segments of liver. Increases in serum γ-glutamyltranspeptidase and alkaline phosphatase usually occur when a sufficiently large amount of the duct system is affected. The ducts undergo progressive cylindrical dilation, which may be extreme. The smallest interlobular ducts and the cholangioles proliferate. There is inflammation in the walls of the ducts and the portal triads, and this is probably due in part to chemical irritation by bile acids but is largely caused by secondary bacterial infections. These infections may be acute and purulent, or low grade; in these cases, bacteria may not be easily cultured. The cholangiohepatitis that almost inevitably follows has been described previously. Rupture of the biliary tract or the gallbladder causes steady leakage of bile into the peritoneal cavity, the omentum being unable to seal even small defects. The bile salts are very irritating and may cause acute chemical peritonitis. The peritoneal effusion that follows may remain sterile; more often it is infected by enteric bacteria, and severe diffuse peritonitis ensues. This may be rapidly fatal, particularly if clostridia are involved. Many perforations of the biliary tract are traumatic in origin; however, in dogs, cholecystitis, gallbladder necrosis/infarction, and gallbladder mucocele have all been associated with gallbladder rupture. Further reading Callahan Clark JE, et al. Feline cholangitis: a necropsy study of 44 cats (1986-2008). J Feline Med Surg 2011;13:570-576. Center SA. Diseases of the gallbladder and biliary tree. Vet Clin North Am Small Anim Pract 2009;39:543-598. Crews LJ, et al. Clinical, ultrasonographic, and laboratory findings associated with gallbladder disease and rupture in dogs: 45 cases (1997-2007). J Am Vet Med Assoc 2009;234:359-366. Day MJ. Immunohistochemical characterization of the lesions of feline progressive lymphocytic cholangitis/cholangiohepatitis. J Comp Pathol 1998;119:135-147. Gabriel A, et al. Suspected drug-induced destructive cholangitis in a young dog. J Small Anim Pract 2006;47:344-348. Gagne JM, et al. Histopathologic evaluation of feline inflammatory liver disease. Vet Pathol 1996;33:521-526. Holt DE, et al. Canine gallbladder infarction: 12 cases (1993-2003). Vet Pathol 2004;41:416-418. Kirpensteijn J, et al. Cholelithiasis in dogs: 29 cases (1980-1990). J Am Vet Med Assoc 1993;202:1137-1142. Mayhew PD, et al. Pathogenesis and outcome of extrahepatic biliary obstruction in cats. J Small Anim Pract 2002;43:247-253. Newell SM, et al. Gallbladder mucocele causing biliary obstruction in two dogs: ultrasonographic, scintigraphic and pathological findings. J Am Anim Hosp Assoc 1995;31:467-472. O'Neill EJ, et al. Bacterial cholangitis/cholangiohepatitis with or without concurrent cholecystitis in four dogs. J Small Anim Pract 2006;47:325-335. Osumi T, et al. A case of recovery from canine destructive cholangitis in a miniature dachshund. J Vet Med Sci 2011;73:937-939. Oswald GP, et al. Campylobacter jejuni bacteremia and acute cholecystitis in two dogs. J Am Anim Hosp Assoc 1994;30:165-169. Ryu SH, et al. Cholelithiasis associated with recurrent colic in a Thoroughbred mare. J Vet Sci 2004;5:79-82. Starost MF, Burkholder TH. Acalculous and clostridial cholecystitis in a pig. J Vet Diagn Invest 2008;20:527-530. van den Ingh TSGAM, et al. Destructive cholangitis in seven dogs. Vet Q 1988;10:240-245. van den Ingh TSGAM, et al. Morphological classification of biliary disorders of the canine and feline liver. In: Rothuizen J, et al., editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Philadelphia: Saunders Elsevier; 2006. p. 61-76. Warren A, et al. Histopathologic features, immunophenotyping, clonality and eubacterial fluorescence in situ hybridization in cats with lymphocytic cholangitis/cholangiohepatitis. Vet Pathol 2011;48:627-641. Weiss DJ, et al. Relationship between inflammatory hepatic disease and inflammatory bowel disease, pancreatitis, and nephritis in cats. J Am Vet Med Assoc 1996;209:1114-1116. Weiss DJ, et al. Inflammatory liver disease. Semin Vet Med Surg (Small Anim) 1997;12:22-27. Infectious Diseases of the Liver Viral infections Various systemic viral diseases may affect the liver. Adenoviral infections of lambs, calves, and goat kids can cause multifocal hepatic necrosis and cholangitis, in addition to pneumonia. Lymphohistiocytic hepatitis, with jaundice and various degrees of apoptosis of hepatocytes, disorganization of hepatic plates, and perilobular stromal condensation, is seen in some cases of porcine circovirus 2–associated disease (Fig. 2-66A, B ). Canid herpesvirus 1 infection in puppies and, more rarely, in adult dogs causes disseminated focal necrosis and hemorrhages in parenchymal organs, including the liver, with formation of amphophilic intranuclear inclusion bodies in epithelial cells of kidney, lung, and liver. Similar microfoci of hepatic necrosis sometimes occur in aborted or newborn foals with congenital equid herpesvirus 1 infections (Fig. 2-67 ), in neonatal calves infected with bovine herpesvirus 1, in piglets with suid herpesvirus 1 infections, and in feline fetuses following intravenous feline herpesvirus 1 inoculation of the pregnant queen. Feline coronavirus infection can cause granulomatous hepatitis in some infected cats, as part of feline infectious peritonitis (Fig. 2-68 ). Multifocal hepatic necrosis has been described in large felids and domestic cats infected with highly pathogenic influenza A virus H5N1. Hepatocellular and Kupffer cell necrosis with mild mononuclear portal inflammation was reported in outbreaks caused by virulent systemic strains of feline calicivirus in cats in the United States and United Kingdom. Systemic cowpox virus infection with foci of hepatic necrosis containing immunoreactive cowpox viral antigen has been reported in a cat. Figure 2-66 A. Hepatitis caused by porcine circovirus 2 (PCV-2) infection in a pig. B. Immunohistochemical stain for PCV-2 antigen. (Courtesy J. DeLay.) Figure 2-67 Focal hepatic necrosis with intranuclear inclusion bodies and syncytial cells in congenital equid herpesvirus 1 infection in a foal. Figure 2-68 Pyogranulomatous hepatitis caused by feline infectious peritonitis virus infection. The viral diseases discussed in more detail later are those in which the liver is the major target organ, causing substantial hepatic injury that sometimes culminates in hepatic failure. Unlike humans, in which several pathogenic hepatitis viruses from various families are well described, few viruses that specifically target the liver have been identified in the major domestic mammals. Hepadnaviruses have been found in members of the squirrel family, including woodchucks and various species of ground squirrels and arctic ground squirrels, as well as in birds, including some species of ducks, geese, and herons. Infection with hepatitis E virus (HEV) genotypes 3 and 4 is common in domestic and wild pigs, and although clinical disease is not apparent in infected swine, they may serve as reservoirs for this potentially zoonotic pathogen. Lesions of mild multifocal lymphoplasmacytic hepatitis with focal hepatocellular necrosis have been reported in pigs experimentally inoculated with HEV, but lesions in natural infections are not recognized. Flaviviruses, including a nonprimate hepacivirus and a GB-virus–like virus (pegivirus) have recently been described in horses, although their potential clinical significance is unknown. A second novel pegivirus species of horses, Theiler's disease–associated virus, is described in more detail later. Infectious canine hepatitis Canine adenovirus 1 (CAdV-1) infection is the cause of infectious canine hepatitis, a severe liver disease in dogs; other canids, including coyotes, foxes, wolves; and in bears. Vaccination has made the disease rare in many countries in which it was endemic. Deaths from infectious canine hepatitis are usually sporadic, although small outbreaks can occur among young dogs in kennels. Fatalities seldom occur among dogs >2 years of age. In areas where the disease is not controlled by vaccination, it is probable that most dogs in the general population contact CAdV-1 in the first 2 years of life and suffer either inapparent infection or mild febrile illness with pharyngitis and tonsillitis. In more severe cases, there is vomiting, melena, high fever, and abdominal pain. There may be petechiae on the gums; the mucous membranes are pale and occasionally slightly jaundiced. Nonspecific nervous signs occur in a few cases. There is also a peracute form of the disease in which the animal is found dead without signs of illness, or after an illness of only a few hours. In convalescence, there may be a unilateral or bilateral opacity of the cornea caused by corneal edema (so-called blue eye, a type III hypersensitivity reaction with immune-complex–mediated corneal injury) (Fig. 2-69 ), which disappears spontaneously. Figure 2-69 Infectious canine hepatitis. Corneal edema or “blue-eye.” (Courtesy North Carolina State University CVM.) CAdV-1 has special tropism for endothelium, mesothelium, and hepatic parenchyma, and it is injury to these that is responsible for the pathologic features of edema, serosal hemorrhage, and hepatic necrosis. The histologic specificity of the lesions depends on the demonstration of large, solid intranuclear inclusion bodies in endothelium or hepatic parenchyma (Fig. 2-70 ). Inclusions are occasionally observed in other differentiated cells but always have the same morphologic and tinctorial features, being deeply acidophilic with a blue tint. Figure 2-70 Infectious canine hepatitis (canine adenovirus 1 infection). Intranuclear inclusion bodies in numerous hepatocytes. (Courtesy University of Guelph.) The morbid picture of spontaneously fatal cases is usually distinct enough to allow a diagnosis to be made at gross postmortem. Superficial lymph nodes are edematous, slightly congested, and often hemorrhagic. Blotchy or paintbrush hemorrhages may be present on intestinal and gastric serosae, and there is usually a small quantity of clear or blood-stained fluid in the abdomen. Jaundice, if present, is slight. The liver is slightly enlarged, with sharp edges, and is turgid and friable, sometimes congested, with a fine, uniform, yellow mottling (eFig. 2-17A). Red strands of fibrin can be found on its capsule, especially between the lobes. In the majority of cases, the wall of the gallbladder is edematous (eFig. 2-17B), and may have intramural hemorrhages; when edema is mild, it may be detected only in the attachments of the gallbladder. eFigure 2-17 Infectious canine hepatitis. A. Mottled, congested liver. B. Edema and hemorrhage of the gallbladder. (Courtesy University of Guelph.) Gross lesions in other organs are inconstant. Small hemorrhagic infarcts may be found in the renal cortices of young puppies. Hemorrhages may occur in the lungs, and occasionally, there are irregular areas of hemorrhagic consolidation in the caudal lobes. Hemorrhages in the brain occur in a small percentage of cases, typically only grossly visible in the midbrain and brainstem. Hemorrhagic necrosis of medullary and endosteal elements occurs in the metaphyses of long bones in young dogs, and the hemorrhages are readily visible through the thin cortex of the distal ends of the ribs. At low magnification, the histologic changes in the liver are of centrilobular (periacinar) zonal necrosis, resembling the zonal pattern of some acute hepatotoxicities. The susceptibility of centrilobular parenchyma to necrosis in this disease is as yet unexplained. Close to the portal triads, the hepatocytes may be nearly normal in appearance, except for loss of basophilia and the presence of a scattering of inclusion bodies. In spontaneously fatal cases, most of the parenchyma of the peripheral and central portions of the lobules (acini) is dead, the hepatocytes having undergone coagulative necrosis, and in some of these, ghosts of inclusion bodies may be detectable. The margin between necrotic parenchyma and viable tissue is usually quite sharp, although in the viable tissue, there are many individual hepatocytes undergoing apoptosis, most of them without inclusion bodies. Fatty change is common. The dead cells do not remain long, so the sinusoids become dilated and filled with blood. The reticulin framework remains intact, an observation in keeping with the fact that, in recovered cases, restitution of the liver is complete. Massive necrosis with collapse does not occur. As is typical of severe centrilobular necrosis, the necrotic zones, initially eccentric areas about hepatic venules, extend and link up to isolate portal units. Intranuclear inclusions can be found in Kupffer cells in variable numbers. Many of the Kupffer cells are dead, others are proliferating, and others are actively phagocytic in the removal of debris. Leukocytic reactions in the liver are mild and are directed against the necrotic tissue; mononuclear cells are present, but neutrophils, many degenerating, predominate. There is some collection of bile pigment, but it is moderate, in keeping with the short course of the disease. Microscopic lesions in other organs are largely the result of injury to endothelium. Inclusion bodies in endothelial cells can be difficult to find and are looked for, with most profit in renal glomeruli, where endothelium is concentrated. Occasionally, they are found in the epithelium of collecting tubules. When areas of hemorrhagic consolidation of the lungs are present, there is hemorrhage, edema, and fibrin formation in the alveoli, and in these consolidated areas, inclusions are often common in alveolar capillaries and even in dying cells of the bronchial epithelium. Changes in the brain are secondary to vascular injury. Hemorrhages, if present, are from capillaries and small venules, and inclusions in endothelial nuclei can usually be found in vessels that have bled. Other endothelial and adventitial cells are hyperplastic and mixed with a few lymphocytes. Small foci of softening or demyelination may be present in relation to the hemorrhages. Lymphoreticular tissues are congested, and inclusions may be found in reticulum cells of follicles, in the red pulp of the spleen, and in macrophages anywhere. Following natural oronasal exposure, viral multiplication occurs in the tonsils and leads to tonsillitis, which may be quite severe with extensive edema of the throat and larynx. Fever accompanies the tonsillitis and precedes the viremic phase, which lasts 4-8 days, accompanied by leukopenia. Hepatic necrosis develops at about day 7 of experimental infection; however, an immune response with adequate neutralizing antibodies may clear the virus from the blood and limit the extent of hepatic damage. In surviving animals, hepatic regeneration occurs rapidly, and there do not appear to be any significant residual lesions. Small foci of hepatocellular necrosis may still be present at 2 weeks, and foci of proliferated Kupffer cells may be detectable for another week or 2. Progressive hepatic injury does not seem to follow the acute phase of the natural disease, although chronic hepatitis has been experimentally reproduced in partially immunized dogs challenged with the virus. Adenoviral antigen has been detected immunohistochemically in occasional Kupffer cells in 5 dogs with a range of chronic hepatic inflammatory lesions and in one dog with a patent ductus venosus; however, a retrospective study of formalin-fixed paraffin-embedded liver from 45 dogs with chronic hepatitis or cirrhosis failed to reveal CAdV-1 by either PCR or immunohistochemistry, although the possibility that the virus initiates hepatic damage by provoking self-perpetuating hepatitis could not be excluded. Focal interstitial nephritis occurs commonly, and the cellular infiltrates are persistent but not functionally significant. They consist of interstitial lymphocytic accumulations, especially about the corticomedullary junction and in the stroma of the pelvis. Corneal edema is a late development, corresponding temporally to increasing neutralizing antibody titer. It may occur as early as day 7 of infection, but is usually delayed to between 14 and 21 days. Viral antigen can be detected in these eyes by fluorescent techniques, but not in the corneal structures. Inflammatory edema is present in the iris, ciliary apparatus, and corneal propria, and inflammatory cells are abundant in the filtration angle and iris. The infiltrates are principally plasma cells, and there is evidence that the ocular lesion is a hypersensitivity reaction to circulating immune complex deposition, with complement fixation, inflammatory cell chemotaxis, and corneal endothelial damage resulting in an anterior uveitis with corneal stromal edema. Originally, it was assumed that the widespread tendency to hemorrhage in this disease was because of leakage from damaged vascular endothelium, coupled with an inability on the part of the damaged liver to replace clotting factors. Although these effects play a role, it is now known that the exhaustion of clotting factors is in large part the result of their accelerated consumption, as the widespread endothelial damage is a potent initiator of the clotting cascade. Wesselsbron disease This disease is caused by Wesselsbron virus, an arthropod-borne flavivirus that, according to serologic surveys, is widespread in Africa in various species of animals and birds. Various Aedes mosquitoes are the vectors. Humans are also susceptible to clinical and inapparent infection. The virus produces outbreaks of abortion and perinatal death in sheep. Susceptible adults rarely show clinical signs but may have a biphasic febrile response to infection; other clinical signs, when present, are of hepatitis and jaundice. The lesions in lambs dying within 12 hours of birth consist mainly of widespread petechiae and gastrointestinal hemorrhage; longer survival allows jaundice to develop, and the liver becomes orange-yellow, enlarged, friable, and patchily congested. The bile in the gallbladder becomes thick and dark in some cases, but this may be due more to hemorrhage into the gallbladder than to hemolysis. Lymph nodes are rather constantly enlarged, congested, and edematous. The most characteristic histologic changes are seen in the liver. There are randomly scattered foci of necrosis, with apoptosis and proliferation of sinusoidal lining cells (Fig. 2-71 ). Mononuclear cells and pigment-filled macrophages accumulate in the portal stroma as well as in the sinusoids. In a variable proportion of cases, hepatocyte nuclei may contain eosinophilic, irregular inclusions. These are not accompanied by as much margination of nuclear chromatin as that associated with conventional viral inclusions, and their significance is obscure. Wesselsbron viral antigen can be demonstrated in necrotic acidophilic and degenerating hepatocytes and rarely in the inclusions. In jaundiced animals, there may be considerable canalicular cholestasis; whether or not this is the result of hemolysis does not appear to have been determined. Hepatocellular proliferation is apparent in the less acute cases. Lymphoid follicles in lymph nodes and spleen show pronounced lymphocyte necrosis and stimulation of lymphoblasts. Figure 2-71 Focal necrosis and apoptosis in Wesselsbron disease in a lamb. (Courtesy S. Youssef.) Rift Valley fever This is an arthropod-borne viral infection of ruminants and humans, in many respects similar to Wesselsbron disease. Rift Valley fever virus (RVFV) is, however, responsible for greater losses. Morbidity and mortality may occur in adult sheep; death sometimes occurs in adult cattle, but it is chiefly a disease of the young, causing heavy mortality among lambs, kids, and calves, and abortion in ewes, does, and cows. The infection in enzootic form is widespread in eastern and southern Africa, but it has, in plague-like proportions, extended to West Africa, Egypt, and the Arabian Peninsula. RVFV is a member of the Phlebovirus genus, 1 of the 5 genera in the family Bunyaviridae. It is transmitted by many species of mosquito of the genera Culex and Aedes, in which transovarial passage can occur. Mosquitoes, once infected, remain so, and in them, the virus is not pathogenic. High levels of viremia occur in sheep and cattle and are maintained for up to 5 days. During epizootics, the virus may be spread by fomites, aerosols, and mechanically by other biting insects. In endemic situations, the disease in adults is usually mild, but in epidemics in sheep and goats, severe illness occurs with fever, mucopurulent nasal discharge, and dysentery. The mortality rate is then very high, reaching 90-100% in lambs and 10-30% in adults. The disease in cattle is less severe, but pregnant animals abort, and the mortality rate in adult animals is 5-10%, whereas in calves it may reach 70%. As in Wesselsbron disease, the gross postmortem picture is dominated by widespread hemorrhage, ranging from serosal petechiae to severe gastrointestinal bleeding. The liver in the acute cases in neonatal lambs is similar to that in cases of Wesselsbron disease, being yellow, swollen, soft, and patchily congested or hemorrhagic. In older animals and in less acute cases, however, the liver tends to be darker and shows scattered pale foci of necrosis 1-2 mm in diameter. There may be fibrinous perihepatitis, edema of the gallbladder wall, and moderate, blood-tinged ascites. Experimental infection of calves produced encephalomyelitis in an animal that survived the initial viremic stage. Within 12 hours of experimental infection of lambs, there are randomly distributed foci of hepatocellular necrosis in the liver. These foci include aggregates of inflammatory cells and prominent apoptotic bodies and initially involve about a half dozen hepatocytes (eFig. 2-18). Within a few hours, however, these primary foci enlarge and may become almost confluent. In the meantime, the remaining parenchyma may rapidly undergo necrosis that spares only a small rim of periportal hepatocytes. In naturally infected calves, the primary foci of necrosis undergo lysis more rapidly than the surrounding parenchyma; these foci thus have a striking “washed-out” appearance. Where the expanding foci of necrosis include portal triads, there may follow fibrinous vasculitis and thrombosis. Fibrin deposition in sinusoids is common, and so is mineralization of necrotic hepatocytes. Cholestasis is apparent in sections but is not a prominent feature. eFigure 2-18 Focal necrosis with hemorrhage in Rift Valley fever in a lamb. (Courtesy S. Youssef.) Eosinophilic intranuclear inclusion bodies, often elongated, are sometimes seen in degenerate hepatocytes; there is associated nuclear vesiculation and chromatin margination. There is necrosis in germinal centers of lymphoid follicles in lymph nodes and spleen similar to that seen in Wesselsbron disease. Renal glomerular hypercellularity and necrosis have been described in the experimental disease. By immunohistochemistry, RVFV antigen can be detected in focal areas in cytoplasm of degenerating and necrotic hepatocytes but not in nonparenchymal cells. Ultrastructural studies have shown condensation of degenerate hepatocytes, abundant apoptosis, and the presence of membrane-bound fragments of hepatocellular cytoplasm, but sinusoidal lining cells are not notably damaged; the Kupffer cells instead participate in the uptake of the necrotic and apoptotic hepatocytes. There is abundant fibrin in sinusoids in the vicinity of the primary foci of necrosis. The intranuclear inclusions are composed not of recognizable viral particles but of filaments. Virus is occasionally discernible in the cytoplasm, associated with tubular membranes. It seems that the primary infection of the liver produces the primary necrotic foci, from which more virus spreads to damage neighboring parenchyma; the virus clearly has a marked preference for hepatocytes. The hemorrhagic component of the syndrome is probably related to consumption of clotting factors; there is no direct evidence for the endothelial damage seen in infectious canine hepatitis. In summary, Rift Valley fever is characterized by obvious focal hepatic necrosis, on which is superimposed an extensive centrilobular and midzonal necrosis; cholestasis is not as prominent as it is in Wesselsbron disease. The liver lesions in the latter disease consist of smaller, randomly distributed foci of hepatocellular necrosis, more active reaction by the sinusoidal lining cells, and more obvious cholestasis. Equine serum hepatitis (Theiler's disease) Equine serum hepatitis is a common cause of acute hepatic failure in horses. The disease was originally described in 1919 by Arnold Theiler, in horses passively immunized with equine serum against African horse sickness, and was later recognized in horses passively immunized against anthrax, tetanus, and equine encephalomyelitis. Various injectable biologics of equine origin have been associated with the disease, including Clostridium perfringens toxoids, tetanus antitoxin, and equine herpesviral vaccines prepared from equine fetal tissue and pregnant mare serum. However, although such an association holds for the majority of cases, there are many, usually sporadic, cases in horses that have not received any injections. It is unknown how many exposed horses get subclinical disease, because the condition is seldom diagnosed before the onset of hepatic failure. However, some horses do recover after transient illness with jaundice, and some can survive with residual neurologic problems. Recent metatranscriptomic analysis of serum from 2 index clinical cases in an outbreak of serum hepatitis in horses passively immunized against botulism, as well as from the equine hyperimmune plasma product administered before the outbreak, identified a previously unknown and highly divergent member of the Flaviviridae family, designated “ Theiler's disease–associated virus ” (TDAV). In this outbreak, 8 of 22 horses developed serum biochemical evidence of liver injury, although only the 2 index cases developed clinical signs of acute hepatic insufficiency. A qRT-PCR–based assay found evidence of TDAV in serum from 15 of 17 horses treated with the botulinum antitoxin, as well as in 1 of the 3 donor horses used to produce the antitoxin, whereas 20 in-contact untreated horses and 20 additional horses from a separate premises were assay negative. Viral load did not, however, appear to be predictive of biochemical or clinical extent of hepatic injury. One of 4 horses experimentally inoculated with the virus developed elevated serum liver enzymes. Four of the original 17 horses continued to be asymptomatically positive for TDAV 1 year later, demonstrating chronic infection, whereas horses that tested negative initially continued to test negative at 1 year, suggesting that horizontal transmission is inefficient. Although Koch's postulates remain to be fulfilled, TDAV appears to be a plausible candidate viral cause of this disease. Further research is necessary to establish the precise role of this virus in the disease. The incubation period of disease is typically 42-60 days, sometimes up to 90 days. Onset of the typical clinical syndrome is sudden, with death occurring in 6-24 hours. Clinically, there is lethargy; jaundice; photosensitivity; hyperexcitability, often with mania; continuous walking and pushing; apparent blindness; and ataxia. Death occurs suddenly without a period of prostration. At autopsy, icterus is present, with moderate ascites; the spleen is normal or congested, and there may be petechial hemorrhages on serous membranes and renal cortices, and some congestion of the intestine with hemorrhage into its lumen. Grossly, the liver usually appears atrophic, small, and flabby because of the acute loss of many hepatocytes (Fig. 2-72A ). The liver may be stained by bile pigments, and its surface mottled with a few strands of fresh fibrin. The mottling is more evident on the cut surface, which may be severely congested with an apparent zonal pattern, and sometimes fatty (Fig. 2-72B). Figure 2-72 Equine serum hepatitis (Theiler's disease). A. Small, limp and flaccid “dishrag” liver. B. The reticular pattern in this slice of liver suggests zonal necrosis. Microscopically, the hepatic lesion is considerably older than the clinical course would suggest. There may be a few surviving swollen and vacuolated periportal hepatocytes or sometimes complete depletion of parenchymal cells in the section. In less-affected regions in animals with adequate dietary or adipose reserves to mobilize, there is severe macrovesicular fatty change in most remaining hepatocytes. Acute necrosis is typically not seen, and there is no significant hemorrhage. Variable numbers of apoptotic hepatocytes are expected. In the periphery of the acini, severely ballooned cells undergo dissolution to leave scattered fatty cysts, but most of them disappear to leave either sinusoids that are dilated and filled with blood or a condensed and distorted reticulin framework. Extensive deposits of bile pigments are present in Kupffer cells and hepatocytes. Leukocytes, including lymphocytes, plasma cells, histiocytes, and a few neutrophils, may infiltrate diffusely, but not in large numbers. There is diffuse but very slight fibroplasia, especially in the portal units. In some livers, there is a ductular reaction, with small irregular clusters of proliferating ductular cells evident in the portal areas. Further reading Baba SS, et al. Wesselsbron virus antibody in domestic animals in Nigeria: retrospective and prospective studies. New Microbiol 1995;18:151-162. Boomkens SY, et al. Hepatitis with special reference to dogs. A review on the pathogenesis and infectious etiologies, including unpublished results of recent own studies. Vet Q 2004;26:107-114. Carmichael LE. The pathogenesis of ocular lesions of infectious canine hepatitis. I. Pathology and virological observations. Pathol Vet 1964;1:73-95. Carmichael LE. The pathogenesis of ocular lesions of infectious canine hepatitis. II. Experimental ocular hypersensitivity produced by the virus. Pathol Vet 1965;2:344-359. Chandriani S, et al. Identification of a previously undescribed divergent virus from the Flaviviridae family in an outbreak of equine serum hepatitis. Proc Natl Acad Sci U S A 2013;110:E1407-E1415. Chouinard L, et al. Use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type I in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis. J Vet Diagn Invest 1998;10:320-325. Coetzer JAW. The pathology of Rift Valley fever. I. Lesions occurring in natural cases in new-born lambs. II. Lesions occurring in field cases in adult cattle, calves and aborted foetuses. Onderstepoort J Vet Res 1977;44:205-212. 1982;49:11-17. Coetzer JAW, et al. Wesselsbron disease: pathological, haematological and clinical studies in natural cases and experimentally infected new-born lambs. Onderstepoort J Vet Res 1978;45:93-106. Coetzer JAW, Ishak KG. Sequential development of the liver lesions in new-born lambs infected with Rift Valley fever virus. I. Macroscopic and microscopic pathology. II. Ultrastructural findings. Onderstepoort J Vet Res 1982;49:103-108, 109-122. Coyne KP, et al. Lethal outbreak of disease associated with feline calicivirus infection in cats. Vet Rec 2006;158:544-550. Fagbo SF. The evolving transmission pattern of Rift Valley fever in the Arabian Peninsula. Ann N Y Acad Sci 2002;969:201-204. Gadsden BJ, et al. Fatal Canid herpesvirus 1 infection in an adult dog. J Vet Diagn Invest 2012;24:604-607. Halbur PG, et al. Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. J Clin Microbiol 2001;39:918-923. Herder V, et al. Poxvirus infection in a cat with presumptive human transmission. Vet Dermatol 2011;22:220-224. Hoover EA, Griesemener RA. Experimental feline herpesvirus infection in the pregnant cat. Am J Pathol 1971;65:173-188. Ikegami T, Makino S. The pathogenesis of Rift Valley fever. Viruses 2011;3:493-519. Kasorndorkbua C, et al. Experimental infection of pregnant gilts with swine hepatitis E virus. Can J Vet Res 2008;67:303-306. Klopfleisch R, et al. Distribution of lesions and antigen of highly pathogenic avian influenza virus A/Swan/Germany/R65/06 (H5N1) in domestic cats after presumptive infection by wild birds. Vet Pathol 2007;44:261-268. Marschall J, Hartmann K. Avian influenza A H5N1 infections in cats. J Fel Med Surg 2008;10:359-365. Moeller RB, et al. Systemic Bovine herpesvirus 1 infections in neonatal dairy calves. J Vet Diagn Invest 2013;25:136-141. Odendaal L, et al. Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep. J Vet Diagn Invest 2014;26:49-60. Pesavento PA, et al. Pathologic, immunohistochemical, and electron microscopic findings in naturally occurring virulent systemic feline calicivirus infection in cats. Vet Pathol 2004;41:257-263. Resendes AR, et al. Apoptosis in postweaning multisystemic wasting syndrome (PMWS) hepatitis in pigs naturally infected with porcine circovirus type 2 (PCV2). Vet J 2011;189:72-76. Rippy MK, et al. Rift Valley fever virus-induced encephalomyelitis and hepatitis in calves. Vet Pathol 1992;29:495-502. Rosell C, et al. Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2. Vet Pathol 2000;37:687-692. Schulze C, Baumgärtner W. Nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies. Vet Pathol 1998;35:209-217. Sinha A, et al. Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs. Res Vet Sci 2012;92:151-156. van der Lugt JJ, et al. The diagnosis of Wesselsbron disease in a new-born lamb by immunohistochemical staining of virus antigen. Onderstepoort J Vet Res 1995;62:143-146. Woods LW, et al. Cholangiohepatitis associated with adenovirus-like particles in a pygmy goat. J Vet Diagn Invest 1991;3:89-92. Bacterial infections Bacterial hepatitis is common, but, with a few important exceptions, is usually focally distributed and of little clinical significance. Bacteria may gain entrance to the liver in various ways: by direct implantation, for example, by foreign-body penetration from the reticulum; by invasion of the capsule from an adjacent focus of suppurative peritonitis; hematogenously via the hepatic artery or portal and umbilical veins; or via the bile ducts. Excepting peracute septicemias, there are few specific bacterial infections that have a sustained or repeated bacteremic phase without producing hepatic lesions. There are, in addition, many cases of nonspecific bacteremia, especially those originating in the drainage field of the portal vein, in which focal hepatitis occurs. Because their differential diagnosis is of some importance, it is probably useful to list here those specific bacterial diseases in which focal hepatitis is expected or characteristic, but not constant. The specific infections may occur as fetal or perinatal infections. The list includes Listeria monocytogenes in fetal and neonatal lambs, calves, foals, and piglets (Fig. 2-73 ); Campylobacter fetus in fetal and neonatal lambs (Fig. 2-74 ); Actinobacillus equuli in foals; A. suis in pigs; Yersinia pseudotuberculosis in lambs and occasionally in dogs and cats; Francisella (Yersinia) tularensis in lambs and cats; Mannheimia haemolytica and Histophilus somni (Haemophilus agni) in lambs; Salmonella spp. in all hosts; Clostridium piliforme (Tyzzer's disease) in foals and dogs; Nocardia asteroides in dogs; and the mycobacteria in all hosts (Figure 2-75, Figure 2-76 ). Figure 2-73 Multifocal hepatitis caused by Listeria monocytogenes infection in a calf. (Courtesy J.L. Caswell.) Figure 2-74 Campylobacter fetus infection in an ovine fetus. (Courtesy P. Stromberg.) Figure 2-75 Tuberculosis in a bovine liver. (Courtesy University of Guelph.) Figure 2-76 Hepatic mycobacteriosis in a Schnauzer dog A. H&E. B. Acid-fast stain. (Courtesy J.L. Caswell.) Hepatic abscess Hepatic abscesses, quite apart from the lesions of the specific infections just given, are common, especially in cattle. They may arise by direct implantation of a foreign body from the reticulum or by direct invasion of the capsule from a suppurative lesion of traumatic reticulitis and may be single or multiple, but in either case, they are often preferentially distributed to the left lobe. They may be hematogenous from portal vein emboli, or by direct extension of omphalophlebitis. Arteriogenic abscesses via the hepatic artery may occur in pyemias but are quite uncommon. Omphalogenic abscesses are more common in calves than in other species but occur in all. The bacterial flora is frequently mixed, but Arcanobacterium pyogenes, Fusobacterium necrophorum, streptococci, and staphylococci usually predominate. Hepatic abscesses are not inevitable sequelae to omphalitis or even to omphalophlebitis, but they do not develop from navel infections in the absence of omphalophlebitis. As there is no flow of blood in these vessels after birth, involvement of the liver is by direct growth along the physiologic thrombus. Omphalophlebitis can be quite severe without extension to the liver. Hepatic abscesses of omphalogenic origin are often restricted to the left lobe (Fig. 2-77 ), but they may be restricted to the right or be generalized in their distribution. Figure 2-77 Omphalophlebitis with miliary metastatic abscesses in the left lobe of the liver in a calf. Hepatic abscesses are also common and of economic importance in feedlot cattle. They are usually found at slaughter but, when numerous, may be fatal after a few days of vague digestive illness. Their pathogenesis and character are discussed with rumenitis, to which they are a sequel (see Vol. 2, Alimentary system and peritoneum). Liver abscesses in feedlot sheep likely have a similar pathogenesis, with F. necrophorum as the primary isolate. A second category includes parasitic granulomas populated by various opportunistic bacteria. Hepatic abscesses of biliary origin occur in all animals. They occur in pigs in which ascarids have migrated into the bile ducts. Cholangitic abscesses in horses, dogs, and cats are usually caused by enterobacteria as part of a fulminating ascending cholangiohepatitis that is fatal after a short course. The sequelae of hepatic abscessation are variable. Usually, they are insignificant and asymptomatic. Sterilization of the focus with either resorption and complete healing or encapsulation is common. Those near the surface of the liver regularly produce fibrinous and then fibrous inflammation of the capsule and adhesion to adjacent viscera. They seldom perforate the capsule but do commonly break into hepatic veins to produce any one or a combination of thrombophlebitis of the vena cava, endocarditis, or pulmonary abscesses or embolism. Acute extension of a hepatic abscess into the major hepatic vein can lead to pulmonary embolism that can be acutely fatal. In adults, death may occur if the hepatic abscesses are multiple and fresh, and especially if they are necrobacillary in origin; death is probably the result of toxemia. Hepatic necrobacillosis Occasionally, F. necrophorum infection of the liver is observed following omphalophlebitis in lambs and calves, or as a complication of rumenitis in adult cattle. In feedlot cattle, both F. necrophorum subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B) have been isolated. The hepatic lesions are multiple slightly elevated, rounded, dry areas of coagulative necrosis, sometimes a few centimeters in diameter (eFig. 2-19) and surrounded by a zone of intense hyperemia. Affected neonates seldom live long enough for the necrotic foci to liquefy and assume the appearance of ordinary abscesses, but this may be seen in adult cattle. The histologic appearance of the foci in the stage of coagulative necrosis is quite characteristic. The necrotic amorphous central area is bordered by a zone of wholesale destruction of leukocytes, whose nuclear chromatin is dissipated in a finely divided form, and among which the filamentous fusobacteria are mostly concentrated. Outside this zone, there is severe hyperemia and hemorrhage, and thrombosis of local vessels is common. The lesion in neonatal lambs is to be distinguished from that caused by Campylobacter fetus. eFigure 2-19 Hepatic necrobacillosis (Fusobacterium necrophorum infection) in an ox. Pale slightly raised areas of dry coagulative necrosis bordered by acute inflammation. (Courtesy University of Guelph.) The pathogenic mechanisms of F. necrophorum involve various toxins, in particular, a high-molecular-weight leukotoxin specifically toxic to ruminant neutrophils. This unique toxin activates neutrophils and induces their apoptosis, consistent with the remarkable abscess-inducing propensity of F. necrophorum in ruminants. However, other toxins, endotoxic lipopolysaccharide (LPS), and hemagglutinins are also implicated as virulence factors. Mixed infections are frequent, and synergism between F. necrophorum and other pathogens, such as Arcanobacterium pyogenes, may also play a role in the pathogenesis of liver necrosis and abscessation. Necrotic hepatitis (black disease) Organisms of the genus Clostridium are notably circuitous in their means of producing disease. This is true of C. novyi; type B strains, which produce potent exotoxins and are the cause of black disease (infectious necrotic hepatitis). The alpha toxin of C. novyi is related to the large clostridial cytotoxins produced by C. difficile and C. sordelli. These toxins enter cells by receptor-mediated endocytosis and inhibit ras and rho guanosine triphosphatases by glycosylation. The beta toxin is a necrotizing and hemolytic phospholipase C (lecithinase). Black disease occurs in nonimmune animals when these exotoxins are released by C. novyi within an anaerobic focus in the liver. These anaerobic sites, which provide a suitable environment for germination of C. novyi spores, are most commonly a result of migrating liver flukes. C. novyi is widely distributed in soil, and the spores are continually ingested by grazing animals in areas where black disease occurs. Some spores cross the mucous membranes, probably in phagocytes, and remain as latent infections in macrophages, mainly in the liver, spleen, and bone marrow. The duration of latency in tissue is not known, but it can be many months. In endemic areas, many healthy sheep, cattle, and dogs harbor latent infections in their livers. Black disease is principally an acutely fatal disease of sheep in regions where the inciting helminths are endemic. The disease is most commonly initiated by migrating larvae of the common liver fluke Fasciola hepatica. In Bessarabia and France, it is endemically related to the distribution of Dicrocoelium dendriticum, the lancet liver fluke. Sporadic cases may be related to Cysticercus tenuicollis infection or may be idiopathic. Deaths in sheep from black disease occur rapidly and usually without warning signs. Illness, if observed, is brief and characterized by reluctance to move, drowsiness, rapid respiration, and quiet subsidence. Affected animals are usually in good nutritional condition. Postmortem decomposition occurs rapidly. The name of the disease is derived from the appearance of flayed skins, the dark coloration being caused by an unusual degree of subcutaneous venous congestion. Frequently, there is edema of the sternal subcutis, and airways contain stable foam. Serous cavities contain an abundance of fluid that clots on exposure to air. The fluid is usually straw colored, but that in the abdomen may be tinged with blood. The volume of fluid in the abdomen and thorax may vary from about 50 mL to 1.5 L. The pericardial sac is distended with similar fluid in amounts up to ~300 mL. Subendocardial hemorrhages in the left ventricle are almost constant. Patchy areas of congestion and hemorrhage may be present in the pyloric part of the abomasum and in the small intestine. The typical and diagnostic lesions occur in the liver and are always present. They are usually clearly evident on the capsular surface, the diaphragmatic surface especially, but the organ may have to be sliced carefully to find them. The liver will be the seat of either the acute traumatic hemorrhagic lesions of acute fascioliasis, the cholangiohepatitis of the chronic disease, or both. The lesion of black disease, and occasionally there are several, is a yellow-white area of necrosis 2-3 cm in diameter, surrounded by a broad zone of intense hyperemia, roughly circular in outline, and extending hemispherically into the substance of the organ. There may be a coagulum of fibrin on the capsular surface overlying the necrotic area. Occasionally, the essential lesions are rectilinear in shape or very irregular. The lesions appear homogeneous on the cut surface, but some contain poorly defined centers of soft or caseous material. The histologic evolution of the hepatic lesions begins with the necrotic and hemorrhagic tracts caused by wandering immature flukes. These are sinuous tunnels ~0.5 cm in diameter that contain blood, necrotic hepatic cells, and the leukocytes, chiefly eosinophils, attracted by the flukes. About the tunnels is a narrow zone of coagulative necrosis, also produced by the flukes. As usual, the necrotic tissue is demarcated by a thin zone of scavenger cells, chiefly neutrophils. If latent spores are present in the necrotic areas, they quickly vegetate and are visible in sections as large, gram-positive bacilli. In nonimmune animals, the vegetative organisms elaborate exotoxins that cause necrosis of the surrounding tissue, including the eosinophils of the fluke tunnel. As the area of necrosis expands, the bacterial proliferation keeps pace so that bacilli can be found in all parts of the necrotic focus but not in the surrounding viable tissue. Usually, bacteria are concentrated at the advancing margin of the lesion, just inside a zone of infiltrated neutrophils. At about the time of death and immediately afterward, the bacilli scatter in the liver and to other organs. Bacillary hemoglobinuria Bacillary hemoglobinuria is a counterpart of black disease. The cause is Clostridium haemolyticum, which is closely related to C. novyi. Both species produce the beta toxin, a necrotizing and hemolytic lecithinase (phospholipase C). The pathogenesis of the 2 diseases is comparable, as both depend on a focus of hepatic injury within which latent spores can germinate. Bacillary hemoglobinuria as an endemic disease exists only in areas where Fasciola hepatica abounds, and it is probable that flukes are the primary cause of the initiating lesion. The disease does occur sporadically where there are no flukes and may be prompted by other parasites or other diverse focal lesions, which are smudged out in the expanding areas of necrosis. There is scant information on the ecology of the organism, but it is clear that it has its own environmental requirements, and the disease will not persist in areas where these requirements are not met. The spores will remain in the livers of cattle for several months after removal from pastures where the disease is endemic. Spores may persist in the bones of cadavers for 2 years. Spores of this and other sporulating anaerobes can frequently be demonstrated in the liver, where they are probably retained in Kupffer cells. Bacillary hemoglobinuria occurs in cattle and sheep. It is characterized clinically by intravascular hemolysis with anemia and hemoglobinuria, but, perhaps reflecting variety in exotoxins between strains of the organism, hemolysis may not be a feature. The essential lesion is hepatic and similar to that of black disease but is much larger and usually single (Fig. 2-78 ). It has been described as an infarct secondary to portal thrombosis, and although this may occur in isolated cases, it is scarcely a creditable pathogenesis for a disease of endemic occurrence. Thrombosis does occur in the affected areas but can be a result, rather than a cause, of the initial lesion and is found more frequently in the hepatic venules than in branches of the portal vein. There is severe anemia, the kidneys are speckled red or brown by hemoglobin, and the urine is of port-wine color. Peritoneal vessels are injected, and in some cases, there is severe, dry, fibrinohemorrhagic peritonitis. Figure 2-78 Large focus of hepatic necrosis in an ox with bacillary hemoglobinuria. (Courtesy University of Guelph.) Clostridium piliforme infection Clostridium piliforme (formerly Bacillus piliformis) infection has long been known as Tyzzer's disease, a cause of severe losses in laboratory rodents; however, it has also been reported in foals, calves, dogs, and cats. Although the disease is probably initiated by an intestinal infection, lesions in the gut are less specific and constant than those in the liver, which consist of focal hepatitis and necrosis. Affected foals usually die between the ages of 1-4 weeks; often they are found dead after a short illness. The liver shows pale foci up to a few millimeters across (Fig. 2-79 ); these are represented microscopically by randomly distributed foci of coagulative necrosis with moderate neutrophilic infiltrate (Fig. 2-80A ). This lesion is not diagnostic in itself; its specificity depends on the presence of the causal organism in hepatocytes in the periphery of the necrotic zones. At present, C. piliforme can only be isolated with difficulty on artificial media, so diagnosis is usually based on demonstration of the large, long bacilli in the cytoplasm of degenerate and also otherwise apparently normal hepatocytes at the periphery of the necrotic zones. The organisms are gram negative and are best delineated with silver impregnation techniques, such as that of Warthin-Starry, but they may be seen with routine stains, such as Giemsa, particularly when the material is fresh. Differentiation from other organisms, including postmortem saprophytes, can be achieved by immunohistochemistry or immunofluorescence. The bacilli tend to lie in sheaves or bundles (Fig. 2-80B). There may also be colitis sufficiently severe to cause diarrhea, but not as severe as that seen in rabbits with this disease. Figure 2-79 Foal liver with multifocal hepatitis in Clostridium piliforme infection (Tyzzer's disease). Figure 2-80 Tyzzer's disease in a foal. A. Foci of necrosis and suppurative inflammation. B. Clostridium piliforme bacilli in bundles in hepatocytes at margin of lesion. Warthin-Starry silver stain. Fewer cases of Tyzzer's disease have been reported in dogs and cats. The liver lesions are essentially the same as those in foals and rodents, and there is also enteritis or enterocolitis. Immunodeficiency predisposes to the disease because it occurs sporadically in dogs that have undergone immunosuppressive or anticancer therapy. Such cases may be complicated by concurrent viral, mycotic, or protozoal infections. Leptospirosis Leptospirosis is a systemic disease characterized by acute jaundice, cholestatic liver injury, and renal failure in dogs, and is discussed in more detail in Vol. 2, Urinary system. The hepatic lesions described following acute experimental infection of dogs with Leptospira kirschneri serovar grippotyphosa include mixed perivascular periportal infiltrates of neutrophils, lymphocytes, and plasma cells, with mild hepatic lipidosis, dissociation of hepatocytes, and intracanalicular bile plugs evident by day 12 postinfection, along with increased hepatocellular mitotic activity. Clinical icterus has been attributed to cholestasis caused by dissociation of hepatocytes (eFig. 2-20). Dogs experimentally infected with Leptospira interrogans serovar pomona developed pulmonary and renal petechial hemorrhages, and friable livers with multifocal 1-2 mm raised white foci corresponding to periportal inflammatory infiltrates of lymphocytes, plasma cells, neutrophils, and macrophages; small foci of hepatic necrosis; and bile plugs in canaliculi, in addition to renal, pulmonary, and cardiac lesions. Organisms were identified by immunohistochemistry at the brush borders of renal proximal convoluted tubules as well as at the luminal surface of bile duct epithelium. In a retrospective study of dogs with supportive clinical signs and microscopic agglutination test titers of ≥320 for one or more serovars tested, including autumnalis, bratislava, canicola, grippotyphosa, icterohaemorrhagiae, and pomona, histologic lesions in the liver were subtle, with sinusoidal neutrophil margination, Kupffer cell hypertrophy, and low levels of hepatocellular single-cell necrosis and mitoses. Some livers had diffuse interstitial lymphocytic hepatitis, with mitotic figures, anisokaryosis, binucleation, and some degree of lobular collapse. Chronic hepatitis has also been experimentally produced by leptospiral infection in dogs; however, clinical cases are rarely documented. eFigure 2-20 Leptospirosis with dissociation of hepatocytes in a dog. Other bacteria A single case of clinical disease associated with Helicobacter canis has been reported in a 2-month-old puppy with peracute disease causing weakness and vomiting before death. Multiple coalescing yellow foci in the liver up to 1.5 cm in diameter consisted of hepatocellular coagulative necrosis with infiltrating mononuclear cells and neutrophils. Spiral bacteria were visualized by Warthin-Starry silver stains in area of necrosis, within bile canaliculi, and occasionally in bile duct lumens. This organism has previously been identified in the blood of diarrheic children, and in the feces of 4% of dogs in an epidemiologic study examining the incidence of Campylobacter-like organisms in 1,000 dogs. Bartonella spp. infection has been associated with a wide variety of granulomatous syndromes and peliosis hepatis in humans, often in association with immunosuppression. Bartonella henselae and B. clarridgeae are associated with self-limiting illness and persistent intravascular infection in cats; mild lymphocytic cholangitis/pericholangitis and lymphocytic hepatitis have been reported with experimental infection of cats with B. henselae. B. henselae has been identified in liver tissue by PCR from a single case of peliosis hepatis in a Golden Retriever and granulomatous hepatitis in a Basset Hound, whereas B. clarridgeae DNA was identified in liver tissue from a Doberman Pinscher with histologic lesions of Doberman hepatitis, however, the extent to which infection induces disease in dogs is currently unknown. Further reading Adamus C, et al. Chronic hepatitis associated with leptospiral infection in vaccinated beagles. J Comp Pathol 1997;117:311-328. Baldwin CJ, et al. Acute tularemia in three domestic cats. J Am Vet Med Assoc 1991;199:1602-1605. Farrar ET, et al. Hepatic abscesses in dogs: 14 cases (1982-1994). J Am Vet Med Assoc 1996;208:243-247. Fox JG, et al. Helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis. J Clin Microbiol 1996;34:2479-2482. Gillespie TN, et al. Detection of Bartonella henselae and Bartonella clarridgeiae DNA in hepatic specimens from two dogs with hepatic disease. J Am Vet Med Assoc 2003;222:47-51. Greenlee JJ, et al. Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa. Am J Vet Res 2004;65:1100-1107. Greenlee JJ, et al. Experimental canine leptospirosis caused by Leptospira interrogans serovars pomona and bratislava. Am J Vet Res 2005;66:1816-1822. Ikegami T, et al. Naturally occurring Tyzzer's disease in a calf. Vet Pathol 1999;36:253-255. Iwanaka M, et al. Tyzzer's disease complicated with distemper in a puppy. J Vet Med Sci 1993;55:337-339. Kordick DL, et al. Clinical and pathologic evaluation of chronic Bartonella henselae or Bartonella clarridgeae infection in cats. J Clin Microbiol 1999;37:1536-1547. Lechtenberg KF, et al. Bacteriologic and histologic studies of hepatic abscesses in cattle. Am J Vet Res 1988;49:58-62. Narayanan S, et al. Fusobacterium necrophorum leukotoxin induces activation and apoptosis of bovine leukocytes. Infect Immun 2002;70:4609-4620. O'Toole D, et al. Tularemia in range sheep: an overlooked syndrome? J Vet Diagn Invest 2008;20:508-513. Paar M, et al. Infection with Bacillus piliformis (Tyzzer's disease) in foals. Schweiz Arch Tierheilkd 1993;135:79-88. Prescott JF, et al. Resurgence of leptospirosis in dogs in Ontario: recent findings. Can Vet J 2002;43:955-961. Rissi DR, Brown CA. Diagnostic features in 10 naturally occurring cases of acute fatal canine leptospirosis. J Vet Diagn Invest 2014;26:799-804. Scanlan CM, Edwards JF. Bacteriologic and pathologic studies of hepatic lesions in sheep. Am J Vet Res 1990;51:363-366. Tadepalli S, et al. Fusobacterium necrophorum: a ruminal bacterium that invades liver to cause abscesses in cattle. Anaerobe 2009;15:36-43. Tan ZL, et al. Fusobacterium necrophorum infections: virulence factors, pathogenic mechanism and control measures. Vet Res Commun 1996;20:113-140. Warner SL, et al. Clinical, pathological, and genetic characterization of Listeria monocytogenes causing sepsis and necrotizing typhlocolitis and hepatitis in a foal. J Vet Diagn Invest 2012;24:581-586. Helminthic infections Various helminths, including cestodes, nematodes, and trematodes, as well as the larval pentastome Linguatula serrata, can produce inflammation of liver and bile ducts. Those parasites that have the biliary system as their final habitat will be discussed in detail later. The others produce hepatic lesions in the course of their natural or accidental migrations, and are discussed under the organ (for most of them, the gut) that is their final habitat. It is useful, however, to describe briefly here the lesions produced by larvae in transit. The initial lesion produced by wandering larvae is traumatic. Sinuous tunnels permeate the parenchyma and often breach the capsule. In the tunnels, there are free red cells, degenerating hepatocytes, and leukocytes, chiefly eosinophils, which react to the parasites. Bordering the tunnel is a narrow zone of coagulative necrosis of parenchyma with neutrophils at its margin. Eosinophils also infiltrate the portal triads. The necrotic parasitic tracts heal by scarification, and the fibroblastic tissue, infiltrated with eosinophils, is eventually incorporated into the portal units. Most larvae escape from the liver, but some eventually become encapsulated in the liver in abscesses containing numerous eosinophils. The abscesses may caseate and come to resemble tubercles, and eventually many are heavily mineralized to form permanent pearly nodules. In sheep, the most common cause of this type of hepatitis (aside from liver fluke) is Cysticercus tenuicollis in its wandering phase. Lambs may die of severe hemorrhagic hepatitis (Fig. 2-81 ) caused by very heavy infections of this parasite, and in pigs, an aberrant host, C. tenuicollis can produce a very intense inflammatory reaction. Figure 2-81 Hemorrhagic subcapsular migration tracks caused by Cysticercus tenuicollis in a lamb. (Courtesy A. Rehmtulla.) In pigs, larvae of Ascaris suum and Stephanurus dentatus produce similar but distinct patterns of focal interstitial hepatitis. The ascarids produce their distinctive accentuation of the stroma (“milk spots”) (Fig. 2-82 ) when quite small larvae are immobilized by the host's inflammatory reaction; thus the foci are relatively small. The fibrotic lesion produced by S. dentatus larvae, on the other hand, is less focal and more in the nature of a track, and there are usually small, inflamed, capsular craters where the larvae have emerged from the liver to migrate to their preferred perirenal site. There will be obvious portal phlebitis at the hepatic hilus when infection by S. dentatus has been by the oral route, and in these livers, the parenchymal lesion is more severe in this vicinity. Figure 2-82 Multifocal interstitial hepatitis (“milk-spot liver”) caused by Ascaris suum migration in a pig. Migration tracks left by larval strongyles can be found under the liver capsule in young horses, and historically, these have been thought to relate to the dense, discrete, capsular fibrous tags and plaques that are almost universally found on the diaphragmatic surface of the liver of mature horses. However, these hepatic fibrotic lesions remain common in horses even after the widespread use of effective anthelmintics, and their precise etiology remains unknown (Fig. 2-83 ). Figure 2-83 Fibrous tags on the diaphragmatic surface of an equine liver. (Courtesy University of Guelph.) Parasites can produce hepatic lesions by other means. The hydatid intermediate stages of Echinococcus encyst in the liver and may destroy much of it; the larvae of Ascaris suum in cattle add to the usual insult by causing portal phlebitis and small areas of infarction; the adults of Ascaris in all species, but especially in pigs, may migrate into the bile ducts; and the eggs of schistosomes enter in the portal blood to lodge in the intrahepatic portal vessels and provoke granulomatous inflammation. Cestodes Stilesia hepatica and Thysanosoma actinioides, the “fringed tapeworm,” are the only cestodes that inhabit the bile ducts. They are parasites of ruminants, Stilesia occurring in Africa, Thysanosoma in North and South America. The life cycles of the parasites are not completely known but may involve oribatid mites as intermediate hosts. T. actinioides may also be found in the pancreatic ducts and small intestine. Usually, the infestations are light but, even when heavy, are not of much significance (eFig. 2-21). Very heavy infestations by S. hepatica occur without signs of illness, although the bile ducts may be nearly occluded, slightly thickened, and dilated. Saccular dilations of the ducts may occur and be filled with worms. The fringed tapeworm is perhaps more pathogenic, and unthriftiness may accompany heavy infestations. eFigure 2-21 Fringed tapeworms Thysanosoma actinioides in the bile ducts of a sheep. (Courtesy A. Reid.) Echinococcus granulosus hydatid cysts occur most commonly in the liver of ruminants in endemic areas (Fig. 2-84 ) but have been reported as incidental postmortem findings in the liver of pigs and horses. The cysticerci and hydatids, which in the intermediate stages invade the liver, are discussed further in Vol. 2, Alimentary system and peritoneum. Figure 2-84 Hydatid liver disease (Echinococcus granulosus infestation) in a sheep. (Courtesy University of Guelph.) Echinococcus multilocularis is found in Europe, parts of northern Asia, and North America, where it has a sylvatic life cycle. Red and arctic foxes are the most important definitive hosts, although coyotes, raccoon-dogs, and wolves can also be infected. In North America, there are few documented cases of intestinal infection by E. multilocularis tapeworms in domestic dogs and even fewer in cats. The adult tapeworm is small, 5 mm, and infective eggs are shed in the feces to be ingested by the intermediate host, which in temperate areas are typically various species of rodents, including voles, lemmings, and deer mice. The hexacanth embryo is released from the egg, travels through the intestinal wall, and migrates to the liver via the hepatic portal circulation. The metacestode stage is an alveolar hydatid cyst, composed of numerous small vesicles lined by a PAS-positive acellular laminated membrane or cuticle layer and a layer of germinal epithelium from which protoscolices develop. Some infections in dogs do not progress to form protoscolices, and instead form irregular large cysts with a PAS-positive laminated membrane (Fig. 2-85 ). Additional exogenous budding results in spread of the metacestodes to other internal locations. The life cycle is completed when the infected intermediate host is consumed by a definitive host, where the protoscolices attach to the intestinal wall and mature. Other species can occasionally act as aberrant intermediate hosts, including pigs, horses, and humans; human alveolar echinococcosis is a serious zoonosis. Dogs can simultaneously act as definitive and intermediate hosts for this parasite. Cases of hepatic alveolar hydatid cysts have been reported in dogs from highly endemic areas in Belgium, Germany, Switzerland, and most recently in Canada (British Columbia, Ontario). Affected dogs display progressive abdominal enlargement, intermittent inappetance, and vomiting, and the cysts may be found in the liver, omentum, abdominal cavity, or lungs. Figure 2-85 Echinococcus multilocularis cysts with acellular laminated membranes in the liver of a dog. PAS stain. (Courtesy A. Brooks.) Nematodes Calodium hepaticum (Capillaria hepatica) is the one nematode that in the adult phase inhabits the liver. It is a slender worm, morphologically resembling the whipworms, and it lives in the parenchyma rather than in the bile ducts. The usual hosts of the adult stage are rodents, but sporadic infestations are observed in dogs. These worms are not highly pathogenic. The adults provoke some traumatic hepatitis, and the eggs, which are deposited in clusters, provoke the development of localized granulomas. The eggs are readily recognized by their ovoid shape and polar caps. The granulomas can be seen through the capsule or in the substance of the liver as yellow streaks or patches. The eggs cannot escape from the liver unless a predator eats them. Predators, however, act only as transport hosts, and the ingested eggs are passed in the feces. Larvae develop in the eggs only in the external environment, and the cycle is completed when a suitable host eats the mature larvae in the eggs. Trematodes Various trematodes (flukes) are parasitic in the livers of animals. They belong to the families Fasciolidae (Fasciola hepatica, F. gigantica, Fascioloides magna), Dicrocoeliidae (Dicrocoelium dendriticum, D. hospes, Platynosomum concinnum), Schistosomatidae (Heterobilharzia americana), and Opisthorchiidae (including Metorchis spp., Opisthorchis felineus, Clonorchis sinensis, Pseudamphistomum truncatum). The diseases produced are known collectively as distomiasis. Fasciola hepatica, the common liver fluke of sheep and cattle, is the most widespread and important of the group. Patent infestations can develop in other wild and domestic animals and in humans. These flukes are leaf shaped and ~2.5 cm long in sheep and slightly larger in cattle. They are found in the bile ducts. Being hermaphroditic, only one fluke is necessary to establish a patent infestation, and each adult may produce 20,000 eggs per day. The longevity of the adult flukes is amazing and is potentially as great as or greater than that of the host; they have been known to survive for 11 years, and it seems that they can produce eggs all this time. The eggs are eliminated in bile, and on pasture, in conditions of suitable warmth and moisture, hatch a larva (miracidium) in ~9 days. If the environmental temperature is low, the incubation period may be delayed for some months. The miracidium can survive only in moisture. It is actively motile and penetrates the tissues of the intermediate host, which is an aquatic snail. Different snails serve this purpose in different countries, but all of them belong to the genus Lymnaea. Each miracidium, on penetrating a snail, develops into a mother sporocyst that reproduces, probably parthenogenetically, giving rise to a small number of the second generation, the redia. Each redia produces either redia or cercariae, or to the 2 successively. Cercariae, the larval stage of the third (sexual) generation, first appear 1-2 months after the miracidium penetrates. Cercariae continue to escape daily for the life of the snail, but even so, total cercarial production is only 500-1,000. They actively escape from the snail and are attracted to green plants, where they encyst and become infective metacercariae in 1 day. These can remain infective for 1 month in summer and up to 3 months in winter. The developmental events from egg to this stage take 1-2 months under favorable conditions. Infestation occurs by ingestion. Excystment occurs in the duodenum. The young flukes penetrate the intestinal wall and cross the peritoneal cavity, attaching here and there to suck blood and penetrate the liver through its capsule; a few no doubt pass in the portal vessels or migrate up the bile duct. They wander in the liver for a month or more before settling down in the bile ducts to mature, which they do in 2-3 months. Some may, by accident, enter the hepatic veins and systemic circulation to lodge in unusual sites; intrauterine infestations are on record. Lesions caused by aberrant flukes are quite common in bovine lung. They consist of resilient nodules just under the pleura of the peripheral parts of the lung. They range in diameter from ~1 to many centimeters and consist of thinly encapsulated abscesses situated at the ends of bronchi. The content is slightly mucoid, unevenly coagulated brown fluid; in some lesions, the reaction is predominantly caseous. The location of the lesion suggests that it begins as a peripheral bronchiectasis that later becomes sealed off. The fluke persists in the debris, but is small and hard to find. The essential lesions produced by F. hepatica occur in the liver and may be described, first, as those produced by the migratory larvae, and second, as those produced by the mature flukes in the bile ducts; the two often intermingle (eFig. 2-22). There is the further incidence of peritonitis, which is produced by the young flukes on their way to the liver and, perhaps also, by some that break out through the capsule. eFigure 2-22 Fasciola hepatica infection in a sheep. (Courtesy J.L. Caswell.) Usually, there is no obvious reaction to the passage of young flukes through the intestinal wall and across the peritoneal cavity, except for small hemorrhagic foci on the peritoneum, where the flukes have been temporarily attached. Few or many parasites may be found in any ascitic fluid and attached to the peritoneum of the diaphragm and the mesenteries. When the infestations are heavy and repeated, such as may be observed in sheep, cattle, and swine, peritonitis occurs. The young flukes at this stage are <1.0 mm long. The peritonitis may be acute and exudative or chronic and proliferative. It is usually concentrated on the hepatic capsule, especially its visceral surface, but may be restricted to the parietal peritoneum or to the visceral peritoneum, including the mesenteries of the gut. In acute cases, there are fibrinohemorrhagic deposits on the serous surfaces, and in chronic cases, there may be fibrous tags, with adhesions or a more or less diffuse thickening by connective tissue. Many young flukes can be found microscopically in the fibrinous deposits, and in the diffuse peritoneal thickenings, there are tortuous migration tunnels containing blood, debris, and the young parasites. In cases with involvement of the visceral peritoneum, young flukes can be found in enlarged mesenteric lymph nodes. The acute lesions in the liver caused by the wandering flukes are basically traumatic, but there is an element of coagulative necrosis, which is possibly related to toxic excretions of the flukes. The migratory pathways are tortuous tunnels that appear on cross-section as hemorrhagic foci 2-3 mm in diameter. If a tunnel is followed, a young fluke <1.0 mm long can be found at the end. When the infestation is heavy, the liver may appear to be permeated by dark hemorrhagic streaks and foci. Older tunnels from which the debris has been cleared may appear as light yellow streaks because of infiltration of eosinophils. Microscopically, fresh tunnels are filled with blood and degenerate hepatocytes and are soon infiltrated by eosinophils. Later, macrophages and giant cells arrange themselves about the debris and remove it, and healing occurs by granulation tissue, which is rich in lymphocytes and eosinophils. In light infestations, the scars may disappear, but in heavy infestations, they may fuse with each other and with portal areas to produce moderate irregular fibrosis. There may, as yet, be no change in the bile ducts. Probably, most of the young flukes reach the bile ducts, but some do not and become encysted in the parenchyma. One or more flukes may be present in each cyst, which consists of a connective-tissue capsule and a dirty brown content of blood, detritus, and excrement from flukes. The cysts ultimately caseate and may mineralize or are obliterated by fibrous tissue. These cysts are most frequent on the visceral surface, where they cause bulging of the capsule. Heavy infestations by immature flukes may cause death at the stage of acute hepatitis. Such an outcome is not common, but occurs in sheep. It is estimated that 10,000 metacercariae ingested over a short period are necessary to produce acute death in sheep. Death may occur suddenly or after a few days of fever, lassitude, inappetance, and abdominal tenderness. This is also the stage of the parasitism in which black disease occurs. Mature flukes are present in the larger bile ducts and cause cholangitis. The relative importance of different factors in their pathogenicity is not known, but they cause mechanical irritation by the action of their suckers and scales, cause obstruction of the ducts with some degree of biliary retention, predispose to bacterial infections, suck blood, and probably produce toxic and irritative metabolic excretions. The biliary changes occur in all lobes but are usually most severe in the left, and the right may be moderately hypertrophied. From the hilus, the bile ducts on the visceral surface stand out as white, firm, branching cords that in extreme instances may be 2 cm in diameter and allow detectable fluctuation over extended segments or in localized areas of ectasia. This dilation of the ducts in sheep, swine, and horses is largely mechanical and is caused by distension by masses of flukes and bile. It is permitted by the relative paucity of new connective-tissue formation in the walls of the ducts in these species; this in turn is probably related to the rather mild catarrhal type of inflammation in the lumina of the ducts. In cattle, desquamative and ulcerative lesions in the large bile ducts are more severe than in other species, and there is a correspondingly greater proliferation of granulation tissue in and about the walls of the ducts (Fig. 2-86 ). The walls of the ducts in cattle are, in consequence, much thickened, and the lumen is irregularly stenotic and dilated and lined largely by granulation tissue. This contributes the typical “pipe stem” appearance to the ducts in cattle; the connective tissue may be, in addition, mineralized, sometimes so heavily that it cannot be cut with a knife. The bile ducts contain dirty dark brown fluid of a mucinous or tough consistency, formed from degenerate floccular bile, pus, desquamated cells and detritus, clumps of flukes, and small masses of eggs in dark brown granular aggregates (eFig. 2-23). Figure 2-86 Thickened, dilated bile ducts, caused by Fasciola hepatica infection in an ox. (Courtesy J.L. Caswell.) eFigure 2-23 Thickened, dilated bile ducts, caused by Fasciola hepatica infection in an ox. (Courtesy J.L. Caswell.) Although the lesions are most obvious in ducts large enough to contain the flukes, there is, with time and severe or repeated infestations, progressive inflammation in the smaller portal units because of direct irritation by the flukes, superimposed infections, and biliary stasis. The course of events is as described earlier for subacute and chronic cholangitis. The proliferating connective tissue and bile ductules in individual portal areas extend to join each other and the scars left over from the migratory phase, so that inflammatory fibrosis may obliterate parenchyma in many foci. In such livers, the left lobe, which is the one most severely affected, may be atrophied, indurated, and irregular. The development of cholangitis of the degree described depends on long-standing or heavy infestations. Lesions of lesser severity, or those less fully developed, are associated with light infestations of short duration. They may then be recognized only by local dilations of the ducts, or even these may not be readily apparent. In such mild infestations, the fact of past or present parasitism may only be suggested by the detection of characteristic black iron-porphyrin pigments, grossly visible in the hilar nodes. It also contributes to the character of the biliary contents. Chronic debility with vague digestive disturbances is common in chronic fascioliasis, and deaths are common among sheep. Clinically and at postmortem, there are, in addition to the essential lesions, more or less severe anemia, moderate anasarca, and cachexia. Jaundice is seldom seen. Fasciola gigantica displaces F. hepatica as the common liver fluke in many parts of Africa and in nearby countries, southeast Asia, and the Hawaiian Islands. It is 2 or 3 times as large as F. hepatica, but its life cycle and pathogenicity are comparable. Fascioloides magna is the large liver fluke of North America; cervids are the definitive hosts. It is a parasite of ruminants and lives in the hepatic parenchyma, not in the bile ducts, although in tolerant hosts (cervids) the cysts in which it localizes communicate with the bile ducts to provide an exit for ova and excrement. The life cycle of this parasite generally parallels that of Fasciola hepatica. The young flukes are very destructive as they wander in the liver. In cattle, they wander briefly, producing large necrotic tunnels before becoming encysted. The cysts, enclosed by connective tissue, do not communicate with bile ducts but form permanent enclosures for the flukes, their excreta, and ova. The cysts, which may be 2-5 cm in diameter, are remarkable for the large deposits of jet black, sooty iron-porphyrin pigment they contain (eFig. 2-24), and except for the flukes and soft contents, they superficially resemble heavily pigmented melanotic tumors. Commonly, these flukes pass from the liver to the lungs of cattle, to produce lesions of similar character. In sheep, this parasite wanders continuously in the liver, producing black, tortuous tracts, which may be 2 cm in diameter, and extensive parenchymal destruction. Even a few flukes may kill a sheep. There is a single case report of natural infection in a horse. eFigure 2-24 Destructive pigmented lesions produced by Fascioloides magna in an ox. (Courtesy University of Guelph.) The dicrocoelid flukes inhabit both biliary and pancreatic ducts. Eurytrema pancreaticum prefers the pancreas (and is described with that organ) but in heavy infestations can be found in the bile ducts. Dicrocoelium and Platynosomum prefer the bile ducts. These are small, narrow flukes, 0.5-1.0 cm long, and may easily be mistaken for small masses of inspissated bile pigment. They are not highly pathogenic, and even in heavy infestations, there may be no signs of the toxemia observed in infestations by F. hepatica. These flukes may occur as mixed infestations. Platynosomum fastosum is a small fluke inhabiting the biliary ducts and gallbladder of cats from southern North America, Central and South America, Malaysia, and the Pacific Islands. The life cycle includes Sublima octona snails as the first intermediate host, and Anolis spp. lizards, marine toads (Bufo marinis), geckos, and various arthropods as second intermediate or paratenic hosts. Cats become infected after ingestion of the second intermediate or paratenic hosts, the cercariae are released into the upper digestive tract, enter the biliary tree, and complete their life cycle. Mature flukes are found in the gallbladder and bile ducts, and after 8-13 weeks, eggs are shed into the feces (Fig. 2-87 ). Although the majority of infections are subclinical, heavy infestations may cause anorexia, vomiting, lethargy, jaundice, and death. Severely affected livers are enlarged, friable, and may be bile stained, with thickened, distended gallbladders, dilated and/or thickened common bile ducts and cystic ducts, and dilated intrahepatic ducts. Histologically, there are various degrees of chronic cholangitis, fibrosing cholangiohepatitis, and cholecystitis, with dilated and hyperplastic ducts surrounded by lymphocytes, plasma cells, and eosinophils, and in some cases neutrophils, with fluke eggs within or adjacent to ducts. Eggs and adults may be difficult to find. Infection has been reported to co-occur with cholangiocarcinoma in some cats. Figure 2-87 Dicrocoelid fluke in the gallbladder of a cat. (Courtesy Purdue University CVM.) Dicrocoelium hospes is found in cattle in countries south of the Sahara. Little is known of it, but it is presumed to be comparable in all respects to the better-known D. dendriticum, which is common in Europe and Asia and sparsely distributed in the Americas and North Africa. Dicrocoelium spp. are found in dry lowland or mountain pastures, whereas Fasciola spp. occur in wetter habitats, so the prevalence of Dicrocoelium is increasing with desertification. This fluke is no more fastidious in its choice of final hosts than many other species of fluke, and, depending on opportunity, it can infest all domestic species, with the possible exception of cats. It is, however, of most importance as a parasite of sheep and cattle, in which it inhabits the bile ducts. Other domestic species and rodents are important as reservoirs. The life cycle of Dicrocoelium dendriticum, the lancet fluke or small liver fluke, differs in some details from that of F. hepatica. The eggs are embryonated when laid and do not hatch until swallowed by one of the many genera of land snails that are the first intermediate hosts. In the snails, the mother sporocyst produces a second generation of daughter sporocysts, which in turn produce cercariae. The cercariae leave the snail in damp weather and are expelled from the snail's lung, clumped together in slime balls. The slime balls are not infective until the cercariae are swallowed by, and encyst in, the ant Formica fusca; other ants may be involved in different countries. The cycle is completed when the definitive hosts swallow the ants. The route of migration of the larvae from the gut to the liver is probably via the bile ducts from the duodenum. The pathologic changes in the liver produced by D. dendriticum are those of cholangiohepatitis that is less severe than that produced by Fasciola hepatica (Fig. 2-88 , eFig. 2-25). The severity and diffuseness of the hepatic lesion are determined by the number of lancet flukes present, and they may be in the thousands. The flukes and their eggs darken the dilated ducts. Even in early infestations, there may be some scarring of the organ at its periphery. In heavy infestations of long standing, there is extensive biliary fibrosis, producing an organ that is indurated, scarred, and lumpy and that at the margins may bear areas that are shrunken and completely sclerotic. The histologic changes are the same as those in fascioliasis, with perhaps more remarkable hyperplasia of the mucous glands of the large ducts (Fig. 2-89 ). Figure 2-88 Dicrocoelium dendriticum, the lancet fluke, in the bile ducts of a sheep. Figure 2-89 Dicrocoelium dendriticum with embryonated eggs in the bile duct of a sheep. eFigure 2-25 Dicrocoelium dendriticum, the lancet fluke, in the bile ducts of a sheep. Heterobilharzia americana, a trematode in the family Schistosomatidae, is the causative agent of canine schistosomiasis in North America, primarily reported from the south Atlantic and Gulf Coast states. Raccoons are the most common definitive hosts, although in addition to dogs, several other diverse mammalian species may become naturally infected, including domestic horses. Eggs shed in feces hatch in water and release motile miracidia, which penetrate the tissues of the intermediate host, lymnaeid freshwater snails. Larval stages multiply in the snail, and free-swimming cercariae are released into freshwater. These penetrate the intact epidermis of the definitive host, migrate to the lung, and finally the liver, where they mature, move to the mesenteric and intrahepatic portal veins to mate, and release eggs that migrate through mesenteric venules, crossing into the bowel lumen by means of proteolytic enzymes. Aberrant migration of eggs results in multifocal eosinophilic and granulomatous inflammation in intestines, pancreas, and liver. Adult trematodes may occasionally be found in hepatic vessels. Clinical signs in affected dogs include diarrhea, vomiting, weight loss, and lethargy, and ~50% of cases in dogs are associated with hypercalcemia, thought to be the result of unregulated calcitriol synthesis by macrophages in the granulomatous lesions. In horses, multiple small fibrosing granulomas scattered throughout the liver, with rare intact or fragmented eggs, are typical and are usually clinically inapparent. The opisthorchid flukes are parasites in the bile ducts of carnivores. They may also occur in swine and humans, and one species, Clonorchis sinensis (Opisthorchis sinensis), is an important human parasite. The life cycles, where known, include mollusks as the first intermediate hosts and freshwater fish as the second. Metorchis conjunctus is the common liver fluke of cats and dogs in North America and is important as a parasite of sled dogs in the Canadian Northwest Territories. The first intermediate host is the snail Amnicola limosa porosa, and the second is the common sucker-fish Catostomus commersonii. The cercariae actively burrow into the musculature of the fish to encyst and become infective. The immature flukes crawl into the bile ducts from the duodenum and mature in ~28 days. Infestations may persist for more than 5 years. Metorchis albidis has been described in a dog from Alaska, Parametorchis complexus in cats in the United States, and Amphimerus pseudofelineus in cats and coyotes in the United States and Panama; the life cycles are not known but are presumed to include fish. Metorchis bilis has been reported in red foxes and occasionally in cats from Germany. Opisthorchis felineus (O. tenuicollis) is the lanceolate fluke of the bile ducts of cats, dogs, and foxes in Europe and Russia. It is particularly common in eastern Europe and Siberia and is more sparse in other areas. Clonorchis sinensis, the Oriental or Chinese liver fluke is an important human pathogen endemic in Japan, Korea, southern China, and southeast Asia; dogs, cats and swine can act as reservoir hosts. There are additional, less well-known, species of Opisthorchis in humans and animals. The first intermediate hosts for the miracidia of O. felineus and C. sinensis are snails of the genus Bithynia, and several genera of cyprinid fishes can act as second intermediate hosts. Pseudamphistomum truncatum occurs in carnivores and humans sporadically in Europe and Asia. Its life cycle is as for Opisthorchis. The opisthorchid flukes, so far as known, resemble Dicrocoelium in migrating up the bile ducts to their habitat. This may be the reason that they are more numerous in the left than in the right lobes of the liver. They can probably live in the liver for as long as the host lives. The pathologic effects are comparable to those of D. dendriticum. Light infestations may be asymptomatic, and heavy infestations may cause jaundice, chronic cholangiohepatitis, and severe biliary fibrosis. Both in humans and animals, adenomatous and carcinomatous changes of the biliary glands have occurred in association with these parasites; the association is probably more than coincidental. Protozoal infections Protozoal hepatitis is due mainly to infection with Toxoplasma (Fig. 2-90 ), Neospora, and Leishmania. Granulomatous hepatitis has been described in dogs associated with systemic infections by Hepatozoon canis. Vascular occlusion by enlarged monocytes bearing Cytauxzoon felis merozoites can be found in the liver of infected cats. Sarcocystis neurona schizonts were reported in the liver of a dog with systemic, multi-organ infection, and schizonts from an unspeciated Sarcocystis were observed in hepatocytes in a horse with suppurative and necrotizing hepatitis. Figure 2-90 Toxoplasma gondii cyst in the liver of a cat. Hepatic coccidiosis causing acute cholangiohepatitis similar to that in mink and rabbits has been observed in isolated cases in the goat, calf, and dog (Fig. 2-91 ). These infections are usually considered aberrant, and often coincide with intestinal coccidiosis. Coccidial meronts and, in some cases, gamonts are present within the cytoplasm of biliary epithelium. The organisms are presently unclassified. Figure 2-91 Hepatic coccidiosis in the bile duct of a pig. Fungal infections Fungal infections of the forestomachs with hematogenous dissemination to the liver occur occasionally in cattle and sheep, usually as a complication of rumenitis. Lesions in the liver are typically hemorrhagic infarcts initially or granulomatous with chronicity, and are associated with infection by Aspergillus fumigatus and various members of the class Zygomycetes (Fig. 2-92 ). Figure 2-92 Fungal hepatitis in a pig. Granulomatous hepatitis associated with disseminated fungal or algal infections has also been reported in dogs and cats. The species involved include Histoplasma capsulatum (eFig. 2-26), Cryptococcus spp., Coccidioides immitis, Sporothrix schenckii, Aspergillus spp., and Prototheca spp. eFigure 2-26 Granulomatous hepatitis caused by infection with Histoplasma capsulatum in a dog. (Courtesy University of Guelph.) Further reading Anderson PJ, et al. Resistance to Fasciola hepatica in cattle. II. Biochemical and morphological observations. J Comp Pathol 1978;88:245-251. Andrade RLFS, et al. Platynosomum fastosum-induced cholangiocarcinomas in cats. Vet Parasitol 2012;190:277-280. Arundel JH, Hamir AN. Fascioloides magna in cattle. Aust Vet J 1982;58:35-36. Chapman BL, et al. Granulomatous hepatitis in dogs: nine cases (1987-1990). J Am Vet Med Assoc 1993;203:680-684. Corapi WV, et al. Multi-organ involvement of Heterobilharzia americana infection in a dog presented for systemic mineralization. J Vet Diagn Invest 2011;23:826-831. Corapi WV, et al. Heterobilharzia americana infection as a cause of hepatic parasitic granulomas in a horse. J Am Vet Med Assoc 2011;239:1117-1122. Corapi WV, et al. Natural Heterobilharzia americana infection in horses in Texas. Vet Pathol 2012;49:552-556. Davis CR, et al. Hepatic sarcocystosis in a horse. J Parasitol 1999;85:965-968. Dubey JP, et al. Sarcocystis neurona schizonts-associated encephalitis, chorioretinitis, and myositis in a two-month-old dog simulating toxoplasmosis, and presence of mature sarcocysts in muscles. Vet Parasitol 2014;202:194-200. Fabrick C, et al. Clinical features and outcome of Heterobilharzia americana infection in dogs. J Vet Intern Med 2010;24:140-144. Goto Y, et al. Frequent isolation of Echinococcus multilocularis from the livers of racehorses slaughtered in Yamagata, Japan. Jpn J Infect Dis 2010;63:449-451. Haney DR, et al. Severe cholestatic liver disease secondary to liver fluke (Platynosomum concinnum) infection in three cats. J Am Anim Hosp Assoc 2006;42:234-237. Hoon-Hanks LL, et al. Hepatic neosporosis in a dog treated for pemphigus folicaceus. J Vet Diagn Invest 2013;25:807-810. Jensen HE, et al. Gastrointestinal aspergillosis and zygomycosis of cattle. Vet Pathol 1994;31:28-36. Martinez-Moreno A, et al. Liver pathology and immune response in experimental Fasciola hepatica infections of goats. Vet Parasitol 1999;82:19-33. McClanahan SL, et al. Natural infection of a horse with Fascioloides magna. J Vet Diagn Invest 2005;17:382-385. Otranto D, Traversa D. Dicrocoeliosis of ruminants: a little known fluke disease. Trends Parasitol 2003;19:12-15. Peregrine AS, et al. Alveolar hydatid disease (Echinococcus multilocularis) in the liver of a Canadian dog in British Columbia, a newly endemic region. Can Vet J 2012;53:870-874. Rezabel GB, et al. Echinococcus granulosus hydatid cysts in the livers of two horses. J Vet Diagn Invest 1993;5:122-125. Rushton B, Murray M. Hepatic pathology of a primary experimental infection of Fasciola hepatica in sheep. J Comp Pathol 1977;87:459-470. Sakui M, et al. Spontaneous Echinococcus multilocularis infection in swine in north-eastern Hokkaido, Japan. Jpn J Parasitol 1984;33:291-294. Schafer KA, et al. Hepatic coccidiosis associated with hepatic necrosis in a goat. Vet Pathol 1995;32:723-727. Taylor D, Perri SF. Experimental infection of cats with the liver fluke Platynosomum concinnum. Am J Vet Res 1977;38:51-54. Watson TG, Croll NA. Clinical changes caused by liver fluke Metorchis conjunctus in cats. Vet Pathol 1981;18:778-785. Wensvoort P, Over HJ. Cellular proliferations of bile ductules and gamma-glutamyl transpeptidase in livers and sera of young cattle following a single infection with Fasciola hepatica. Vet Q 1982;4:161-172. Xavier FG, et al. Cystic liver disease related to high Platynosomum fastosum infection in a domestic cat. J Feline Med Surg 2007;9:51-55. Toxic Hepatic Disease Hepatic susceptibility The liver is particularly vulnerable to toxic injury because it is exposed to virtually everything that is absorbed. The portal vein that drains the stomach and intestines flows directly to the liver. This facilitates high hepatic concentrations of ingested foreign chemicals or drugs termed xenobiotics, as well as many naturally occurring substances with toxic potential. Most xenobiotics are unable to directly enter the hepatocyte and require specific transporters to pass through the lipid bilayer of the hepatocyte membrane. The uptake of exogenous and endogenous compounds from the sinusoidal blood is facilitated by a number of basolaterally located transporters. These transporters are members of a large group of sodium-independent solute transporters known as organic anion transporting polypeptides (OATP). Xenobiotics may be concentrated in hepatocytes to various degrees by mechanisms that remain obscure, but which are associated with special binding proteins of hepatocytes, attachment to enzymatic sites where metabolic conversions occur, and inhibition of export into the bile. The liver is exposed to high concentrations of toxic metabolites because of its role as the primary site of biotransformation for many therapeutic agents and endogenous substances. Some metabolites may produce hepatocellular injury, and others may cause biliary injury once they are transported into the canaliculi. Certain drug metabolites may be reabsorbed in the enterohepatic circulation in a fashion similar to bile acids, facilitating repeated exposure to the drug in question. In addition, many drug administration regimens may achieve relatively high millimolar concentrations, resulting in depletion of conjugating cofactors such as glutathione, which can reach the lower limits required for other cytoprotective roles. Role of hepatic biotransformation in hepatotoxicity An understanding of hepatic biotransformation is essential for an appreciation of the hepatotoxic potential of foreign compounds. Hepatocytes are the major site of metabolism of endogenous substances and xenobiotics, including plant- or fungal-derived secondary metabolites consumed in food, environmental chemicals and drugs. Given the numerous enzymes and the magnitude of their expression, the liver easily exceeds the metabolic capacity of all other organs. A major metabolic function of the liver is to transform lipophilic substances, including endogenous steroid hormones and most xenobiotics, into more water-soluble polar molecules to be excreted in bile or urine. Relevant hepatic enzymes involved in biotransformation and their associated activities can be grouped into 3 major categories. • Phase 1 reactions promote oxidation, reduction, hydrolysis, cyclization, and decyclization of the parent compounds. This is typically accomplished through addition of oxygen or removal of hydrogen, carried out by mixed-function oxidases that are usually CYP enzymes (also known as cytochrome P450 monooxygenases), using NADPH and O2. Most of these phase 1 enzymes are found in the smooth endoplasmic reticulum of the hepatocyte. • Phase 2 reactions are typically conjugation reactions in which a polar molecule, such as glucuronic acid, sulfate, or glutathione, is added to carboxyl, hydroxyl, amino, or sulfhydryl groups on phase 1 metabolites, making them typically less toxic and more water soluble. Most of these enzymes are found in the cytosol. • In phase 3 reactions, conjugated molecules are transported by various transporter molecules across the modified hepatocyte membrane that lines the canaliculus. Phase 1 reactions may either increase (bioactivate) or eliminate the biological activity of the xenobiotic substrate. However, bioactivation of molecules poses a potential risk. Although this step is necessary to prepare the substrate to form a covalent bond with polar compound in phase 2, it creates, if only transiently, reactive intermediates, such as free radicals and epoxides. These reactive intermediates can bind to cellular macromolecules, such as the CYP enzymes, that produced them, other cellular enzymes or structural proteins, or RNA and DNA, leading to hepatocyte injury, death, or neoplastic transformation. The CYPs can be found in all parts of the hepatic lobule, but the hepatocytes of centrilobular region have a higher content than those of the periportal region, which apparently accounts for the centrilobular predominance of injury produced by compounds metabolized by this system, including carbon tetrachloride and acetaminophen. In contrast, hepatocytes in the periportal zone are more susceptible to direct-acting toxicants, such as metal salts, because of their proximity to incoming portal and arterial vascular flow. So-called “drug-drug” interactions can arise when 2 drugs are metabolized by the same CYP, so that metabolism of one or both of the compounds is altered by competition or interference with the relevant CYP enzyme function. Phase 2 reactions inactivate the phase 1 metabolite through conjugation with a polar molecule and facilitate its export by transforming the lipophilic molecule into a water-soluble molecule that can be transported out of the hepatocyte and into the bile or the circulation for removal via the kidney. Reduced glutathione is a major substrate for phase 2 reactions mediated by glutathione S-transferases. Active metabolites of many compounds, including acetaminophen are detoxified by conjugation with glutathione. Depletion of glutathione can greatly enhance the toxicity of many compounds. Hepatic glutathione is also important in the removal of various free radicals and reactive oxygen species generated by normal metabolic processes as well as detoxification pathways through the action of glutathione peroxidase. Different species and different breeds of animals possess a divergent array of phase 1 and phase 2 types with differing levels of activity and target substrates that contribute to the differences in metabolism and toxicity of xenobiotics observed in different species. Acetaminophen toxicity in the cat is a relevant example. Felids have limited phase 2 metabolism caused, in part, from diminished activity of uridine diphosphate (UDP)-glucuronosyl transferase, an enzyme involved in glucuronidation of bioactivated molecules. Limited glucuronidation of acetaminophen metabolites leads to saturation of other detoxification pathways and depletion of glutathione. The lack of glutathione then enables a highly toxic metabolite of acetaminophen, N-acetyl-para-benzoquinoneimine (NAPQI) to bind to cellular proteins and membranes, causing cell injury and death. NAPQI is also responsible for the prominent methemoglobinemia seen in intoxicated cats. Toxic hepatic injury depends on the balance between the production of reactive metabolites and their detoxification by conjugation and other protective mechanisms. The enzyme complex involved in bioactivation is not limited to metabolism of foreign substances, but is also involved in the metabolism or synthesis of a number of lipophilic endogenous substances. The principal endogenous substrates include arachidonic acid, eicosanoids, cholesterol, bile acids, steroids, and vitamin D. Phase 3 reactions involve movement of conjugated substrates across the membrane of the canaliculus. As with the processes involved in uptake of substances from the plasma, several transporters embedded in the canalicular membrane are responsible for the movement of the water-soluble conjugated substrates from the cytoplasm into the lumen of the canaliculus. These transporters have a range of occasionally overlapping substrates, including bile acids and conjugates of glutathione, glucuronate, and sulfonate. These water-soluble metabolites are excreted in the bile via members of the ATP-binding cassette (ABC) superfamily of transport proteins located on the apical canalicular membrane. These include the most significant transporter in humans, the multidrug-resistance protein-1 (MDR1). Multidrug-resistance–associated protein-2 (MRP2) is responsible for export of glutathione conjugates, such as bilirubin conjugates, and shows a striking species difference in expression, with very low expression in the dog and very high expression in the rat. This influences biliary excretion of drug conjugates from the liver and may influence toxicity in the liver or elsewhere. The mechanism by which xenobiotics and some endogenous substances can cause CYP enzyme induction in the liver, as well as induction of phase 2 reactions, and hepatocellular hypertrophy is mediated by the activation of nuclear receptors that function as transcription factors. These transcription factors include aryl hydrocarbon hydroxylase receptor (AHR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator–activated receptor-α (PPAR-α). These nuclear receptors are also important mediators of hepatocellular metabolism, including hepatic lipid metabolism, bile acid homeostasis, as well as liver regeneration, inflammation, fibrosis, cell differentiation, and tumor formation. Each of these receptors may be directly activated by the binding of the xenobiotic (or its metabolite) to the receptor, although some activators of CAR do not bind to the receptor but rather phosphorylate the receptor, which results in nuclear translocation. The nuclear receptor genes vary among species, and there are many species differences and gene polymorphisms that affect the receptors or the genes that they stimulate. In summary, a broad variety of factors can influence the mechanisms and extent of drug-induced liver injury (DILI), the term for hepatic injury from xenobiotics. There is a variety of alternative pathways for metabolism, supported by a variety of isoforms of different gene families, particularly the CYP enzymes and the many genetic polymorphisms within individual alleles that code for the different isoforms. Other factors such as age, nutritional status, sex, diet, prior or concurrent exposure to environmental compounds, or intercurrent disease all influence the response to toxic injury. Some of these variables contribute to the variations seen in responses among species and individuals to liver injury. Role of inflammation in hepatotoxicity The extent of liver injury following exposure to xenobiotics is not, however, determined by the chemistry of drug metabolism alone. Following exposure to injurious compounds, the inflammatory response, particularly the innate immune response, can significantly influence the extent of hepatocellular injury. Studies using acetaminophen have provided most relevant information on this issue; however, the effects of inflammation are complex, and not all studies are in agreement. There are conflicting studies demonstrating either proinflammatory or anti-inflammatory effects of Kupffer cells following drug-induced injury. Some clarification of this controversy has been facilitated by the identification of subsets of intrahepatic macrophages following injury. In addition to the resident Kupffer cells, a population of infiltrating macrophages can be detected quite soon (within 12 hours) after acetaminophen intoxication. There are 2 major phenotypes of intrahepatic macrophages. M1 macrophages are proinflammatory, a source for tumor necrosis factor-α, interleukin-1β (IL-1β) and nitric oxide, which stimulates hepatocellular injury, and these macrophages constitute the majority of the Kupffer cell population. M2 macrophages are anti-inflammatory, secreting IL-10-, IL-6- and IL-18–binding proteins, which modulate inflammation, and comprise the majority of the infiltrating macrophages. Stimulation of other members of the innate immune system, such as natural killer and natural killer T cells can accentuate acetaminophen toxicity. Neutrophils recruited to the liver by the release of damage-associated molecular pattern (DAMP) molecules from necrotic hepatocytes also augment tissue injury. Thus it is clear that altered inflammation can influence drug-induced hepatic injury, but the subtleties remain to be unraveled. Mechanisms of injury There are many mechanisms underlying hepatotoxicity. These include (1) covalent binding of cellular proteins by bioactivated metabolites, which leads to intracellular dysfunction manifested by loss of normal intracellular ionic gradients altering intracellular calcium homeostasis, by actin filament disruption, and by cell membrane damage, such as cell blebbing or swelling and total disruption. A consequence of actin filament disruption can be cholestasis because of the loss of pulsatile contractions that drive canalicular bile flow; (2) drug-induced disruption of canalicular transport pump function, leading to cholestasis and jaundice; (3) inhibition of cellular enzyme pathways of drug metabolism because of covalent binding of CYPs by bioactivated metabolites; (4) covalent binding of the drug to cell proteins, which creates new adducts that serve as immune targets for cytotoxic T-cell attack or antibody formation when transported to the cell surface in vesicles, inciting an immunologic reaction; (5) programmed cell death (apoptosis), occurring through tumor necrosis factor and Fas pathways; and (6) inhibition of mitochondrial function, limiting β-oxidation of fat and ATP generation, leading to accumulation of reactive oxygen species and lipid peroxidation, microvesicular fat accumulation, lactic acidosis, and inability to generate ATP. These are discussed in detail in other texts. The liver is composed of several cell types, not only hepatocytes, and similar types of injury can also affect the biliary epithelium and sinusoidal endothelium, and to a lesser extent, hepatic stellate cells, Kupffer cells, and other immune cells. Classification of hepatotoxins Hepatotoxins can be classified as intrinsic or idiosyncratic, and these categories apply as well to the types of drug-induced hepatotoxicity that are observed in humans and animals. The effects of intrinsic hepatotoxins are considered to be dose related, predictable, and reproducible in experimental animals, and the underlying mechanisms are typically at least partially understood. Intrinsic hepatotoxins can cause liver injury in overdose situations in most normal recipients but are also capable of inducing similar liver damage at lower doses in individuals with genetic or acquired abnormalities in drug metabolism. The majority of intrinsic hepatotoxicants are converted to reactive metabolites, including lipoperoxidative free radicals. The analgesic acetaminophen represents a well-characterized example of an intrinsic hepatotoxin, with species differences in metabolism and associated susceptibility to oxidative and hepatic injury. Dogs and cats can tolerate doses within therapeutic levels; however, higher doses may saturate the glucuronidation and sulfation detoxification pathways, resulting in increased formation of the reactive benzoquinone-imine metabolite NAPQI via a third cytochrome P450–mediated pathway. These can be scavenged by glutathione conjugation; however, massive doses can cause lethal acute hepatic failure, associated with overproduction of reactive metabolites, and depletion of hepatic and erythrocyte glutathione. As discussed previously, clinical toxicity occurs at lower doses and is more severe in cats because they express fewer hepatic isoforms of glucuronyltransferase as part of phase 2 hepatic metabolism and are unable to accelerate excretion of excess metabolites through glucuronide conjugation. This is compounded by a propensity for hemoglobin oxidation, resulting in methemoglobinemia. Toxicity results when reduced glutathione levels drop below a threshold level, resulting in marked oxidative stress, and allowing reactive metabolites to bind covalently to cellular macromolecules. By comparison, idiosyncratic hepatotoxins are typically less dose related (although there is likely a minimum threshold), more unpredictable, and importantly, occur in only a very small proportion of exposed individuals (i.e., <1 in 10,000-100,000). The mechanisms of idiosyncratic hepatotoxicity are generally not known, but reflect an unusual susceptibility of individual recipients to effects that are unrelated to the drug's therapeutic action or overdose toxicity. Idiosyncratic hepatotoxicity is more likely a series of rare drug-related toxicities with different forms of pathogenesis than a single entity. There are 2 main categories of idiosyncratic hepatotoxicity recognized in humans: hypersensitivity-related drug-induced liver injury (drug allergy) and toxic metabolite-dependent drug-induced liver injury. Hypersensitivity-related idiosyncrasies are characterized by a latency period before toxicity is evident and reoccur promptly upon re-exposure. Typically, they involve immune-mediated hypersensitivity responses to the drug metabolites that covalently bind to liver proteins, forming neoantigens that may be recognized as foreign by the immune system, with further liver injury resulting from the ensuing specific immune response or upregulation of components of the innate immune system. In humans, a genetic component to hypersensitivity-related idiosyncratic hepatic toxicity is strongly suspected. Single nucleotide polymorphisms in the human leukocyte antigen (HLA) region or particular HLA haplotypes have been associated with idiosyncratic drug toxicity in humans for several drugs, reflecting the development of pharmacogenomics. Toxic metabolite-dependent idiosyncrasies involve excessive generation of a regular toxic metabolite, or altered metabolism to unusual hepatotoxic metabolites. There is accumulating evidence of considerable genetic diversity (polymorphisms) in hepatic drug metabolism among individual humans and domestic animals, and these may explain many unusual responses to drugs. This variation could be qualitative, with the production of a toxic metabolite not normally produced, or quantitative, with overproduction of a normally minor hepatotoxic metabolite. Morphology of toxic injury to the liver The morphologic forms of hepatic injury produced by hepatotoxins are varied. Acute toxic injury to the liver can be cytotoxic (hepatocellular), cholestatic, or mixed. Cytotoxic hepatocellular injury results in hepatic degeneration, zonal necrosis, focal and nonzonal necrosis (apoptosis), or lipidosis, accompanied by the clinicopathologic features of acute hepatic injury. In general, necrosis produced by intrinsic hepatotoxins is zonal. Acute cytotoxic injury is often manifested as steatosis (lipidosis), a result of impairment of movement of triglycerides through the liver, interference with very-low-density lipoprotein synthesis or transport, or impaired hepatic consumption of fatty acids by mitochondrial oxidation. Cholestatic injury is a reflection of failure of bile excretion associated with biliary epithelial or canalicular injury or other alteration of bile secretion, and displays the features of obstructive jaundice. The typical histologic manifestation consists of bile casts in canalicular spaces, with variable parenchymal injury. An exception to this pattern can be seen in some plant intoxications, such as those produced by steroidal sapogenins and Lantana spp., which can cause cholestasis with minimal evidence of canalicular plugging. Mixed toxic insults display the morphologic and clinical features of both hepatocellular and obstructive injury. Chronic toxic injury to the liver may be manifested as chronic hepatitis, with fibrosis progressing to cirrhosis; vascular injury, such as veno-occlusive disease; and neoplasia. The histologic changes in acute toxic hepatic injury are rather stereotyped. They range from apoptosis, through confluent coagulative and zonal necrosis, to massive hemorrhagic destruction that includes sinusoidal lining cells (Fig. 2-93 ). The histology of these acute intoxications is often characterized by centrilobular necrosis, usually coagulative (Fig. 2-94 ). Rarely, the pattern of necrosis may be periportal or midzonal. Depending on the nutritional status of the animal, there may be variably severe fatty or hydropic change in hepatocytes adjacent to the necrotic zones. The necrotic cells may accumulate calcium. Variations on the general process of hepatocellular necrosis have little diagnostic or pathogenetic specificity. In sublethally injured cells, there may be prominent but nonspecific clumping of smooth endoplasmic reticulum, particularly in the centrilobular zones. Figure 2-93 Massive necrosis following ingestion of Amanita phalloides by a cat. Figure 2-94 Acute centrilobular hepatic necrosis following ingestion of Cycad sp. in a dog. (Courtesy J. Cooley.) The clinical and gross characteristics of fatal acute intoxications that destroy liver parenchyma are rather consistent, regardless of the origin of the toxin. The animal dies after a brief period of dullness, anorexia, colic, and various neurologic disturbances, including convulsions; these are attributed to hepatic encephalopathy. Postmortem examination reveals a slight excess of clear, yellow abdominal fluid, which contains sufficient fibrinogen to form a loose, nonadherent clot. The appearance of the liver depends on the severity and stage of the injury. Severe acute toxicity that destroys endothelium typically results in a hemorrhagic zonal pattern, in which case the liver may be deep red-purple and obviously swollen and turgid. In less severe injury without hemorrhage, the liver tends to be lighter brown because of a combination of edema (exclusion of sinusoidal blood), destruction of cytochrome pigments, and accumulation of bile pigments and/or fat. If the animal survives for several days, the liver develops a typical zonal yellow fatty change as triglyceride accumulates in sublethally injured hepatocytes. In acute fatal hepatotoxicities, there may be widespread hemorrhage resulting from lack of coagulation factors. Petechiae and ecchymoses are seen most consistently on serous membranes, especially on the epicardium and endocardium and abdominal viscera. Diffuse hemorrhage into the gut, particularly the duodenum in ruminants, is also common, as are hemorrhages into the wall of the gallbladder. Hemorrhages are largely the result of excessive consumption of clotting factors and platelets within the areas of necrosis in the liver, although the concurrent failure of the damaged liver to replace those coagulation factors undoubtedly becomes important when these factors are consumed. Gross lesions, such as icterus, and photosensitization, which reflect failure of biotransformation or excretion of endogenous materials, develop too slowly to be a feature of acutely fatal hepatotoxicities. However, the rate of accumulation of bilirubin increases when hemorrhage occurs in the necrotic liver or as a consequence of coagulopathy. Chronic hepatotoxic injury may manifest in many patterns, as previously described. These include areas of necrosis with primarily mononuclear inflammatory infiltrates, steatosis, cirrhosis, atrophy with nodules, hepatic vein thrombosis, veno-occlusive disease, peliosis hepatis, cholangitis, ductular reaction (biliary hyperplasia), and carcinogenesis. In contrast to the acute intoxications, chronic hepatotoxicities are more likely to display a mix of these responses, and this can provide more diagnostic specificity. For example, agents that impair hepatocellular regeneration might not be potent necrogens but tend to produce hepatic apoptosis and atrophy, fibrosis of various patterns, compensatory bile duct hyperplasia, nodular regeneration, some degree of cholestasis, and frequently megalocytosis (polyploidy). Such hepatotoxins are potential carcinogens because they favor the selective growth of hepatocellular nodules that are resistant to mitoinhibitory effects. Clinical signs of chronic hepatotoxicity are usually problems resulting from inadequate detoxification and excretion; these include jaundice, photosensitization, and hepatic encephalopathy. Most of the toxins responsible for chronic hepatotoxicity may produce acute nonspecific zonal or massive necrosis if experimentally administered at dose rates higher than those to which animals are likely to be exposed in the field, although such acute toxicity is only occasionally observed in field cases of the diseases. Toxic agents The range of substances that can cause hepatotoxicity is so broad that it encompasses virtually all categories of natural and synthetic chemicals. These include metals (iron, copper), drugs (e.g., acetaminophen), plant components (phytotoxins), fungal metabolites (mycotoxins), bacterial products (e.g., cyanobacterial microcystin-LR), and various industrial products (especially aromatic solvents). Many drugs are also hepatotoxic. Differences in individual susceptibility to hepatotoxic responses likely occur for all classes of chemicals that are metabolized in the liver. In the following sections, toxic hepatic disease will be separated into drug- or pharmaceutical-induced hepatotoxicity (so-called adverse drug reactions), and hepatotoxicity associated with exposure to plant or environmental toxins, including metals. The latter have been somewhat arbitrarily divided into acute and chronic, and although it is recognized that the difference between acute and chronic hepatotoxicity is often simply a matter of dose rate, it is convenient to categorize the sources according to the syndrome of liver damage that they most commonly produce. Various plants and moldy feeds are hepatotoxic, and some phytotoxins (e.g., pyrrolizidine alkaloids) and mycotoxins (e.g., aflatoxins) are notorious hepatotoxins and hepatocarcinogens. Many phytotoxins and mycotoxins target other organ systems or have physiologic rather than pathologic effects. Some of those that cause diagnostic lesions are discussed under the respective target tissues elsewhere. Generally, these areas of toxicology are better accessed in comprehensive references on phytotoxins or mycotoxins. Adverse drug reactions: drug-induced liver injury (DILI) The definition of an adverse drug reaction is any injurious or unintended response to a drug that occurs at a normal dose for normal use. DILI is the most common form of adverse drug reaction in humans, and although hepatotoxic drug reactions are recognized in dogs and cats, the true prevalence of DILI in domestic animals is unknown. DILI should not be considered as a single disease, given the diverse array of drugs that can trigger injury. Acute hepatic disease has been attributed to adverse reactions to a wide variety of therapeutic drugs, particularly in companion animals. Submassive to massive hepatic necrosis and cholestatic hepatitis have been reported in dogs as an idiosyncratic reaction to trimethoprim-sulfonamide combination therapy and the related sulfonamide-based anticonvulsant drug zonisamide, although zonisamide is in a different chemical category, is less likely to form toxic adducts, and is not likely to share a similar pathogenesis of liver injury. Severe lobular to massive hepatic necrosis may occur in cats associated with repeated oral administration of diazepam at recommended doses. Severe centrilobular hepatic necrosis has been associated with use of the anthelmintic mebendazole in dogs. Hepatotoxicity has been reported in 4 dogs treated with amiodarone, a class III antiarrhythmic agent. This is one of the more commonly reported adverse effects of amiodarone in humans and is related to the drug's effect on lipid metabolism. Administration of the anabolic steroid stanozolol has also been associated with elevated alanine aminotransferase, coagulopathy, and development of hepatic lipidosis with cholestasis in cats. Adult beef cattle testing positive for stanozolol in urine also had hepatic changes, including cholestasis, periportal fibrosis and inflammation, and focal necrosis, although the changes could not conclusively be attributed to anabolic steroid administration. Other pharmacologic agents associated with acute hepatic injury include thiacetarsemide, methoxyflurane, halothane, oil of pennyroyal, intravenous injection of manganese chloride and inadvertent subcutaneous injection of intranasal Bordetella bronchiseptica/canine parainfluenza vaccine in dogs, methimazole, glipizide, and the photodynamic therapy agent aluminum phthalocyanine tetrasulfonate in cats. Xylitol is an artificial sweetener used commonly in baked goods or chewing gum intended for use by diabetics or dieters. Although high doses can be tolerated by most species, there is a particular sensitivity in dogs. Dogs that ingest >0.5 g/kg can be at risk to develop acute severe hepatic necrosis. Dogs exposed to fatal doses have centrilobular to massive lytic necrosis of hepatocytes and widespread hemorrhage. Affected dogs also develop hypoglycemia and hyperinsulinemia. There may be an element of idiosyncratic toxicity involved as dogs have tolerated higher doses in experimental settings, so there may be breed-related or other factors influencing acute toxicity. The mechanism of toxicity is not currently known, but intracellular ATP depletion and generation of reactive oxygen species have been suggested as possible mechanisms for hepatocyte injury. Carprofen is a nonsteroidal anti-inflammatory drug used in the treatment of degenerative joint disease and management of acute pain in dogs primarily. Diffuse and massive hepatocellular injury, characterized by hepatocellular vacuolar change, lytic necrosis, apoptosis, and bridging necrosis, with mild secondary inflammation and cholestasis, has been reported in dogs administered therapeutic dosages of carprofen. The injurious response is likely to be idiosyncratic as a chronic experimental study did not produce injury. A related nonsteroidal anti-inflammatory drug, diclofenac, causes idiosyncratic liver injury in humans. The anticonvulsant drugs primidone, phenytoin, and phenobarbital have been associated with the development of chronic hepatic disease and cirrhosis in dogs. These drugs, used either alone or in combination, can cause biochemical and clinical signs of hepatic dysfunction in up to 14% of dogs treated for >6 months, but only a small percentage of cases progress to cirrhosis and hepatic failure. Currently, monitoring blood levels and adjusting dosages of these drugs to a nontoxic level has significantly reduced the incidence of liver injury. The most consistent histologic finding in the majority of dogs treated with phenobarbital is proliferation of the hepatocellular smooth endoplasmic reticulum resulting from induction of microsomal enzymes, including various subfamilies of cytochrome P450. This results in hepatocellular swelling with fine diffuse granularity, a so-called “ground-glass” appearance to hepatocyte cytoplasm. This histologic change is an adaptive response, reflected clinically by increases in serum concentrations of alkaline phosphatase, alanine aminotransferase, and γ-glutamyl transpeptidase, but is not indicative of hepatocellular injury. Actual hepatotoxicity may represent an idiosyncratic reaction in a small percentage of treated dogs, although the possibility of dose-dependent intrinsic hepatotoxicity with long-term treatment has not been ruled out. It is also possible that the enzyme induction associated with anticonvulsant therapy may alter the ability of the liver to detoxify other nonspecified compounds that could be the effectors of liver damage. Certainly, enzyme induction may alter the pharmacokinetics of other co-administered drugs. Chronic hepatitis associated with anticonvulsant therapy is characterized by bridging portal fibrosis, biliary hyperplasia, nodular regeneration, and mild inflammatory cell infiltrates. A separate syndrome of cholestatic hepatotoxic injury with jaundice has also been described in dogs receiving high doses of phenytoin in combination with primidone or phenobarbital. This is characterized by intrahepatic cholestasis, with hepatocellular swelling, vacuolation, and small multifocal areas of hepatocellular necrosis, and has been suggested to represent a metabolic disturbance rather than direct cytotoxic hepatocellular injury. Hepatocutaneous syndrome (superficial necrolytic dermatitis) associated with typical hepatic pathology of parenchymal collapse, vacuolation, and nodular regeneration has also been reported as a separate syndrome in dogs with a history of chronic phenobarbital therapy. Acute and chronic hepatic disease, characterized by periportal hepatitis, periportal fibrosis, and biliary hyperplasia, have been reported in dogs treated with oxibendazole-diethylcarbamazine combination therapy for prevention of hookworm and heartworm. Chronic hepatic disease has also been reported following administration of mibolerone, methotrexate and CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) in dogs, ketoconazole in dogs and cats, and megestrol acetate and griseofulvin in cats. Hepatotoxic plants Hepatotoxic plants occur in botanical families as diverse as the relatively primitive Cycadaceae through the Compositae and Solanaceae. The evolutionary relationships between herbivores and toxic plants are complex; in some situations, consumption of plants by herbivores has competitive or neutral advantage for the plant, especially those that are lush and prolific, so toxicity is counterproductive or unnecessary. However, in arid or semiarid habitats, plants must put up more resistance to herbivores that could obliterate them. Indigenous herbivores are typically reluctant to do this, and are either resistant or reluctant to graze some plants. Plants sometimes protect critical parts at particular stages of growth. For example, Xanthium pungens (Noogoora burr) concentrates its toxin in the cotyledons. The seeds are rarely eaten, even by cattle, but intoxications can occur when this plant is eaten shortly after germination. Phytotoxic liver disease is therefore more frequently encountered in animals grazing pastures at particular times of the year, with limited choice or supply, or when hungry animals are introduced to plants for which their natural or induced resistance is low. The patterns of liver disease resulting from consumption of toxic plants are for the most part quite consistent in comparison with what is seen with exposure to other classes of hepatotoxins. An exhaustive review of the toxicity of all such plants would be repetitive. However, some plants that do produce more distinctive patterns of liver lesions will be discussed in more detail. Further reading Adams DH, et al. Mechanisms of immune-mediated liver injury. Toxicol Sci 2010;115:307-321. Bunch SE. Hepatotoxicity associated with pharmacologic agents in dogs and cats. Vet Clin North Am Small Anim Pract 1993;23:659-670. Center SA, et al. Fulminant hepatic failure associated with oral administration of diazepam in 11 cats. J Am Vet Med Assoc 1996;209:618-625. Court MH, Greenblatt DJ. Molecular genetic basis for deficient acetaminophen glucuronidation by cats: UGT1A6 is a pseudogene, and evidence for reduced diversity of expressed hepatic UGT1A isoforms. Pharmacogenetics 2000;10:355-369. Dayrell-Hart B, et al. Hepatotoxicity of phenobarbital in dogs: 18 cases (1985-1989). J Am Vet Med Assoc 1991;199:1060-1066. Dunayer EK, Gwaltney-Brant SM. Acute hepatic failure and coagulopathy associated with xylitol ingestion in eight dogs. J Am Vet Med Assoc 2006;229:1113-1117. Harkin KR, et al. Hepatotoxicity of stanozolol in cats. J Am Vet Med Assoc 2000;217:681-684. Holt MP, et al. Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury. J Leukoc Biol 2008;84:1410-1421. Jacobs G, et al. Hepatopathy in 4 dogs treated with amiodarone. J Vet Intern Med 2000;14:96-99. Kaplowitz N, DeLeve LD. Drug-Induced Liver Disease. 3rd ed. New York: Academic Press; 2013. Khan KN, et al. Toxicity of subacute intravenous manganese chloride administration in beagle dogs. Toxicol Pathol 1997;25:344-350. Kristal O, et al. Hepatotoxicity associated with CCNU (lomustine) chemotherapy in dogs. J Vet Intern Med 2004;18:75-80. Kumar V. NKT-cell subsets: Promoters and protectors in inflammatory liver disease. J Hepatol 2013;59:618-620. Leach MW, Peaston AE. Adverse drug reaction attributable to aluminum phthalocyanine tetrasulphonate administration in domestic cats. Vet Pathol 1994;31:283-287. MacPhail CM, et al. Hepatocellular toxicosis associated with administration of carprofen in 21 dogs. J Am Vet Med Assoc 1998;212:1895-1901. March PA, et al. Superficial necrolytic dermatitis in 11 dogs with a history of phenobarbital administration (1995-2002). J Vet Intern Med 2004;18:65-74. Marques PE, et al. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology 2012;56:1971-1982. Miller ML, et al. Apparent acute idiosyncratic hepatic necrosis associated with zonisamide administration in a dog. J Vet Intern Med 2011;25:1156-1160. Navarro VJ, Senior JR. Drug-related hepatotoxicity. N Engl J Med 2006;354:731-739. Polzin DJ, et al. Acute hepatic necrosis associated with the administration of mebendazole to dogs. J Am Vet Med Assoc 1981;179:1013-1016. Raekallio MR, et al. Evaluation of adverse effects of long-term orally administered carprofen in dogs. J Am Vet Med Assoc 2006;228:876-880. Sudekum M, et al. Pennyroyal oil toxicosis in a dog. J Am Vet Med Assoc 1992;200:817-818. Toshach K, et al. Hepatocellular necrosis associated with the subcutaneous injection of an intranasal Bordetella bronchiseptica-canine parainfluenza vaccine. J Am Anim Hosp Assoc 1997;33:126-128. Twedt DC, et al. Association of hepatic necrosis with trimethoprim sulfonamide administration in 4 dogs. J Vet Intern Med 1997;11:20-23. Wagner M, et al. Nuclear receptors in liver disease. Hepatol 2011;53:1023-1034. Acute hepatotoxicity: plant-derived and environmental toxins Cyanobacteria (blue-green algae) These primitive highly toxic microorganisms can flourish as a seasonal bloom on lakes and ponds that have accumulated phosphates and nitrates as runoff from fertilized soils. Outbreaks of poisoning are not common, but they occur in many countries and may be responsible for heavy mortality among mammals or birds that drink from affected bodies of water. Most well-documented cases have involved Microcystis aeruginosa, which may poison stock after the bloom has been piled by wind against the shores of expanses of water. Other toxic species are included in the genera Anabaena and Aphanizomenon. Some deaths are too sudden to be caused by liver damage and are probably the result of the “fast-death factor” that has been found in some blooms. The syndrome is one of collapse and prostration, with hyperesthesia that may be manifested as convulsions and death within a few minutes; there are no specific lesions in this form of the toxicosis. The most well-understood cyanobacterial hepatotoxin is microcystin-LR, a highly potent cyclic heptapeptide protein phosphatase inhibitor. Other species of cyanobacteria, such as Nodularia spumigenia found in New Zealand and the Baltic region, contain different, but still potent toxins such as nodularin. Ruminants are most commonly poisoned, but poisoning has been reported in horses, sheep, dogs, and domestic poultry. The cyanobacterial toxin is released when the microorganisms disintegrate, which may occur spontaneously in bodies of water or after the application of copper sulfate for algae control, or in the rumen or stomach after ingestion. Microcystin-LR is very stable and persists in water at typical ambient conditions, with a 10-week half-life. It is stable following boiling as well. The toxin is taken into hepatocytes by organic anion-transporting polypeptide (OATP) membrane transporters and causes cell damage by inhibiting cytoplasmic protein phosphatase 1 and 2A. This leads to disorganization of hepatocyte and endothelial cytoskeletal actin filaments, and disruption of their shape and integrity, leading to necrosis, apoptosis, and perisinusoidal hemorrhage. The distribution of the necrosis is usually centrilobular to massive. The pattern may vary within the individual liver and from case to case. In subacute intoxications, the liver is severely fatty, and necrosis is limited to individual hepatocytes or small groups, rather than being zonal. Phagolysosomes and bile pigments accumulate in the cytoplasm, and there is slight biliary proliferation and fibrosis. Necrosis of the renal tubules can also occur. Diagnosis can be made by detection of microcystin in vomitus or liver. Toxic fungi Amanitins are potent hepatotoxins found in several mushroom genera, including Amanita, Galerina, and Lepiota. The genus Amanita contains several species, including A. phalloides, A. verna, A. virosa, and A. ocreata, which are considered extremely toxic. These mushrooms are mycorrhizal with various species of deciduous and coniferous trees, and may be found in urban, suburban, and rural areas. The amanitins are bicyclic octapeptides, ingestion of which is responsible for gastroenteritis, hypoglycemia, and fulminant liver failure in humans, dogs, cats, cattle, and other animals. They are stable compounds and persist in the acid environment of the stomach and following cooking. Amanitin is transported from the systemic circulation to hepatocytes by a specific OATP transporter on the hepatocyte surface. Toxicity is accentuated by enterohepatic circulation of the toxin, causing repeated hepatic exposure. Amanitin inhibits nuclear RNA polymerase II, thus interfering with transcription and inhibiting protein synthesis, resulting in cell death. Dogs or cats dying after ingestion of Amanita spp. have massive hepatocellular necrosis with focal areas of hemorrhage (see Fig. 2-93). Surviving periportal hepatocytes have vesicular nuclei with loss or fragmentation of nucleoli, consistent with ultrastructural reports of chromatin condensation, dissolution of the nucleolus, and decline in nucleolar RNA content. Necrosis of the proximal convoluted tubules often accompanies the hepatic injury and can aid in the index of suspicion for amanitin intoxication. Because the histologic lesions are nonspecific, and previously, it was difficult to obtain a definitive diagnosis of amanitin intoxication, under-diagnosis was likely. Confirmatory testing is available, and the liver, bile, and kidney are the preferred organs for testing. Cycadales Members of this order have been responsible for chronic hepatotoxicity and neurotoxicity in cattle. Acute hepatotoxicity has been reported in cattle and sheep that have eaten the seeds or young leaves of species of Cycas or Zamiaceae. Dogs are intoxicated by eating the seeds of Cycas spp. usually raised as ornamental plants in warmer climates. The toxin responsible is methylazoxymethanol, which is the aglycone of various nontoxic glycosides, including cycasin and macrozamin, in these plants. The toxin is split from the glycoside by bacterial metabolism in the gut, and its hepatotoxicity is the result of further metabolism by hepatic CYPs; the pattern of necrosis is thus centrilobular (see Fig. 2-94). The metabolites of the aglycone are apparently potent alkylating agents, and the chronic liver lesions reflect this; there is megalocytosis (which is not as persistent as that of pyrrolizidine alkaloid poisoning), nuclear hyperchromasia, cholestasis, fatty change, and various degrees of diffuse fibrosis. There is fairly consistent acute renal tubular injury. Chronic exposure leads to cancer development in the liver, kidney, and intestinal tract of laboratory rodents. Cycads also produce a neurotoxic amino acid, β-N-methylamino-L-alanine (BMAA). BMAA is excitotoxic, activating neurotransmission mediated by glutamate receptors, which in excess can lead to cytotoxic influx of calcium ions. Chronic cycad poisoning of cattle causes a chronic nervous disorder characterized by a progressive proprioceptive deficit. This is the result of axonopathy in upper spinocerebellar and lower corticospinal tracts. The axonopathy, morphologically subtle at first, may eventually progress to frank Wallerian degeneration. Solanaceae Toxic species of the genus Cestrum (jessamine) include Cestrum diurnum, a cause of enzootic calcinosis in cattle attributed to active vitamin D analogs; the other known toxic species all produce similar hepatic disease. Speciation within the genus is uncertain, partly because of hybridization; however, the species named as hepatotoxic are C. parqui, C. laevigatum, and C. aurantiacum. Cestrum spp. cause acute hepatotoxicity in the field in South America, southern and central Africa, and Australia. Cattle are more frequently poisoned than other species, but sheep and goats are susceptible, and fowl may be if they eat the fruit. The young leaves and unripened berries are the most toxic parts of the plant. In acute hepatoxicity, there is marked centrilobular and midzonal coagulative necrosis and hemorrhage. There are no records of chronic liver disease caused by this plant, and photosensitization is rarely seen. The toxin is water soluble and has been identified as an atractyloside. Compositae/Asteraceae Xanthium pungens (Noogoora burr) in Australia and Xanthium strumarium (rough cocklebur) in the United States, as well as X. cavanillesii, (the cocklebur) in Brazil and South Africa, have been reported to be hepatotoxic while in the seedling stage because the toxins are concentrated in the cotyledons. The burrs are also toxic, and although usually too coarse to be grazed, they can be consumed if ground into feeds. Cattle, swine, and sheep are susceptible, and toxicosis typically occurs following a period of feed scarcity, after flooding or rain has allowed germination. The clinical signs and lesions are not specific, being those described for acute hepatotoxins in general. The toxic principle is a diterpenoid glycoside, carboxyatractyloside, although there may be other closely related toxic glycosides in some plants. Wedelia glauca also contains atractyloside and causes acute hepatotoxicity in cattle and sheep in Uruguay and Argentina. The condition has been reproduced experimentally in sheep and cattle and rats. Atractyloside toxins inhibit exchange of ATP from the mitochondria with adenosine diphosphate (ADP) in the cytosol, a process essential for oxidative phosphorylation. Atractylosides inhibit the ADP/ATP carriers (AACs), including the form expressed in the liver. Carboxyatractyloside blocks exchange by binding to a cationic functional domain of bovine AAC1. Lack of ATP and mitochondrial pore leakage lead to apoptosis and necrosis, with ion pump failure, lipid peroxidation, and glutathione depletion. The extent of necrosis depends on dosage. In lethal poisoning with hepatic failure, there is typically midzonal vacuolation and centrilobular necrosis, but it can be panlobular. Other plants produce similar lesions, but the toxins have not been identified. Helichrysum blandowskianum is hepatotoxic to cattle and sheep in southern Australia and has caused sudden deaths with centrilobular necrosis. The condition has been reproduced experimentally. High mortality with acute centrilobular liver necrosis has been reported in cattle grazing sprouting plants of Vernonia rubricaulis in Brazil. Similar lesions were reproduced with 3 g/kg of sprouting plants. Various species of Asteraceae in South Africa, including Asaemia axillaris, Athanasia trifurcata, Lasiospermum bipinnatum, Hertia pallens, and Pteronia pallens, have been associated with field outbreaks of acute hepatotoxicity in grazing sheep or cattle. Similar liver lesions have been reproduced experimentally. Experimental intoxications by all of these species have, in some animals, produced centrilobular as well as midzonal necrosis. Asaemia axillaris, Athanasia trifurcata, and Lasiospermum bipinnatum are also associated with more chronic liver toxicity with photosensitivity. Ulmaceae Trema tomentosa (T. aspera), the poison peach, has caused severe losses of cattle in Australia. Similar disease has been reported in goats and horses ingesting Trema micrantha in Brazil, and hepatotoxicity has been reproduced in rabbits. The syndrome is acute, there is no photosensitization, and mildly intoxicated animals may recover completely. The toxic principle is a glycoside, designated trematoxin. The pattern of necrosis is consistently centrilobular and is identical in appearance to that of Cestrum and Xanthium poisoning. The gross and microscopic pathology of experimental poisoning by Trema, Xanthium pungens, and Cestrum parqui has been shown to be identical in all morphologic respects in the same group of sheep. Myoporaceae Hepatotoxic species of Myoporaceae so far incriminated are Myoporum deserti, M. acuminatum, M. insulare, and M. tetrandum of Australia, and M. laetum in New Zealand, southern Brazil, and Uruguay. The toxic oils are contained in the leaves and branchlets, but within the species, there is variation in the chemical characters and toxicity of the oils; not all strains of toxic species are toxic. There is some delay between ingestion and absorption of the furanosesquiterpenoid oils, the best known of which is ngaione, which are responsible for intoxication. Twenty-four to 48 hours may elapse before signs of toxicity appear. Gross lesions include widespread hemorrhage and a pale yellow liver. Histologically, the pattern is unusual, as the injury to the hepatocytes occurs in periportal or midzonal regions accompanied by biliary hyperplasia (Fig. 2-95A, B ). Some animals live long enough to become photosensitized; others may die much more rapidly, with pulmonary edema. The edema appears to be a direct effect of the toxin after its metabolism by the club cells (formerly Clara cells) of the airway. Figure 2-95 Midzonal necrosis caused by ingestion of Myoporum sp. in a donkey. A. Low-power view. B. Higher magnification. (Courtesy R. Kelly.) Livers from intoxicated sheep may show a striking, broad pattern of variable congestion and even infarction, which is superimposed on the more regular lobular pattern of periportal necrosis. Sections of these livers reveal acute fibrinoid necrosis of portal vessels, which suggests that the coarser lesions may have a vascular basis. Sawfly larvae Sawfly larval poisoning is an acute hepatotoxicosis documented in cattle and to a lesser extent sheep and goats in various locations, including Australia, Denmark, and South America. Ingestion of the larval stage of the “sawfly,” 1 of 3 insect species that are members of the order Hymenoptera and the suborder Symphyta, produce hepatic injury. In Australia, the insect species is Lophyrotoma interrupta (Pergidae) or L. zonalis (Pergidae); in Denmark the insect is Arge pullata (Argidae), and in South America a third species, Perreyia flavipes (Pergidae), is incriminated. In parts of northeastern Australia, the larvae are parasitic on the leaves of the tree Eucalyptus melanophloia, and heavy infestations may occur. L. zonalis has been introduced to Florida to control the spread of Melaleuca quinquenervi. In Denmark, sawfly larvae feed on birch trees. There is a short clinical course following ingestion, and often, affected cattle are found dead. Animals that survive typically develop icterus and secondary photosensitization. At postmortem examination, ascites, petechiae, and ecchymoses are evident. The liver is enlarged with an accentuated lobular pattern. Mural edema of the gallbladder may be apparent. Sawfly larvae can be found in the rumen and, on occasion, through to the abomasum. Histologically, there is centrilobular to massive acute hepatic necrosis. Outbreaks tend to be seasonal, related to the life cycle of the insects. The toxic principle is believed to be D-amino acid–containing peptides. The major toxin is an octapeptide, lophyrotomin, present in the in the larvae of Australian and Danish sawflies. There is a different toxin in the South American sawflies, identified as the heptadecapeptide pergidin. Halogenated hydrocarbons Various halogenated hydrocarbons, such as carbon tetrachloride (used historically as a fasciolicide), bromobenzene, hexachloroethane, tetrachloroethylene, and chloroform, are activated by cytochromes P450 to hepatotoxic or nephrotoxic metabolites. They have similar hepatotoxic properties, but these agents are little used now, so few animals encounter them in toxic doses. However, some of these chemicals have been used experimentally to investigate the mechanisms of hepatotoxicity, so they warrant brief consideration here. Carbon tetrachloride is metabolized to trihalomethane, a necrogenic free-radical metabolite, and reactive oxygen radicals are concurrently generated. Centrilobular hepatic necrosis with midzonal hydropic change caused by membrane peroxidation occurs within 30 hours of experimental exposures, but lethal toxicity occurs later, when steatosis and the early stages of tissue repair responses are manifest. Phosphorus White phosphorus has historically been used for vermin control, and has been implicated in the accidental exposure and deaths of wild waterfowl. It may be present in some incendiary devices such as fireworks. As a rodenticide, it is mixed with fat to promote absorption, and much of the dose is transported to the liver shortly after ingestion. A small amount may be lost by vomition, as elemental phosphorus is directly irritating to the gastrointestinal tract. The mechanism of phosphorus hepatotoxicity is uncertain. It is apparent that metabolism to a toxic intermediate is not necessary, but there is some dispute on the involvement of lipoperoxidation in the hepatocellular injury. There is evidence that protein synthesis is impaired early and that this is responsible for the lipid accumulation that is a prominent feature. A few hours after ingestion of phosphorus, there is severe colic and vomition. If the animal survives this acute phase, there may be apparent recovery for a few days, followed by jaundice and other signs of liver failure, and death by about the fifth day. At postmortem, there is severe icterus and fatty liver, the latter sometimes being predominantly periportal in distribution. Hepatocellular necrosis is not often a prominent feature histologically, notwithstanding the evidence of liver failure. Fatty change is also seen in the myocardium and distal nephrons. Iron Iron-dextran complexes have been widely used in the prevention and treatment of anemia in suckling swine. Very occasionally, severe losses may occur in animals with marginal vitamin E–selenium deficiency; in these cases, there is, apparently, iron-catalyzed lipoperoxidation in hepatocytes and muscle. The result is sudden massive hepatic necrosis similar in many respects to that of hepatosis dietetica. Large amounts of potassium escape into the circulation from the liver and muscle, and sudden death may result from the cardiotoxicity of this ion. At postmortem, there is staining of subcutaneous tissues and lymph nodes near the site of injection, and there are lesions in the liver or skeletal muscles. The liver is of normal size and of normal color or pale, depending on whether or not the animal is anemic. The presence of underlying necrosis may be indicated only by the numerous small or large hemorrhages present on the capsular and cut surface. Insoluble iron compounds with the staining reactions of hemosiderin are found in mesenchymal cells in many tissues, the largest amounts being in macrophages of the local lymph nodes and in Kupffer cells. Death in piglets from hepatic necrosis occurs ~10 hours after administration. Saccharated iron may produce acute widespread muscle necrosis at ~24 hours, rather than hepatic necrosis in piglets with marginal vitamin E–selenium status. The myocardium is not affected. Acute hepatotoxicity was reported in young foals because of administration of a proprietary paste of iron and yeast products, given as a dietary supplement within a few hours of birth. Not all foals so treated became sick, but those that did developed severe acute centrilobular hepatocellular necrosis, resembling the disease in piglets, and dramatic ductular reaction (biliary hyperplasia). Acute iron intoxication has also been reported in young cattle administered injectable hematinics containing elemental iron, and rarely in adult horses administered oral vitamin supplements containing ferrous fumarate or ferrous sulfate. Oversupplementation, low vitamin E or selenium concentrations, or concurrent disease may have contributed to these cases. Consumption of elemental iron produces a periportal or panlobular pattern of necrosis. Chronic hepatotoxicity: plant-derived and environmental toxins Aflatoxin The aflatoxins are a group of bisfuranocoumarin compounds produced as metabolites mainly by Aspergillus flavus, A. parasiticus, and Penicillium puberulum. The metabolites are designated by the blue or green color they fluoresce when viewed under ultraviolet light and migration patterns during chromatography, and the major ones are B1, B2, G1, and G2. A less toxic metabolite, M1 is found in milk and other dairy products from cattle that ingest B1. Many others may be produced in minor amounts in fungal colonies or as metabolic products of the major toxins in animals. The most significant and best studied of the aflatoxins is B1 because of its relative abundance and its potency as a hepatotoxin. Strains of Aspergillus differ in the varieties and amounts of individual toxins produced, indicating that the biosynthesis of the toxins is genetically determined. The production of toxins also varies under different conditions of fungal growth, which is influenced by the quality of the substrate, temperature, relative humidity, moisture content of the substrate, and microbial competition. Thus the toxicity of moldy feedstuffs is impossible to assess without measurement of toxin production. Aflatoxins can be produced on growing crops in the field, but much greater levels are likely to accumulate in stored or unharvested mature grains, particularly if they are damaged by moisture. Various feeds other than grains, ranging from legume stubbles to bread, may be the substrate in outbreaks of aflatoxicosis. Contaminated grain has been incorporated into commercial dog food, leading to outbreaks of acute toxicity. Aflatoxins are metabolized by the hepatic mixed-function oxidase system to various toxic and nontoxic metabolites, the proportions of which vary with the species and age of the animal involved. The most potent of these is the 8,9-epoxide metabolite of aflatoxin B1; this binds to a variety of cellular proteins causing acute toxicity, and its carcinogenic activity derives primarily from adduct formation with guanine in nucleic acids in sensitive species that lack adequate glutathione S–transferase–mediated resistance. The mutational consequence is a G to T transversion. Acute toxicity is most evident in dogs, rats, ducks, guinea pigs, and calves, and each species may be fatally intoxicated by a dose rate of <1.0 mg/kg body weight. Although cats are also sensitive, they rarely ingest contaminated food. Acute, fulminating liver necrosis is sometimes seen in dogs that eat contaminated bread, dog food, or garbage, which may contain very high concentrations of the toxin. Younger animals of all species are much more susceptible and may die within a few hours. The gross postmortem picture is dominated by widespread hemorrhage and massive hepatic necrosis, changes also seen at the microscopic level (eFig. 2-27). Large animal species rarely are exposed at sufficient doses by normal dietary intake to develop acute toxicity. Sheep and adult cattle are quite resistant to the toxin. eFigure 2-27 Acute aflatoxin injury in the liver of a dog, characterized by acute necrosis and hepatic lipidosis in surrounding hepatocytes. Prolonged exposure to low concentrations of the toxin is a more common problem than acute toxicity in large animal species, and may merely produce reduced growth rates and moderate enlargement of the liver without any significant hepatic signs. The enlargement may be partly the result of hypertrophy of hepatocellular smooth endoplasmic reticulum and some degree of fatty change. As the level of aflatoxin in the ration increases (in young pigs, e.g., to 1.0 mg/kg ration), the liver may show all or none of the following changes: pallor, enlargement, bile staining, increased firmness because of fibrogenesis, and fine nodular regenerative hyperplasia. There may also be edema of the gallbladder and bile-tinged ascites in more severe cases. Even under experimental conditions, some individuals may show minimal liver lesions, whereas others, under the same levels of exposure, die of liver failure. Histologically, affected livers show obvious increase in size of some hepatocytes and their nuclei (megalocytosis) with focal necrosis or apoptosis. Bile ductules proliferate early, and reticulin and collagen deposition occurs throughout the acinus according to no distinct pattern (Fig. 2-96 ). Fatty change in affected livers is variable in extent and occurrence, and bile pigments accumulate in canaliculi and hepatocytes in more severely affected livers. Minor degrees of megalocytosis may be seen in proximal tubular epithelium in the kidney. The changes produced resemble those of pyrrolizidine alkaloid toxicosis. This can be attributed to the fact that aflatoxin and pyrrolizidine alkaloids inhibit hepatocellular regeneration such that nodules and ductules regenerate as the liver becomes atrophic. These are also genotoxic and carcinogenic, so they might be involved in the occurrence of liver cancers in animals, as they are in humans. Figure 2-96 Hepatocellular steatosis and ductular proliferation in canine chronic aflatoxicosis. At higher dose rates of aflatoxin, most centrilobular hepatocytes disappear and are replaced by a mixture of inflammatory cells, fibroblasts, and primitive vascular channels. The liver may be much smaller than normal, particularly in young animals, presumably because of mitotic inhibition, and focal hepatocellular necrosis is more obvious or may be supplanted by zonal (centrilobular) necrosis. Fatty change in these livers may be severe and uniformly distributed. Fumonisin Fumonisin B1, a mycotoxin elaborated by certain strains of Fusarium verticillioides (previously moniliforme) and F. proliferatum in infected corn, induces a pulmonary edema syndrome in pigs, hepatocellular carcinomas in laboratory rats, and leukoencephalomalacia. Hepatotoxicity occurs in horses and swine. Hepatic toxicity has also been reported in sheep and baboons. In horses, hepatoxicity occurs less often than leukoencephalomalacia. The clinical course is relatively short, with death likely within 5-10 days of the onset of clinical signs, such as anorexia, depression, icterus, and edema of the head. Bilirubin and liver enzymes are typically elevated. At postmortem, the liver is typically firm, yellow, and small, with an accentuated lobular pattern. Histologic lesions include abundant apoptosis that may progress to centrilobular necrosis and variable amounts of fibrosis in the portal tracts. Cardiotoxicity is also evident in swine and horses. Fumonisin B1 is also a primary hepatotoxin in pigs. Experimental feeding trials with pigs have shown dose-related differences in pathology. Pigs intubated with a minimum of 16 mg fumonisin B1/kg body weight per day developed interlobular edema, variable hydrothorax, and pulmonary edema, whereas pigs intubated with 8 mg/kg per day for 7-8 days, or fed diets containing 200 mg fumonisin B1/kg of feed for 21 days, developed marked icterus. Histologic changes included hepatocellular necrosis without a zonal distribution, depletion of centrilobular hepatocytes, lobular disarray, and megalocytosis characterized by large numbers of swollen hepatocytes with abundant granular eosinophilic cytoplasm and occasional large nuclei, randomly interspersed with small angular hepatocytes and scattered necrotic cells. These pigs showed no evidence of pathologic changes in the lungs, whereas pigs given higher doses showed hepatic necrosis in addition to pulmonary edema. The liver changes appear to be reversible upon cessation of exposure. Gilts fed low levels of fumonisin B1 for 90 days developed hepatic nodular hyperplasia, along with hyperkeratosis and parakeratosis and hyperplasia of the distal esophageal mucosa. Fumonisins are inhibitors of sphingosine and ceramide synthetase, leading to inhibition of sphingolipid biosynthesis. As a consequence, there is an accumulation of bioactive intermediates of sphingolipid metabolism (sphinganine and other sphingoid bases and derivatives), as well as the depletion of complex sphingolipids, which interfere with the function of some membrane proteins and may be involved in the toxic hepatic effects. Phomopsin There are 2 distinct manifestations of toxicity associated with Lupinus spp.; discussed here is the condition formerly known as “lupinosis,” which is a true mycotoxic liver disease. The teratogenic and neurotoxic effects of some of the isoquinoline alkaloids from the plants themselves are discussed in Vol. 1, Bones and joints and Vol. 1, Nervous system. The fungus Diaporthe toxica (formerly Phomopsis leptostromiformis) is parasitic on green Lupinus plants, but it becomes saprophytic after the host plant dies. Phomopsins (A or B) are produced if the lupin stubble is moistened, and such stubbles may remain toxic for months. Additional toxins are suspected. Severe acute liver damage has been described in sheep on very toxic stubbles in Western Australia, but in many of these cases, it has been difficult to separate the toxicity of the lupins from that of copper, which in this area is often concentrated in ovine livers (see later section on Copper). The usual syndrome of phomopsin poisoning is subacute to chronic. Inappetance occurs soon after experimental administration of the toxin is begun; liver damage is clinically inapparent for several days. The gross pathologic abnormalities of acutely intoxicated sheep include icterus and modest ascites. The main abnormalities are evident in the liver, which varies from a yellow discoloration because of lipidosis in more acute cases, to a variable ochre to orange discoloration with a firm texture with increasing chronicity (Fig. 2-97A ). Histologically, there is early hepatocyte swelling and accelerated cell death among hepatocytes. Increased mitotic activity is soon apparent, although by this time, the liver has become smaller. The mitotic activity is in fact largely ineffectual, as close examination reveals that many mitotic figures are abnormal. There is either clumping or dispersal of chromatin, and there appears to be mitotic arrest at late metaphase (Fig. 2-97B- D). Remaining hepatocytes swell, their cytoplasm becomes granular, and their nuclei vesicular and may contain vesicular intranuclear pseudoinclusions. There is variably severe fatty change, dependent to a large degree on the fat reserves of the animal. There is also accumulation of complex pigment in macrophages in portal stroma and about hepatic venules; this granular material contains lipofuscin, ferric iron, and copper at least. Bile duct proliferation is also a prominent feature of the chronic disease. With progression, hepatic fibrosis occurs, predominantly diffuse in distribution, and by this stage, there is usually clinical icterus and anorexia. Figure 2-97 Phomopsin poisoning (lupinosis) in sheep. A. Affected sheep have pale livers with prominent icterus. B. Numerous, and abnormal, mitotic figures in subacute phomopsin poisoning. C. Brown pigment within Kupffer cells. D. Clear intranuclear pseudoinclusions can be found in affected hepatocytes. (Courtesy J.G. Allen.) The liver continues to shrink, presumably as a result of continued mitotic inhibition and progressive fibrosis. The organ is small, tough, and has a finely granular surface and texture. It is pale gray-orange but usually retains its shape. Fibrosis is initially portal to periportal, but central areas can also become fibrotic. Bridging fibrosis between portal areas and portal to central regions can develop. In naturally occurring cases, however, discontinuous intake of the toxin may produce a liver grossly distorted by asymmetrical nodular regeneration and fibrosis. The atrophic changes are most severe in the left lobe. Photosensitization occurs in phomopsin-poisoned sheep; it may be severe if the animals have access to green feed while under the influence of the toxin. Lupinosis in sheep has also been experimentally observed to induce mild skeletal myopathy, resembling nutritional myopathy. Phomopsin poisoning in cattle causes most losses when the animals are lactating or heavily pregnant; in these animals, the syndrome is essentially one of ketosis, to which such cows would be predisposed by the anorexia that is an obvious clinical feature of this intoxication. Pregnant sheep are less likely to have access to toxic lupin roughage during late gestation; otherwise, ketosis triggered by phomopsin would be expected just as often as in cattle. Chronic hepatic fibrosis with fine, nodular regeneration may occur infrequently in cattle as the result of chronic phomopsin poisoning. Similar liver changes may also be seen in horses, in which there may also be hemolytic anemia of unknown pathogenesis. Sporidesmin The mycotoxin sporidesmin is produced by the fungus Pithomyces chartarum and concentrated in the conidia (spores) ; the most important substrate is dead ryegrass (Lolium perenne) that has been moistened in warm weather. Intoxication causes chronic liver damage and severe hepatogenous photosensitivity (“facial eczema”) and is a serious cause of loss of sheep and, to a lesser extent, cattle, goats, and farmed deer on the North Island of New Zealand. Sporadic and subclinical intoxication occurs irregularly on the South Island, in southern Australia, and South Africa. Although the fungus is found throughout the world, toxigenic strains appear to be limited to New Zealand. Sporidesmin intake in conjunction with ingestion of Tribulus terrestris causes another hepatogenous photosensitivity (geeldikkop); this is similar to but distinct from facial eczema, and is described later. Sporidesmin is concentrated in the fungal spores, and the toxigenicity of pasture is related to the density of the spores in it. Sporidesmin is not specifically hepatotoxic. Administration of the toxin does produce rapid disorganization of hepatic cell organelles and triglyceride accumulation, but these are mild and nonspecific changes. If administered in suitable dosage, the toxin causes permeability alterations in many tissues and will, for example, produce corneal edema on local application. The hepatobiliary lesions are caused by the excretion of unconjugated sporidesmin in bile, where its concentration may initiate oxidative injury, likely mediated by the production of a hydroxyl radical. Sporidesmin is also excreted in urine, and if the dose is high enough, edema and mucosal hemorrhage occur in the bladder. The hepatic lesion is the result of irritation of mesenchymal tissues in the portal triads and surrounding the bile ducts. A high concentration of sporidesmin can injure biliary epithelium, allowing diffusion of the bile duct contents. Release of toxin, possibly accentuated by the release of bile acids as well, produces irritative lesions and necrosis in the adjacent blood vessels. The liver in acute forms of the disease is enlarged, with rounded edges, and is finely mottled and discolored yellow-green by retained bile pigments, although the discoloration may be blotchy. There is mild edema and congestion of the wall of the gallbladder, which may be distended with bile of normal quality or with mucin (white bile). The extrahepatic ducts are thickened and prominent, and there is edema of the adventitia. The ductal changes may extend to the papilla of Vater and can be traced by the naked eye deeply into the parenchyma. In more chronic cases, alterations of size and pigmentation of the liver are variable. Pale areas of capsular thickening, which may be elevated or depressed, are visible. On cut surfaces, they extend deeply as wedge-shaped areas in which biliary fibrosis has produced an exaggerated acinar pattern, and the parenchyma is pale and atrophic; these areas are related to occluded bile ducts. The liver is firm and cuts with increased resistance. The medium and large caliber intrahepatic ducts are conspicuous. There is irregular stenosis of their lumens, some are occluded by cellular debris and inspissated bile or mucin, and in some, cicatrization of the new fibrous tissue causes complete atresia. Occlusion of the ducts causes the parenchyma served by them to undergo atrophy, necrosis, and fibrosis (Fig. 2-98 ). The livers of animals that have survived an attack of cholangitis of this genesis are distorted in shape and size by large nodules of regeneration and persistent areas of atrophy and fibrosis. The atrophy and fibrosis may affect either lobe, but usually, the left is most severely affected. Figure 2-98 Chronic sporidesmin intoxication (“facial eczema”) in sheep. Liver lobe atrophy and fibrosis. (Courtesy K.G. Thompson.) Histologically, the changes are those of acute cholangitis or cholangiohepatitis to which there is minimal leukocytic reaction. There is extensive necrosis of the lining of the larger intrahepatic ducts and the extrahepatic ducts, the epithelium being cast off as debris mixed with a few leukocytes. There is edema of the adventitia of the ducts, with active fibroplasia and scarring. Inflammatory cells are present, but not in large numbers, and they are chiefly lymphocytes and histiocytes. Injury to the smaller radicles of the bile ducts is less severe, but fibrosis is active. The portal tracts are enlarged by fibrous tissue and by the active generation of new bile ducts that follows and which is more prominent in chronic intoxication (eFig. 2-28). Affected camelids appear to be susceptible to florid hyperplasia of the bile ducts. eFigure 2-28 Chronic sporidesmin toxicity in sheep causes prominent ductular fibrosis of larger ducts and damage to the biliary epithelium. (Courtesy R. Kelly.) In more severe intoxications, there may be coagulative necrosis of blood vessel walls in the portal triads; when this is incomplete, the most damaged segment of the vessel tends to be that adjacent to the nearest injured bile duct. Both arteries and veins may be affected, and it is possible that necrosis is related to vascular insufficiency as well as to impaired bile drainage. Changes in the hepatic parenchyma are minimal and secondary to those in the portal triads. In acute cases, there may be extensive pigmentation of hepatocytes and Kupffer cells by bile pigments, but this is irregular in distribution in the liver. Inspissated bile can be found in the bile ducts and canaliculi. There is some necrosis of hepatocytes adjacent to inflamed portal areas, and other areas of necrosis, focal in type and distribution and probably the result of biliary obstruction, may be numerous. Other morbid alterations include great enlargement of the adrenals produced by cortical hypertrophy; sclerotic intimal plaques in the arteries, veins, and lymphatics in the hilus of the liver; and a tendency for the newly formed bile ductules to recanalize occluded ducts. Pyrrolizidine alkaloids Pyrrolizidine alkaloids have been identified in nearly 3% of all plant species, from >6,000 species of 3 families: the Asteraceae (Compositae), Leguminosae (Fabaceae), and Boraginaceae. The main genera responsible for plant toxicoses in domestic mammals are Senecio, Crotalaria, Heliotropium, Cynoglossum, Amsinckia, Echium, and Trichodesma, and are widely distributed around the world. Intoxication is relatively infrequent because most plants containing pyrrolizidines are unpalatable. Contamination of baled or cubed forage with toxic plants is a common route of exposure. The seeds are also toxic, and the small seeds of Amsinckia and Crotalaria may, depending on harvesting technique, heavily contaminate other harvested grains used in prepared pig and poultry feeds. More than 350 pyrrolizidine alkaloids have been identified chemically, and most of the toxic plant species contain more than one of the alkaloids. So far, only a small proportion of the known alkaloids have well-characterized toxicity; these are all esters of 1 of 3 amino alcohol bases (necines) or acids (necic acids). The retronecine group includes monocrotaline, retrorsine, retronecine, ridelliine, senecionine, and jacobine, and the heliotridine group includes lasiocarpine and heliotrine. These toxins must be metabolized to more reactive forms for toxicity. Cytochromes P450 mediate the N-oxidation of the necine bases. They can also mediate the 2-step hydroxylation of the necine bases at the C3 and C8 positions; this is followed by spontaneous dehydration to the highly reactive dehydropyrrolizidine (DHP) alkaloids. The toxic DHP alkaloids are electrophilic and can bind covalently to amino acids, proteins, and nucleic acids at guanine and adenine residues. Acute toxicity derives from damage to cellular proteins. Antimitotic effects can be exerted via damage to microtubules. The DNA-binding activity is responsible for genotoxicity; the DHP-derived DNA adducts are a common pathway for carcinogenic activity of pyrrolizidine alkaloids. The DHP metabolites can be detoxified by glutathione conjugation by glutathione S–transferases. Ester linkages at the C7 and C9 positions can be hydrolyzed by carboxylesterases to generate the necine or acid moieties; this is generally considered to be a detoxification pathway. The toxicity of a pyrrolizidine alkaloid-containing plant depends on many factors. The content of toxin varies by species of plant; the stage of growth, as new growth tends to have more toxin in general; and the time of year and environmental factors such as rainfall. For an individual pyrrolizidine alkaloid, toxicity depends on the amount of alkaloid that can be converted to reactive metabolites, the rate of cytochrome P450–mediated generation of the DHP, and the efficiency of detoxification by glutathione conjugation. Toxicity also depends on the species exposed, age and sex of the animal intoxicated, and the metabolic activity and the mitotic stage of its target cells. Young animals are generally much more susceptible than adults. Grazing animals are more likely to consume plants that contain pyrrolizidine alkaloids, but because the toxins are produced by the plants to deter herbivores, it is unusual for an animal to consume large amounts. Ruminants, especially sheep and goats, are much less susceptible than pigs, in part because the toxins can be degraded in the rumen. However, sheep can graze out stands of Senecio that would be lethal to cattle. Horses and cattle have similar, intermediate susceptibilities. Most pyrrolizidine alkaloids are hepatotoxic because they are metabolically activated to DHPs in the liver. There are 3 common morphologic expressions of pyrrolizidine poisoning. First, acute centrilobular to massive necrosis occurs in animals ingesting large amounts of these alkaloids. Because the plants responsible are unpalatable, naturally occurring outbreaks of acute poisoning are generally restricted to circumstances where animals face starvation, such as grazing pastures affected by prolonged drought. Ingested dosages in grazing circumstances are usually too low for acute effects; however, acute hemorrhagic centrilobular necrosis has been described after experimental exposures to high doses of pyrrolizidine alkaloids. The centrilobular pattern of necrosis produced is similar to that caused by many other hepatotoxins that are bioactivated by hepatic cytochromes P450. Second, phasic (usually seasonal) repetitive exposure to these alkaloids leads to hepatic atrophy with formation of regenerative nodules. This is the most common expression of field exposure to pyrrolizidine alkaloids, and affected livers show a characteristic pattern of hepatocellular polyploidy known as megalocytosis (Fig. 2-99 ). Third, prolonged exposure exclusively to Heliotropium induces firm, fibrotic atrophic livers without nodular regeneration. If seasonal exposure to these toxic alkaloids declines, then a remarkable degree of recovery is possible. Figure 2-99 Megalocytosis in Senecio ragwort poisoning in an ox. Pyrrolizidine alkaloids inhibit DNA synthesis and mitosis in hepatocytes, but some are able to replicate their DNA without undergoing mitosis, resulting in greatly enlarged hepatocytes with large convoluted polyploid nuclei. Some enlarged nuclei have cytoplasmic invaginations that can become entrapped as intranuclear inclusions. However, many hepatocytes in an affected liver do not become megalocytic. Those that are completely inhibited do not replicate DNA at all, whereas those that are more resistant can replicate more normally and give rise to nodular populations of smaller, more normal hepatocytes. Inhibited hepatocytes and megalocytes are long lived but eventually many undergo apoptosis. In chronic pyrrolizidine alkaloid poisoning, the liver can become atrophic as hepatocytes are lost faster than they can be replaced. The atrophy can be compensated to various degrees by megalocytosis and regeneration of small, less inhibited hepatocytes that proliferate in a nodular pattern. Concurrently, there is usually proliferation of bile duct epithelial cells (termed ductular reaction) in the portal triads. This is largely explained by the propensity of hepatic progenitor cells to proliferate forming ductules when adult hepatocytes cannot respond to the regenerative stimuli that prevail when liver mass is inadequate. There may also be some periportal fibroplasia that varies with species and exposure; typically, it is minimal in sheep, moderate in horses, and may be marked in cattle (eFig. 2-29). In cattle, the fibrous tissue can infiltrate along the sinusoids to dissect lobules, separate individual cells, and link up with the walls of efferent veins. In acute poisoning, in which there is centrilobular necrosis, fibrosis can also develop in a “veno-occlusive” pattern around and sometimes obliterating the hepatic venules. There is evidence that pyrrolizidine metabolite injury is not restricted to hepatocytes and that endothelial cells are also involved, leading to additional vascular insult and fibrosis in the liver. Direct endothelial injury from pyrrolizidines may occur, but an alternative explanation proposes a form of “bystander” injury, where toxic metabolites are formed in hepatocytes but are able to damage adjacent endothelial cells. This pattern of fibrosis may occur in some field cases in cattle, but is more commonly observed in humans exposed to pyrrolizidine alkaloids in herbal preparations or “bush teas,” and it can be produced experimentally after sublethal acute experimental hepatotoxicity. In fatal cases in cattle, the livers are very tough and nodular, and the nodules have variably efficient biliary drainage and cytochrome expression, so there may be a very striking color pattern, ranging from fatty yellow through green and brown. Hepatic fibrosis can result in portal hypertension with ascites, severe mesenteric edema, and diarrhea. Excretory insufficiency with moderate jaundice and photosensitization can also occur. eFigure 2-29 Diffuse fibrosis of liver in chronic Senecio poisoning in an ox. (Courtesy P. Stromberg.) The disease in sheep is always protracted as a consequence of the relative resistance of this species; indeed, clinical signs may not be seen until after a second season of exposure. The plants most often implicated are Heliotropium europaeum and Echium plantagineum. The shape of these failed livers is normal, but they are small, gray-yellow, fairly smooth, and toughened by condensation of normal stroma rather than by fibroplasia. If the liver copper content was high before intoxication, toxicity can culminate in copper release and an episode of intravascular hemolysis. In this case, the carcass will be intensely jaundiced, and the kidneys are stained with methemoglobin and bilirubin. The relationship of chronic copper poisoning to pyrrolizidine alkaloid poisoning is discussed later. Horses are susceptible to both acute and chronic toxicosis, and liver failure produced in horses by these alkaloids is similar to that seen in cattle. Horses are more likely to manifest signs of hepatic encephalopathy with head-pressing and compulsive walking; in some places, these nervous signs give rise to colloquial names such as “walkabout” and “walking disease.” Administration of a high dose of Cynoglossum officinale resulted in severe liver disease within 7 days after dosing, with elevated serum enzymes, altered bile acid metabolism, and extensive hepatocellular necrosis with minimal periportal fibrosis and biliary hyperplasia, and little or no megalocytosis. Edema and infarction of the cecum and colon were also present at postmortem. Administration of a low dose to horses for 14 days resulted in transient clinical depression and weight loss, transient elevations of serum enzymes and bile acids, and minimal periportal hepatocellular necrosis with fibrosis, developing extensive megalocytosis by week 14. The megalocytosis became the most prominent change 6 months after exposure. Metabolites of pyrrolizidine alkaloids are formed by any cells with adequate cytochrome P450 activity. Although the liver is the main site of bioactivation, other cells such as proximal convoluted renal tubules and club cells (formerly termed Clara cells) in the lung can generate metabolites that cause local injury. Death in some instances may be the result of renal damage and in others the result of pulmonary vascular and interstitial lesions. Variation in the source of the toxin and in the species-based metabolic differences in the affected animals account for the differences in susceptibility of the different tissues. Alkaloids from Crotalaria affect the widest range of tissues in most animals; most notably, monocrotaline causes diffuse lung injury, leading to pulmonary edema progressing to fibrosis. Respiratory difficulty has been described in horses eating C. dura, and C. crispata produces similar lesions. Sheep develop pulmonary signs after eating C. globifera and C. dura, and pigs after eating Senecio jacobaea. The experimental feeding of C. spectabilis to rats or the injection of monocrotaline, extracted from the plant, produces progressive pulmonary disease, pulmonary hypertension, and cor pulmonale, with necrotizing vasculitis of the pulmonary arterioles. Emphysema occurs in pigs and is an outstanding feature of the pulmonary disease of horses. The essential reactive lesion is diffuse fibrosis of alveolar and interlobular septa, with patchy epithelialization occurring more slowly. Lantana camara Lantana camara is an attractive ornamental shrub native to the Americas and Africa that grows readily in tropical and subtropical habitats. It contains various toxic pentacyclic triterpenes, the most abundant of which are lantadene A, lantadene C, and icterogenin, although metabolites may also be toxic. Lantadene A appears to be the most toxic; at high doses experimentally, it causes severe acute centrilobular necrosis of the liver. The mechanism of toxicity is not known, but may be related to the effects of lantadene A on mitochondrial energetics. However, L. camara poisoning in grazing animals is manifested as subacute or chronic cholestasis, characterized by severe icterus and photosensitization, most commonly seen in cattle, but rarely in sheep, goats, and horses. Goats are quite susceptible to the toxin, but are less likely to eat the plant. Neonatal ruminants appear resistant suggesting the rumen may retain the plants and provide longer exposure to the toxins. Photosensitization is usually severe after 2 days, but jaundice is more severe in chronic cases. Heavily intoxicated cattle can die within 2 days, but most fatal cases run a course of ~2 weeks. Ruminal stasis and anorexia appear early, and the large bowel contains dark, dry feces. The rumen is a reservoir of toxin that remains active and can perpetuate the intoxication after access to the plant is curtailed. The liver is enlarged, pale, and stained yellow, orange, or green-gray by bile pigment. The gallbladder is greatly distended with pale, sometimes slightly mucoid, bile. The severity of the liver changes may be much less than expected from the intensity of the icterus and photosensitization. The most consistent histologic finding in the liver is hepatocellular enlargement and fine cytoplasmic vacuolation, together with some degree of bile accumulation in canaliculi, hepatocyte cytoplasm, and Kupffer cells. The canalicular cholestasis is usually more severe in the centrilobular zones, whereas the cytoplasmic vacuolation is often more pronounced in the periportal hepatocytes. There is usually some bile duct proliferation, and in some cases, there will be a high incidence of periportal apoptosis, focal coagulative necrosis, or hepatocellular dissociation. Electron microscopy reveals an apparent increase in volume of smooth endoplasmic reticulum and a quite characteristic form of collapse of many bile canaliculi. Other canaliculi are distended and have damaged microvilli. Because much of the bilirubin that accumulates in the plasma of such animals is conjugated, it seems that the cholestasis is due in large measure to direct interference with canalicular transport of bile. The mechanisms of cholestasis have not been determined, but damage to the contractile pericanalicular cytoskeleton or its associated cell adhesion molecules required for canalicular integrity are plausible targets (see previous section on Cholestasis and jaundice). Animals are usually polyuric because of acute tubular injury, resulting in severe dehydration, in part related to a disinclination to drink. The kidneys are slightly enlarged and wet on section, and, especially in the more chronic cases, the cortex is pale and the medulla hyperemic. The renal lesion is nonspecific acute tubular injury, ranging in severity from mild vacuolar change to patchy tubular necrosis and extensive tubular cast formation. The role of the hyperbilirubinemia in the production of the renal damage has not been assessed. Severe myocardial necrosis can be produced in sheep by Lantana poisoning, and may be responsible for the early deaths in cattle. Intoxication by steroidal sapogenins: tribulosis and related toxicoses Consumption of Tribulus terrestris, particularly by the grazing of young wilted plants or sometimes hay, causes cholangitis and photosensitization in sheep. This plant has caused enormous loss of sheep in South Africa, where the toxicosis is known as geeldikkop (“yellow bighead,” because of icterus and marked edema of ears and face). The disease has been reproduced by oral administration of crude extracts of steroidal sapogenins or their glycosides, saponins, of T. terrestris, but which of the various saponins in the plant are responsible for the disease is unknown. The disease has also been reproduced experimentally by co-administration of sporidesmin and T. terrestris, but the liver lesions are histologically distinct from those produced by sporidesmin alone (facial eczema). In geeldikkop, the most characteristic gross finding is the presence of a white, semifluid accumulation of fine, crystalline material that can be expressed from the cystic duct and larger intrahepatic ducts. The gallbladder mucosa is also partly covered with a fine, crystalline deposit. The gross lesions of geeldikkop are similar to those of facial eczema in that there is generalized icterus, and the liver is discolored by bile pigment and either slightly swollen or distorted, according to the duration of the disease. In poisoning by sporidesmin alone, however, there is more obvious edema and fibrosis of bile ducts, and bile infarcts in the parenchyma are more common. The acute lesion consists of swelling and feathery vacuolation of hepatocytes, and marked hyperplasia of Kupffer cells that may show similar cytoplasmic changes. In these acute cases, the presence of acicular crystals may be very difficult to detect (Fig. 2-100A, B ). With more chronic intoxication, the most consistent histologic abnormality is the presence in bile ducts of various amounts of crystalline material (Fig. 2-100C). The crystals are fine, flat, and are deposited in affected ducts, and, less commonly, in hepatocytes themselves and in renal tubules. There may be associated bile ductular proliferation in severe cases, but often the degree of histologic hepatocellular damage is mild compared to the severity of the photosensitization. The cholestasis is not solely the result of mechanical obstruction by the crystals, because photosensitization can occur in such outbreaks in animals whose livers show very little cholangitis and contain very few crystals. The severity of peribiliary fibrosis is more variable and probably depends on the relative contribution of sporidesmin to the intoxication. There is some hepatocellular degeneration and apoptosis, and there is fairly uniform swelling of cytoplasm. Bile pigment accumulates in Kupffer cells and hepatocyte cytoplasm, but not to any marked extent in canaliculi. Focal necrosis of the gallbladder mucosa is often present. The severity of the hepatic lesion increases with the duration of exposure. Figure 2-100 A. Crystal deposition within bile duct epithelium in a goat grazing Panicum sp. B. Foamy material within macrophages in an ox ingesting Brachiaria sp. (Courtesy R. Kelly.) C. Fine crystalline material in expanded Kupffer cells in a goat grazing Panicum sp. The biliary crystals from sheep with geeldikkop have been determined to be composed principally of calcium salts of steroidal sapogenins present in T. terrestris. Plant saponins are metabolized in the rumen and liver to episapogenin glucuronides, which in the presence of calcium may precipitate, forming the characteristic biliary crystals. Cholestasis is likely related to reduced bile acid secretion into the lumen of the canaliculi, rather than biliary occlusion by these plant sapogenins. This explains the evidence of cholestasis before crystals can be seen histologically. Other unidentified plant components may also play a role in hepatocellular and biliary injury. It has also been suggested that the toxins responsible may act primarily on the membranes of the bile canaliculi, in a manner similar to that of Lantana poisoning. Hepatocellular damage likely also plays a role in phytoporphyrin (phylloerythrin) retention. Switchgrass (Panicum virgatum), Kleingrass (P. coloratum), and other Panicum species are associated with hepatogenous photosensitization in grazing animals, including sheep, goats, and horses in the United States, Australia, and other areas of the world. The toxic agent(s) are steroidal sapogenins, and their type and concentration vary with the part of the plant (highest concentrations being in the young growing leaf) and with the environmental conditions. Hot and dry conditions, have been associated with outbreaks. Younger animals appear to be more susceptible. Crystals may be produced in the biliary tree, hepatocytes, sinusoids, or Kupffer cells. Crystal-associated cholangiohepatopathy is not specific for Tribulus or Panicum spp. intoxication. Similar changes occur in the hepatogenous photosensitivity disease, alveld, in sheep grazing pastures in Norway, the British Isles, and in the Faeroe Islands containing Narthecium ossifragum. Similar disease has been reported in ruminants intoxicated by Agave lecheguilla, Nolena texana, and the pasture species Brachiaria decumbens (signal grass) in Brazil. In New Zealand, crystal deposition in the biliary tree of cattle has been reported following ingestion of Phytolacca octandra (inkweed). In those cases studied (T. terrestris, P. dichotomiflorum, P. schinzii, Narthecium ossifragum), the characteristic crystalloid material deposited in the bile is principally composed of calcium salts of steroidal sapogenins. In some cases, crystal formation may not be apparent in bile ducts, and affected Kupffer cells and macrophages are more evident. There may be differences in the histologic responses of cattle and sheep grazing various sapinogenin-containing plants. Foamy Kupffer cells and hepatocytes along with foamy macrophages in mesenteric and hepatic lymph nodes may be a more common response in cattle versus crystal formation within bile ducts in sheep (Fig. 2-101 ). To complicate diagnostic efforts, foamy macrophages in the mesenteric and hepatic lymph nodes are also found in healthy cattle and sheep grazing B. decumbens in Brazil. With the exception of Nolena texana, steroidal sapogenins have been demonstrated in all of these plants. As with T. terrestris, extracts of B. decumbens containing steroidal saponins produced typical lesions of cholangitis with crystal deposition when orally administered to lambs, with Pithomyces chartarum spore counts below detectable levels. Similarly, analysis of samples of B. decumbens and P. dichotomiflorum on which cattle and goats had recently been photosensitized showed only low levels of P. chartarum spores, and all isolates obtained failed to produce sporidesmin. Despite this, it is also apparent that neither Tribulus nor Panicum stands are at all times dangerous. Levels of saponins have been shown to vary greatly within a species from site to site, and with the age of the plant. Additionally, the role of sporadic environmental conditions, such as wilting, which might concentrate the saponins or other unidentified toxins, or the synergistic involvement of endophytic fungi, remains to be determined. Figure 2-101 Chronic cholangitis and plugging of bile duct by lipophilic crystalloid material, in photosensitivity disease of sheep grazing pangola grass. Similar to reaction seen in geeldikkop. (Courtesy J.G. Allen.) Other plants reported occasionally to produce unexpected photosensitivity include Digitaria spp., Cooperia pedunculata, Nidorella foetida, and Chloris spp., and such valuable pasture genera as Medicago, Trifolium, Avena, and Biserrula pelecinus, although the latter species may cause primary photosensitization. Nitrosamines Epizootics of poisoning by dimethylnitrosamine occurred in Norwegian cattle, sheep, and fur-bearing animals, from 1957-1962. The toxin was present in herring meal and was thought to be a reaction product of trimethylamine and other lower amines with sodium nitrite, added as a preservative, the reacting amines being products of decomposition. Animals consumed several pounds of the toxic meal each day before becoming ill. This intoxication has little importance in livestock now that it is easily prevented. At postmortem, there was moderate anasarca and signs of hemorrhagic diathesis. Livers affected acutely were enlarged and firm, with mottled discoloration and sometimes a nutmeg appearance. In chronic intoxication, the liver was small, granular, and very firm. A number of cases recovered after prolonged convalescence, and in these, there was atrophy and fibrosis of the left and caudate lobes, and the right lobe was hyperplastic and hemispheric. Histologically, in acute cases, there was widespread hemorrhagic centrilobular necrosis and an unusual degree of intimal and subendothelial reaction in sublobular and hepatic veins. The chronic lesion was dominated by extensive centrilobular fibrosis, with obliterative changes in many central and sublobular veins. Sporadic cases of portal hypertension in Greyhound dogs have been recognized where hepatic veno-occlusive disease is the predominant lesion. In these cases, a history of feeding meat treated with large amounts of nitrites or sulfites in an attempt to preserve the color of fresh meat is common, but a causal relationship has not been established. The hepatocellular changes are not specific for dimethylnitrosamine or other hepatotoxic nitrosamines. Nitrosamines are metabolized in the liver and other tissues to reactive alkyl groups that bind covalently to various macromolecules, especially guanine in nucleic acids. Hepatotoxicity develops more slowly than in response to toxicants that induce membrane peroxidation, as many hepatocytes with DNA damage undergo apoptosis when stimulated to replicate. There is some megalocytosis, nuclear vesiculation and nucleolar prominence, cytoplasmic intranuclear inclusions, and variable fatty change and cytoplasmic bile accumulation. The typical effects of chronic experimental nitrosamine hepatotoxicity, such as hepatocellular fibrosis, nodules, and neoplasms, have not been evident in the accidental poisonings. Dimethylnitrosamine-induced hepatotoxicosis in dogs has been proposed as a model of toxin-induced progressive hepatic disease in this species. Indospicine Legumes of the genus Indigofera have long been known to contain the toxic amino acid indospicine (6-amidino-2-hexanoic acid), which is a structural analogue of arginine, and which was shown in early experimental work to be hepatotoxic for rats and other species. The dose rates necessary to cause chronic liver injury in these species were, however, quite high, and field cases of intoxications were only seen in cattle grazing I. spicata, which has the highest naturally occurring concentrations of indospicine. In Australia, a serious outbreak of fatal liver disease occurred in dogs that had been fed meat from horses that had been grazing I. linnaei, a plant native to the arid zones of Australia that has been known to produce, in horses, a chronic neurologic disorder known as “Birdsville horse disease.” The disease in horses is not associated with liver damage, although these animals do accumulate indospicine in most tissues, including muscle. Dogs fed indospicine-contaminated meat for several weeks may develop progressive liver damage. Affected livers may be small, firm, and pale, or they may be nodular because of hypertrophy of surviving hepatocytes (eFig. 2-30A). Histologically, lesions begin as vacuolation of a narrow band of centrilobular hepatocytes, followed shortly by accumulation of mononuclear inflammatory cells in this zone and in the stoma of the hepatic venules (eFig. 2-30B). Progression of the lesion is marked by scattered necrosis of a widening zone of centrilobular hepatocytes, disorganization, vacuolation because of fatty change, and accumulation of ceroid pigment in macrophages. Moderate centrilobular fibrosis is seen in later stages, as well as pronounced canalicular cholestasis. By this stage, affected animals begin to show icterus, inappetance, and depression. Bile ductular proliferation is not a marked feature. Death is attended by the usual signs of hepatoencephalopathy and tendency to bleed spontaneously. There is no evidence of direct neurologic damage as is seen in horses. More recently, dogs ingesting canned commercial camel meat developed serious and sometimes fatal liver disease. This may be a concern for dogs with food allergies that are fed nonstandard protein sources in special diets. eFigure 2-30 A. Liver from a dog with indospicine toxicity with a zonal pattern of pallor. (Courtesy R. Kelly.) B. Mononuclear inflammatory infiltrates, biliary stasis, irregular vacuolation of hepatocytes, and disorganization of hepatic plates in a dog. This intoxication is remarkable by virtue of its unpredictability. Experimental intoxication by pure indospicine has validly been shown to reproduce the naturally occurring intoxication caused by horsemeat feeding; however, liver failure can be produced by either means in only a small proportion of dogs so exposed. On the other hand, milder degrees of liver damage are reliably produced by both methods. Thus it seems an idiosyncratic response is superimposed upon a more consistent effect of the toxin; the nature of the accompanying inflammatory response suggests that the former may be immunologically mediated. This has yet to be established, as has the proposal that the mechanism of the intoxication is the result of competitive inhibition of arginine. Senna (Cassia) occidentalis Ingestion of large amounts of seeds from Senna occidentalis in the family Leguminosae can produce acute hepatic necrosis in cattle, pigs, goats, and horses. Hepatic injury is characterized by lesions ranging from vacuolation, to scattered apoptosis and centrilobular necrosis. In all species, except for horses, there is significant cardiac and skeletal muscle injury and myoglobinuria that are the predominant lesions, rather than hepatic injury. The toxic agent is not known currently. Trifolium hybridum (alsike clover) Alsike clover is a legume that grows well on wet clay soils in North America, where it is sometimes responsible for outbreaks or single cases of cholangitis in horses that consume it as pasture or hay. The toxic principle has not been isolated, but it likely is in highest concentrations in the flowering stage used for hay. However, there are evidently contributing factors because, although alsike clover is widespread in cultivation, toxicity is rare. Toxicity usually occurs when alsike clover is a major component of pastures or hay, but some horses are believed to graze it selectively in a mixed pasture. Clinical signs of poisoning initially include mild colic, ill-thrift, and anorexia, with increasing occurrence of signs of hepatotoxicity with cholestasis. In some there can be icterus and photodynamic dermatitis. Exposure to the plant can occur for a year or more before signs of hepatic insufficiency develop, but cholestatic liver disease can be detected biochemically before horses become ill. More severely affected horses display neurologic disturbances, either excitement or mania in irregular episodes, or long periods of extreme dullness, anorexia, apparent blindness, forced wandering, head pushing, and yawning. Clinical problems related to hypoproteinemia, such as coagulopathy or ascites, are not a feature of this disease, probably because most of the hepatocytes are not directly affected. The lesions of alsike clover poisoning are those of diffuse subacute to chronic biliary hyperplasia (ductular reaction) and fibrosis, with the major lesions within and adjacent to all intrahepatic biliary tracts (Fig. 2-102 ). The liver may be enlarged, sometimes greatly so, or shrunken, pale, and tough or rubbery in consistency. The surface is smooth, but its appearance is mottled. The mottling is clear on the cut surface and is caused by bands of gray fibrous tissue, distinctly visible to the naked eye, surrounding and compressing each lobule. Near the margins of the liver and in some other areas, scar tissue may completely replace the parenchyma. Areas of parenchymal atrophy may occur upstream of some occluded ducts. Microscopically, there is pronounced proliferation of fibrous tissue in and around the portal tracts associated with hyperplasia of well-formed bile ducts (ductular reaction). Some areas have prominent neutrophil infiltration into ducts, but in many areas this does not occur. In the early stages, the proliferation of bile ducts is often greater than the proliferation of the fibrous tissue, but later the proportions are reversed. The proliferating tissue extends slowly to connect adjacent portal triads, circumscribing areas that correspond to conventional lobules. The fibrous tissue does not permeate along the sinusoids, and there is gradual and uniform constriction of the parenchyma. Figure 2-102 Chronic cholangiohepatitis caused by alsike clover poisoning in a horse. The distribution of liver lesions suggests that the harmful factor in alsike clover is excreted into the bile in a form that damages the ducts. However, the biliary proliferation and fibrosis is similar to that observed in areas of the liver that are drained by chronically obstructed bile ducts, so it seems likely some of the later changes are secondary to cholestasis. Agents that interfere with bile excretion have the potential to interfere with elimination of various potentially injurious substances. Thus it is possible that alsike clover ingestion might increase toxicity of other factors in the forage. Tephrosia cinerea Tephrosia cinerea is a member of the Leguminosae family and is found in Brazil. Prolonged ingestion causes chronic liver disease characterized clinically by wasting, and dramatic ascites in affected sheep. Hydrothorax and hydropericardium can also be present. Livers are pale with multiple small, ~1-mm diameter nodules. The livers are firm, and there is gallbladder edema. Histologically, there is portal fibrosis and biliary hyperplasia that is typically more severe in the subcapsular region. Most hepatocytes are enlarged because of vacuolation. Scattered individual necrotic cells are present, and focally, there is evidence of bile stasis. Inflammation is modest. Brassica rapa Turnip (Brassica rapa) or other Brassica family forage ingestion by cattle on the North Island of New Zealand is incriminated as a cause of hepatogenous photosensitization and biliary injury. The injury can mimic sporidesmin toxicity clinically with elevated γ-glutamyl transferase (GGT) and phytoporphyrin plasma levels. Histologically, bile ducts of smaller caliber than those injured by sporidesmin may be affected following turnip forage ingestion. Copper The heavy-metal copper is an essential trace element that plays an important role in numerous essential biological processes, including mitochondrial respiration (cytochrome C oxidase), connective-tissue maturation cross-linking (lysyl oxidase), antioxidant defense (superoxide dismutase), melanin synthesis (tyrosinase), iron metabolism (ceruloplasmin), and neurotransmitter biosynthesis (dopamine β hydroxylase). However, copper is toxic at excess concentrations because of its 2 redox states that can mediate free-radical production, resulting in direct oxidation of cellular components. The liver is the major organ involved in the regulation of copper levels, and homeostasis is maintained by the balance of dietary intake and copper excretion via the bile. Dietary copper is taken up by the enterocytes of the small intestine by dedicated transporters, divalent metal transporter (DMT1) and copper transporter 1 (Ctr1), and conveyed in the portal blood bound to carrier proteins ceruloplasmin and transcuprein, or in a nonspecific fashion to albumin, to the liver for uptake. In the hepatocyte, copper is sequestered in metallothionein or glutathione. Excretion into the bile or blood is regulated by a series of specific chaperone proteins, such as the copper-transporting ATPase ATP7A. Excess copper is stored in lysosomes. Copper-induced injury is produced by oxidative processes that initially reduce available glutathione and eventually damages lipids, proteins, and nucleic acids. Cellular metabolism, structural integrity, and energy generation are affected. Hepatic copper toxicosis can result from a primary metabolic defect in hepatic copper metabolism, altered hepatic biliary excretion of copper, or from excess dietary intake of the element. There is significant variation in species susceptibility to copper toxicosis. Sheep as a species are most prone to copper poisoning, because of reduced biliary excretion of copper. Some sheep breeds are more susceptible than others, reflecting differences in the efficiency of intestinal absorption of copper, rather than differences in efficiency of biliary excretion. Altered biliary excretion of copper in sheep does not appear to be associated with alteration in the structure or expression of the gene for ATP7B, the copper transporting P-type ATPase that is defective in Wilson disease, the human autosomal recessive disorder of hepatic copper metabolism. Excessive hepatic copper accumulation is important in chronic hepatitis in dogs, and is discussed in further detail in the section Chronic hepatitis in dogs. In cases of chronic hepatitis caused by copper accumulation, the liver is usually small, often with an accentuated lobular pattern; severely affected livers are characterized by architectural distortion, which ranges from a coarsely nodular texture to an end-stage liver. Chronic hepatitis, depending on the duration of inflammation and injury, is characterized by portal and periportal mononuclear cell inflammation and fibrosis of portal areas that may extend into adjacent periportal areas of the lobule, leading to the prominent lobular pattern. Small aggregates of pigmented macrophages, containing copper and lipofuscin, surrounded by mononuclear inflammatory cells are a reliable feature of copper excess. With progression, hyperplastic nodules and bridging fibrosis develop. In dogs and sheep, toxic amounts of copper can accumulate in the liver, although dietary copper levels are not excessive by standards for other species. Copper poisoning does occur in cattle and pigs, but in these species, it is because of abnormally high intake of the element. Acute copper poisoning can occur following either the ingestion or injection of excess copper, and animals deficient in vitamin E or molybdenum appear especially susceptible to acute copper poisoning. Chronic copper toxicosis in sheep occurs as a result of the presence of 3 environmental factors acting alone or in concert. First, excessive copper intake may occur as a result of contamination of water (naturally occurring or through the use of copper piping or fixtures), pasture or prepared feed; the latter is difficult to avoid when feed mills are preparing rations for different species and is probably partly responsible for the observation that housed sheep are more prone to copper poisoning than animals at pasture. Second, increased copper accumulation occurs as a result of increased availability of dietary copper; this happens when dietary levels of molybdenum are unusually low. Molybdenum, in the presence of sufficient sulfate, forms insoluble complexes with copper in the gut and liver, making the copper biologically inert. Subterranean clover growing on calcareous soils in southern Australia may be relatively deficient in molybdenum, and in these areas, British breeds of sheep are known to be more susceptible than Merinos to chronic copper poisoning. Other hepatotoxins constitute the third environmental factor that predisposes sheep to outbreaks of chronic copper poisoning. The most important of these are pyrrolizidine alkaloids (from Heliotropium or Echium) in eastern Australia, and phomopsin from lupins in western Australia and, possibly, South Africa. The basis for chronic copper poisoning in sheep is the peculiar avidity of the liver for copper, coupled with the very limited rate at which this species can excrete the element in the bile. After intraportal injection of a copper isotope, practically all the radioactivity is removed during the first passage through the liver. Most of the copper is sequestered in hepatocellular lysosomes, where it does little damage at concentrations of up to 200-300 µg/g dry weight. As the concentration rises, there is presumably more interaction between other cell components and the copper. There is some evidence that lysosomal membranes lose integrity and allow copper and lysosomal hydrolases to damage the rest of the cytoplasm. By the time the liver copper concentration has reached 300 µg/g or more, there is a histologically apparent increase in hepatocellular turnover, with single hepatocytes undergoing apoptosis within a dense knot of neutrophils. At still higher copper levels, the apoptotic rate increases, while all cells become swollen and their nuclei vesicular. The mitotic rate increases, presumably to keep pace with the accelerated loss of hepatocytes, and large macrophages appear in the sinusoids and stromal spaces about the vessels. These cells contain eosinophilic or somewhat brown, granular debris, which consists of copper-containing lipofuscins. Sheep with liver copper concentrations >1,000 µg/g may be clinically and hematologically normal, so long as the increasing mitotic rate produces enough new hepatocytes to take up the copper released by dying cells. At this stage, however, there will be elevated levels of liver-specific enzymes in the plasma. If the rate of hepatocellular loss exceeds the capacity of the liver to phagocytose and sequester cell debris quickly, the plasma copper levels can rise to levels that are high enough to damage circulating erythrocytes, and intravascular hemolysis ensues. The effect of the hemolysis and anemia on the liver is to accelerate the rate of hepatocellular necrosis; thus copper enters the circulation at an increasing rate and acute copper toxic crisis is manifest. The lethal clinical syndrome is then one of paroxysmal intravascular hemolysis and liver failure, in which a sheep may pass from apparent good health to death within ~6 hours. Stresses, such as brief starvation, may also precipitate the crisis in susceptible sheep, but the mechanisms involved are unknown. During the hemolytic crisis, some of the copper is lost from the disintegrating liver; some passes into the urine, and kidney copper concentration rises to 1,000 µg/g or more. Blood or kidney copper levels therefore give a truer indication of a prior hemolytic crisis caused by chronic copper poisoning than does elevation of liver copper alone. The gross lesions of fatal chronic copper poisoning are those of acute copper toxicity. The carcass is discolored by marked icterus, superimposed on which is the red color imparted by free hemoglobin. Often, there is a brown hue as well, because a proportion of the hemoglobin is oxidized to methemoglobin. The spleen is engorged, dark, and soft. The kidneys are deep red-brown to black and the urine deep red, as a result of hemoglobinuria with oxidation to methemoglobin, and concurrent icterus. The liver is often slightly soft and swollen, and deep orange, but if the condition arises after long-term liver injury, atrophy and fibrosis may be evident. The spectrum of liver lesions can be complicated by other causes of liver necrosis, including hypoxia caused by anemia, heart failure, and shock. A breed of sheep from the Hebridean island of North Ronaldsay has apparently adapted to a seaweed diet low in both copper and molybdenum but rich in zinc. Zinc is also capable of interfering with copper uptake, and these sheep, although avoiding copper deficiency, are exquisitely susceptible to chronic copper poisoning when transferred to normal pasture. Hepatic disease associated with copper toxicity in this breed appears to differ morphologically from other domesticated sheep breeds. Copper accumulation begins in periportal hepatocytes, accompanied by a mixed inflammatory infiltrate and cholangiolar proliferation. Characteristic pericellular fibrosis, initially confined to the portal tracts and periportal zones, later extends to diffuse fibrosis and cirrhosis, associated ultrastructurally with numerous hepatic stellate cells. North Ronaldsay sheep have been proposed as a possible animal model for human non-Wilsonian hepatic copper toxicosis and cirrhosis of infancy, and for investigation of copper-associated hepatic fibrogenesis. The events described in sheep also occur in chronic copper poisoning in pigs and cattle, and acute intravascular hemolysis may be seen, especially in calves. Usually, however, there is less of the acute terminal chain reaction in these species, and there is more evidence of chronic liver damage with extensive portal fibrosis and biliary hyperplasia within the triads. Acute copper poisoning is most often seen in ruminants after accidental administration of single large doses of copper, by either the oral or parenteral routes. Iatrogenic copper toxicosis has been induced by administering copper oxide boluses to neonatal calves, and by injection with copper disodium edetate in weanling calves. Doses of 20-100 mg/kg can produce acute poisoning in sheep. Copper toxicosis has also been reported in veal calves fed milk replacer supplemented with various copper-containing hematinics. Affected animals develop severe gastroenteritis, abdominal pain, diarrhea, and dehydration. The liver lesion varies with chronicity of exposure, from nonspecific acute centrilobular necrosis, to cholangiohepatitis with periportal fibrosis. Intravascular hemolysis may occur if plasma copper levels are sufficiently elevated. Acute bovine liver disease Acute bovine liver disease is a poorly understood entity that occurs in southern Australia. Outbreaks occur primarily in the spring and the autumn, and all ages of cattle can be affected. Sudden death or photosensitization may occur. There is an association with ingestion of Cynosurus echinatus (rough dog's tail). The characteristic histologic lesion involves a periportal hepatocellular necrosis with bile duct proliferation (eFig. 2-31). The presence of mycotoxin(s) produced by the fungus Dreschlera biseptata growing in some plants has been suggested to be the principal toxicant or cofactor in the syndrome. eFigure 2-31 The acute bovine liver disease syndrome is characterized by periportal necrosis of hepatocytes and bile duct proliferation. (Courtesy R. Kelly.) Further reading Albretsen JC, et al. Cycad palm toxicosis in dogs: 60 cases (1987-1997). J Am Vet Med Assoc 1998;213:99-101. Allen JG, et al. The toxicity of Myoporum tetrandrum (boobialla) and myoporaceous furanoid essential oils for ruminants. Aust Vet J 1978;54:287-292. Allen JG, Hancock GR. Evidence that phomopsins A and B are not the only toxic metabolites produced by Phomopsis leptostromiformis. J Appl Toxicol 1989;9:83-89. Allen JG, Randall AG. The clinical biochemistry of experimentally produced lupinosis in the sheep. Aust Vet J 1993;70:283-288. Aslani MR, et al. In vitro detection of hepatocytotoxic metabolites from Drechslera biseptata: a contributing factor to acute bovine liver disease? Aust J Exper Agric 2006;46:599-604. Bandarra PM, et al. Trema micrantha toxicity in horses in Brazil. Equine Vet J 2010;42:456-459. Bridges CH, et al. Kleingrass (Panicum coloratum L.) poisoning in sheep. Vet Pathol 1987;24:525-531. Brum KB, et al. Intoxication by Vernonia rubricaulis in cattle in Mato Grosso do Sul. Pesq Vet Bras 2002;22:119-128. Casteel SW, et al. Chronic toxicity of fumonisin in weanling pigs. J Vet Diagn Invest 1993;5:413-417. Cerqueira VD, et al. Colic caused by Panicum maximum toxicosis in Equidae in northern Brazil. J Vet Diagn Invest 2009;21:882-888. Cesar ASJ, et al. Toxic hepatopathy in sheep associated with the ingestion of the legume Tephrosia cinerea. J Vet Diagn Invest 2007;19:690-694. Collett MG. Bile duct lesions associated with turnip (Brassica rapa) photosensitization compared with those due to sporidesmin toxicosis in dairy cows. Vet Pathol 2014;51:986-991. Collett MG, et al. Photosensitisation, crystal-associated cholangiohepatopathy, and acute renal tubular necrosis in calves following ingestion of Phytolacca octandra (inkweed). N Z Vet J 2011;59:147-152. Colvin BM, et al. Fumonisin toxicosis in swine: clinical and pathologic findings. J Vet Diagn Invest 1993;5:232-241. Cornick JL, et al. Kleingrass-associated hepatotoxicosis in horses. J Am Vet Med Assoc 1988;193:932-935. Coulton MA, et al. Sporodesmin toxicosis in an alpaca. Aust Vet J 1997;75:136-138. Cruz C, et al. Experimentally induced cholangiohepatopathy by dosing sheep with fractionated extracts from Brachiaria decumbens. J Vet Diagn Invest 2001;13:170-172. Edens LM, et al. Cholestatic hepatopathy, thrombocytopenia and lymphopenia associated with iron toxicity in a Thoroughbred gelding. Equine Vet J 1993;25:81-84. Ferguson D, et al. Survival and prognostic indicators for cycad intoxication in dogs. J Vet Intern Med 2011;25:831-837. FitzGerald LM, et al. Hepatotoxicosis in dogs consuming a diet of camel meat contaminated with indospicine. Aust Vet J 2011;89:95-100. Fu PP, et al. Pyrrolizidine alkaloids—genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metab Rev 2004;36:1-55. Garcia AF, et al. Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics. Toxicon 2010;55:1331-1337. Glastonbury JRW, et al. A syndrome of hepatogenous photosensitization, resembling geeldikkop, in sheep grazing Tribulus terrestris. Aust Vet J 1984;61:314-316. Hamar DW, et al. Iatrogenic copper toxicosis induced by administering copper oxide boluses to neonatal calves. J Vet Diagn Invest 1997;9:441-443. Haywood S, et al. The greater susceptibility of North Ronaldsay sheep compared with Cambridge sheep to copper-induced oxidative stress, mitochondrial damage and hepatic stellate cell activation. J Comp Pathol 2005;133:114-127. Holland PT, et al. Isolation of the steroidal sapogenin epismilagenin from the bile of sheep affected by Panicum dichotomiflorum toxicosis. J Agric Food Chem 1991;39:1963-1965. Hooser SB, et al. Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa. Vet Pathol 1991;28:259-266. Howell JM, et al. Experimental copper and heliotrope intoxication in sheep: morphological changes. J Comp Pathol 1991;105:49-74. Ishmael J, et al. Experimental chronic copper toxicity in sheep. Biochemical and haematological studies during the development of lesions in the liver. Res Vet Sci 1972;13:22-29. Jago MV, et al. Lupinosis: response of sheep to different doses of phomopsin. Aust J Exp Biol Med Sci 1982;60:239-251. Jerrett IV, Chinnock RJ. Outbreaks of photosensitisation and deaths in cattle due to Myoporum aff. Insulare R. Br. toxicity. Aust Vet J 1983;60:183-186. Kellerman TS, et al. Photosensitivity in South Africa. VI. The experimental induction of geeldikkop in sheep with crude steroidal saponins from Tribulus terrestris. Onderstepoort J Vet Res 1991;58:47-53. Koppang N. Toxic effect of dimethylnitrosamine in cows. J Natl Cancer Inst 1974;52:523-531. Lockhart PJ, et al. Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue. Biochim Biophys Acta 2000;1491:229-239. Mackie JT, et al. Lupinosis in yearling cattle. Aust Vet J 1992;69:172-173. McLennan MW, Kelly WR. Cestrum parqui (green cestrum) poisoning in cattle. Aust Vet J 1984;61:289-29. Mendez MC, et al. Intoxication by Xanthium cavanillesii in cattle and sheep in southern Brazil. Vet Hum Toxicol 1998;40:144-147. Mercer HD, et al. Cassia occidentalis toxicosis in cattle. J Am Vet Med Assoc 1967;151:735-741. Miles CO, et al. Identification of insoluble salts of the β-D-glucuronides of episarsasapogenin and epismilagenin in the bile of lambs with alveld and examination of Narthecium ossifragum, Tribulus terrestris, and Panicum miliaceum for sapogenins. J Agric Food Chem 1993;41:914-917. Miles CO, et al. Photosensitivity in South Africa. VII. Chemical composition of biliary crystals from a sheep with experimentally induced geeldikkop. Onderstepoort J Vet Res 1994;61:215-222. Munday R. Studies on the mechanism of toxicity of the mycotoxin, sporidesmin. V. Generation of hydroxyl radical by sporidesmin. J Appl Toxicol 1987;7:17-22. Nation PN. Hepatic disease in Alberta horses: a retrospective study of “alsike clover poisoning” (1973-1988). Can Vet J 1991;32:602-607. Oelrichs PB, et al. Isolation and identification of the toxic peptides from Lophyrotoma zonalis (Pergidae) sawfly larvae. Toxicon 2001;39:1933-1936. Oliveira-Filho JP, et al. Hepatoencephalopathy syndrome due to Cassia occidentalis (Leguminosae, Caesalpinioideae) seed ingestion in horses. Equine Vet J 2013;45:240-244. Patamalai B, et al. The isolation and identification of steroidal sapogenins in Kleingrass. Vet Hum Toxicol 1990;32:314-318. Peterson JE, et al. The toxicity of phomopsin for sheep. Aust Vet J 1987;64:293-298. Puschner B, et al. Diagnosis of amanita toxicosis in a dog with acute hepatic necrosis. J Vet Diagn Invest 2007;19:312-317. Quinn JC, et al. Secondary plant products causing photosensitization in grazing herbivores: Their structure, activity and regulation. Int J Mol Sci 2014;15:1441-1465. Raposo JB, et al. Experimental intoxication by Myoporum laetum in sheep. Vet Hum Toxicol 1998;40:132-135. Simola O, et al. Pathologic findings and toxin identification in cyanobacterial (Nodularia spumigena) intoxication in a dog. Vet Pathol 2012;49:755-759. Steffen DJ, et al. Copper toxicosis in suckling beef calves associated with improper administration of copper oxide boluses. J Vet Diagn Invest 1997;9:443-446. Stegelmeier BL. Pyrrolizidine alkaloid–containing toxic plants (Senecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium, and Echium spp.). Vet Clin North Am Food Anim Pract 2011;27:419-428. Stegelmeier BL, et al. Pyrrole detection and the pathologic progression of Cynoglossum officinale (houndstongue) poisoning in horses. J Vet Diagn Invest 1996;8:81-90. Sullivan JM, et al. Copper toxicosis in veal calves. J Vet Diagn Invest 1991;3:161-164. Tan ET, et al. Determination of hepatotoxic indospicine in Australian camel meat by ultra-performance liquid chromatography-tandem mass spectrometry. J Agric Food Chem 2014;62:1974-1979. Tapia MO, et al. An outbreak of hepatogenous photosensitization in sheep grazing Tribulus terrestris in Argentina. Vet Hum Toxicol 1994;36:311-313. Tegzes JH, Puschner B. Amanita mushroom poisoning: efficacy of aggressive treatment of two dogs. Vet Hum Toxicol 2002;44:96-99. Thiel C, et al. The enterohepatic circulation of amanitin: kinetics and therapeutical implications. Toxicol Lett 2011;203:142-146. Tokarz D, et al. Amanitin toxicosis in two cats with acute hepatic and renal failure. Vet Pathol 2012;49:1032-1035. Van Der Lugt JJ, et al. Experimentally-induced Cestrum laevigatum (Schlechtd.) poisoning in sheep. Onderstepoort J Vet Res 1992;59:135-144. Voss KA, Riley RT. Fumonisin toxicity and mechanism of action: overview and current perspectives. Food Safety 2013;1:2013006-2013006. Wisløff H, et al. Accumulation of sapogenin conjugates and histological changes in the liver and kidneys of lambs suffering from alveld, a hepatogenous photosensitization disease of sheep grazing Narthecium ossifragum. Vet Res Commun 2002;26:381-396. Yee MM, et al. Amanitin intoxication in two beef calves in California. J Vet Diagn Invest 2012;24:241-244. Hyperplastic and Neoplastic Lesions of the Liver and Bile Ducts Remarkably, the occurrence of fatal liver malignancies is rather uncommon in aged dogs and cats; this suggests that they are either less exposed, or they are more resistant to the etiologic agents responsible for liver neoplasms in humans. Domestic species are not subject to significant chronic oncogenic viral infections, such as the hepatitis B and hepatitis C viruses that account for the majority of human hepatocellular carcinoma. Ectopic, metaplastic, and hyperplastic lesions Ectopic and metaplastic lesions in the gallbladder have been reported. Ectopic tissue includes hepatocyte nodules attached to or within the wall, and pancreatic islands, which may include islets of Langerhans. Gastric ectopia, readily distinguishable by the presence of chief and parietal cells, may form plaques or large polyps. Ectopia of cardiac or pyloric mucosa is mimicked by metaplastic changes. Nodular hyperplasia of hepatocytes is common in old dogs, but rare in other species. The lesions in dogs do not have a breed or sex predisposition, but their incidence increases sharply with age. Hyperplastic nodules typically develop in livers of normal mass, whereas regenerative nodules arise as a result of compensatory hyperplasia of surviving hepatocytes in a background of hepatic injury, atrophy, and fibrosis (see Responses of the liver to injury). Nodular hyperplasia often occurs as multiple randomly distributed masses throughout the lobes. The grossly visible nodules are spherical, well circumscribed, varying in size from 2 mm to 3 cm or more, and may bulge from the capsular surface or be entirely hidden within the parenchyma (Fig. 2-103 ). They may be sharply distinct from the surrounding parenchyma because of color differences, either lighter than the surrounding liver because of increased lipid or glycogen content within hepatocytes comprising the nodule, or darker because the sinusoidal vessels are distended with blood. Some nodules are the same color as the surrounding tissue and can only be identified by examination of a washed or blotted slice under a strong light. Figure 2-103 Nodular hyperplasia in the liver of a dog. The larger hyperplastic nodules grow expansively; they do not induce a fibrous capsule, but they can compress the surrounding parenchyma. The hepatocytes comprising nodular hyperplasia are often phenotypically different from those in adjacent parenchyma (Fig. 2-104 ). Old canine livers often have microscopic focal hyperplasia of similarly altered hepatocytes that can be distinguished from the surrounding parenchyma, but they evidently grow more slowly and do not compress their boundaries. These can be regarded as examples of the numerous atypical focal hyperplasias that arise as altered foci, nodules, plaques, or polyps in various tissues of old dogs. In more prominent paler nodules, hepatocytes can be enlarged and vacuolated with lipid or glycogen accumulation. Mitotic figures are uncommon. The sinusoids of the nodules are often dilated, and foci of hematopoiesis are occasionally observed. Necrosis and hemorrhage in hyperplastic nodules are rare. Figure 2-104 Nodular hyperplasia in the liver of a dog. These nodules have a discrete border with adjacent parenchyma and often are vacuolated. The significance of hyperplastic nodules in old dogs is usually negligible. After acute liver necrosis, these atypical hepatocytes within pre-existing hyperplastic foci and nodules may have phenotypic alterations that confer a survival advantage, and can survive and flourish as larger nodules amid regions of necrosis, so in these instances, they can be beneficial. Although hyperplastic nodules rarely progress to lesions with detrimental impact, they are often found during liver imaging, laparotomy, or biopsy and need to be differentiated from other focal lesions of more significance. Because they are atypically differentiated, they are the potential source of secreted products of clinical or diagnostic significance, but their potential in this regard is unexplored. The hallmark of nodular hyperplasia, in contrast to the hepatic adenoma and hepatocellular carcinoma, is that it largely retains normal liver architecture, including a modified lobular structure with recognizable central veins and portal triads. However, this feature may be difficult to appreciate in small biopsies. The biological distinction between atypical hyperplasia and neoplasia is sometimes equivocal; in the latter, continued growth that is independent of promoting influences is responsible for enlargement, and the designation of adenoma should apply to lesions that are enlarging. Cystic mucinous hyperplasia of the mucus-producing glands in the mucosa of the gallbladder has been reported as an incidental lesion in dogs and sheep. These cystic hyperplastic nodules are often numerous, may be sessile or polypoid, and contain mucin (Fig. 2-105 ). Figure 2-105 Cystic mucinous hyperplasia of the mucosa of the gallbladder in a dog. Dilation of the gallbladder with accumulated mucoid secretion (gallbladder mucocele) has been reported in dogs and infrequently in cats. Histologically, affected gallbladders display proliferated mucosa with characteristic projecting fronds of epithelium that extend into the lumen of the gallbladder, as well as formation of mucus-filled cysts. Severely affected gallbladders are distended with abnormal semisolid accumulations of mucus or other secretions, and the wall may undergo ischemic necrosis and rupture (Fig. 2-106 ). Bile-laden mucus may also extend into the cystic, hepatic, and common bile ducts, resulting in variable degrees of extrahepatic biliary obstruction. The etiology of this condition is unknown, and may be related to decreased gallbladder motility, bile stasis, and altered bile composition and viscosity. Figure 2-106 Gallbladder mucocele in a dog. Mucosal hyperplasia in the large bile ducts is frequently observed in the long-standing mild cholangiohepatitis of fluke infestation. The hyperplasia is of microscopic dimensions, but in some instances, it appears histologically to be atypical. Localized, polypoid foci of cystic hyperplasia are specific changes in cattle poisoned by highly chlorinated naphthalene. Hepatocellular tumors Primary epithelial neoplasms of the liver include tumors of either hepatocellular or cholangiocellular origin, and they are uncommon in most species, representing <1% of all neoplasms in cats and dogs. Pot-bellied pigs are a possible exception. Proportionately, hepatocellular neoplasms are more common in dogs, whereas cholangiocellular tumors predominate in cats. Unlike the situation in humans, there are no clear associations in domestic animals between hepatocellular tumors and viruses, chemical carcinogens, mycotoxins, or drugs, such as synthetic steroids. With the exception of the occasional association of liver tumors with certain flukes or tapeworm larvae, and the rare adenoma seen in the bovine liver with chronic pyrrolizidine poisoning, hepatobiliary tumors in domestic animals are not obvious successors to recognized antecedent liver diseases. Hepatocellular adenomas are benign neoplasms of hepatocytes. Hepatocellular adenomas are reported in dogs, cats, cattle, sheep, and pigs, but likely occur in all species. They are rarely of any clinical significance. Hepatocellular adenomas are typically solitary, but they can be multiple. They range from 2-12 cm in diameter and are roughly spherical masses. Typically, they are well demarcated from the adjacent parenchyma because of the compression caused by their expanding growth, but not encapsulated. Hepatocellular adenomas often bulge from the capsular surface, or they can be pedunculated, and in some instances, they may be found entirely within the hepatic parenchyma. The color of hepatocellular adenomas varies from yellow-brown to dark mahogany red. Paler adenomas contain lipid or glycogen, and these lesions often have a soft and friable consistency compared to normal liver (Fig. 2-107 ). Figure 2-107 Hepatocellular adenoma in a dog. Histologically, the hepatocytes in hepatic adenomas do not differ markedly from normal hepatocytes or those in hyperplastic nodules, and often contain lipid, glycogen, or occasionally protein secretory vacuoles. Minimal anisocytosis and increased basophilia of the cytoplasm and prominence of nucleoli can be seen, but mitotic figures are uncommon. Cells are arranged in cords or trabeculae that may be several cells thick, but the width tends to be consistent. There is a discrete well-defined border with adjacent parenchyma (Fig. 2-108 ). Adenomas lack normal lobular architecture. Essentially all of the blood supplying adenomas comes from the hepatic artery. As a consequence, rather than portal tracts, isolated arteries, bile ducts, and hepatic veins can be found coursing through the mass. Usually, no more than a single portal tract may be present, likely entrapped by the expanding hepatocyte population. Extramedullary hematopoietic foci may sometimes be found in the adenomas, as well as ectatic vascular spaces. Focal necrosis may occur. Figure 2-108 Histologic appearance of a canine hepatocellular adenoma. Compression of the adjacent parenchyma is apparent, and a capsule is evident at this site. Hepatocellular carcinomas are uncommon, but occur in all veterinary species; they may be single and massive, nodular or diffuse. Several features suggest malignancy, including the absence of pedunculation or clear demarcation from the adjacent parenchyma and the presence of varied coloration of the cut surface produced by hemorrhage and necrosis (eFig. 2-32). Venous invasion is typical of the hepatocellular tumors, and intravascular spread may extend to the large hepatic veins and vena cava. Carcinomas occasionally penetrate the capsule to implant on the peritoneum. Metastasis is uncommon in all domestic species but, when present, may be evident in the hepatic lymph node most often. Hematogenous metastases occur first in the lungs. Spontaneous rupture is common and may cause fatal blood loss. This consequence is a much more frequent cause of morbidity and mortality than metastatic disease. Some hepatocellular carcinomas in cattle are scirrhous, hard, and white. Paraneoplastic hypoglycemia may occur in animals with hepatocellular carcinoma. eFigure 2-32 Hepatocellular carcinoma in a dog. Hepatocellular carcinomas may be quite variable in histologic appearance, and multiple patterns may occur within a single neoplasm. Trabecular carcinomas are the most common histologic pattern (Fig. 2-109 ). Neoplastic cells resemble normal liver, and grow in irregularly thick plates or trabeculae that vary from few to many cells thick. Necrosis may be present, and sinusoids may be dilated, and there may be large ectatic blood-filled spaces. Other patterns include pseudoglandular (adenoid) carcinomas, characterized by lobular arrangement of neoplastic hepatocytes within scant connective-tissue stroma, and solid carcinomas, characterized by solid sheets of poorly differentiated, often pleomorphic cells that do not form sinusoids (Fig. 2-110A, B ). A scirrhous variant has been described in dogs, characterized by multiple foci of cytokeratin 19–positive ductular structures embedded in abundant fibrous stroma (Fig. 2-111A, B ). A feature of some hepatocellular tumors is the presence of giant cells, quite conspicuous by virtue of a large nucleus, multilobed nuclei, or multiple nuclei. Mitotic figures are more frequent in carcinomas than adenomas, but may not be a prominent feature of well-differentiated carcinomas. Immunohistochemical staining using antibodies that bind a hepatocyte specific antigen, HepPar1, identifies a large proportion of canine and feline hepatocellular carcinomas. However, more aggressive forms of canine hepatocellular carcinoma may not stain with HepPar1, but instead >5% of the cells can be stained with antibodies that recognize cytokeratin 19, a marker of hepatic progenitor cells, immature hepatocytes, and biliary epithelial cells. These canine hepatocellular carcinomas have an aggressive growth pattern, including a markedly increased incidence of intrahepatic and extrahepatic metastasis. Figure 2-109 Trabecular hepatocellular carcinoma from a dog. Figure 2-110 A. A solid pattern in a poorly differentiated hepatocellular carcinoma from a dog. B. Invasion at the margin of a hepatocellular carcinoma in the liver of a dog. Figure 2-111 A. The scirrhous variant of hepatocellular carcinoma has areas of ductular proliferation and abundant connective tissue within areas of neoplastic hepatocytes. B. Ductular structures staining with cytokeratin 19. Hepatoblastomas, rare benign tumors putatively originating from primitive hepatic precursor cells, have been reported most often in horses, but also in sheep, dogs, cats, and a llama. Hepatoblastomas in humans are neoplasms of infancy and childhood. Cases in domestic species have been reported in both young and adult animals, but are most frequently reported in foals. Hepatoblastomas are typically single, firm, lobulated masses with areas of necrosis and hemorrhage. Compression of adjacent parenchyma is evident and, on occasion, invasion can be seen. The histologic patterns of hepatoblastomas can be epithelial, either fetal or embryonal, or mixed epithelial and mesenchymal. Fetal hepatoblastomas are composed of large, polygonal cells, approximately the size of adult hepatocytes, with round to oval nuclei and granular to vacuolated, eosinophilic to amphophilic cytoplasm (Fig. 2-112A ). Embryonal hepatoblastomas form ribbons or rosettes of smaller basophilic cells with scant cytoplasm (Fig. 2-112B). Mixed epithelial-mesenchymal hepatoblastomas contain variable amounts of fibrous connective tissue or other mesenchymal tissues, including cartilage or bone, in addition to epithelial cells. Combinations of these patterns are common. Portal tracts are absent. Immunohistochemical staining for α-fetoprotein is typically positive, and HepPar-1 staining is usually absent, supporting the view that they arise from precursor cells. Figure 2-112 A. Hepatoblastoma with a fetal pattern from a foal. B. Hepatoblastoma with an embryonal pattern from a foal. Cholangiocellular tumors Cholangiocellular adenomas are uncommon benign neoplasms of biliary epithelium. Cholangiocellular adenomas are usually solitary, pale gray to white, and well-circumscribed roughly spherical masses that tend to grow by expansion. They may distend the normal outline of the liver, although they can occur as intrahepatic masses. Cholangiocellular adenomas are composed of tubules lined with a single layer of well-differentiated biliary epithelium and a moderate amount of intervening stroma. The tubules may have narrow lumens, and there can be variable amounts of stroma within the mass (Fig. 2-113 ). Adjacent hepatocytes are usually compressed at the margins, but are not found between tubules. The tubules contain a clear watery to viscous fluid. Cuboidal or flattened neoplastic biliary epithelial cells have a moderate amount of pale eosinophilic cytoplasm. Nuclei are round to oval, vesicular, and oriented centrally. Nucleoli are small or inapparent. There are typically no mitotic figures. Figure 2-113 Biliary adenomas are composed of uniform arrays of well-differentiated biliary epithelium forming uniform small tubules. Extrahepatic cholangiocellular adenomas are rare, with the exception of gallbladder adenomas reported in abattoir surveys of cattle and rarely in domestic carnivores (Fig. 2-114 ). The smaller specimens may be solid on cut surface and white, but the large specimens, and many of the small ones, are cystic. Figure 2-114 Gallbladder adenoma in a dog. (Courtesy J. Tobias.) Lesions formerly termed biliary cystadenomas, a subtype commonly seen in cats and occasionally in dogs, are most likely developmental anomalies of the embryonal biliary tree, termed ductal plate anomalies, which is more clearly appreciated when biliary cysts are coincident with cystic renal developmental lesions, although either lesion may occur independently. The ductal plate anomalies can be multilocular and lined by bile duct epithelia that may be flattened by pressure, or in some areas papillary. The septal stroma is collagenous. Their development later in life may be the result of progressive secretion by the lining epithelial cells, creating cysts only when sufficient fluid has been produced. Entrapped hepatocytes are often present. Cholangiocarcinomas (cholangiocellular carcinomas) are reported in dogs, cats, sheep, cattle, horses, and goats. Affected livers are usually otherwise normal, with no suggestion as to an underlying cause, although there are associations with chronic fluke infestations in humans and rarely in carnivores. Cholangiocellular tumors can usually be distinguished from the hepatocellular variety by their multiplicity, firmness, pale beige color produced by more or less abundant stroma, and the typical umbilicate appearance of those that involve the capsule (Fig. 2-115 ). The central depressed area can be the result of necrosis or cavitation-associated collapse of tumor vessels in the central parts of the tumor nodules. Even multiple nodules may not cause much enlargement of the liver. In dogs and cats, the tumors are almost always multiple or diffuse. The multiple nodules of tumor might represent intrahepatic lymphogenous metastases, but the possibility of multicentric origin must be entertained. Hematogenous metastases are unusual, but metastases to the regional nodes are common. In cats especially, there is a tendency to invade Glisson's capsule and implant on the peritoneum; the diffuse variety may cause great enlargement, although with retention of shape. Figure 2-115 A cholangiocarcinoma in the liver of a dog. Umbilication is prominent. (Courtesy J.L. Caswell.) Microscopically, cholangiocellular carcinomas form ductules and acini, and sometimes papillary formations within the lumen of the neoplastic ducts (Fig. 2-116 ). The cells are cuboidal or columnar, with a small amount of clear or slightly granular cytoplasm. The nuclei are small and fairly uniform, and nucleoli are not prominent. Mitotic figures are often abundant. The tubules do not contain bile, but in well-differentiated specimens may contain mucins. The epithelial components are separated by fibrous connective-tissue stroma, which may have pronounced collagen deposition, the so-called scirrhous response that gives the tumor a firm texture. In poorly differentiated cholangiocarcinomas, pleomorphic to anaplastic cells can be observed. It is not uncommon to encounter areas of poorly differentiated cells within a mass that is predominantly well differentiated. Cholangiocellular carcinomas have a highly invasive growth pattern, and frequently metastasize to hepatic lymph nodes, lungs, and the peritoneal cavity. Figure 2-116 Cholangiocarcinoma from a cat with characteristic crude acini and tubules separated by abundant connective tissue. Primary intrahepatic cholangiocellular carcinoma can be difficult, and often impossible, to distinguish from metastatic adenocarcinomas, especially those of pancreatic or mammary epithelial origin. Mucus secretion and intrasinusoidal permeation are more typical of biliary origin. Distinction from hepatocellular carcinoma of adenoid pattern may be assisted by demonstration of mucin, abundant mitotic figures, and presence of a prominent connective-tissue stroma. Extrahepatic cholangiocellular carcinoma (or biliary cholangiocarcinoma) of the gallbladder or extrahepatic bile ducts is much less frequent than the intrahepatic form, but has been reported in dogs, cats, cattle, and swine. Mixed hepatocellular and cholangiocellular carcinomas Rare hepatic carcinomas have the histologic and cytologic characteristics of both hepatocellular carcinoma and cholangiocellular carcinoma. These tumors likely arise from bipotential hepatic progenitor cells. The hepatocytic nature of some tumor cells can be confirmed with the monoclonal antibody HepPar-1, and cells of biliary phenotype can be stained by immunohistochemistry using antibodies that bind to cytokeratin 7, a typical intermediate filament of biliary epithelium (Fig. 2-117A-C ). Figure 2-117 A. Mixed hepatocellular and biliary carcinomas contain neoplastic cell populations with both biliary and hepatocellular phenotypes. H&E. B. HepPar1-stained cells. C. Cytokeratin 7–stained cells. Hepatic carcinoids Hepatic carcinoids, presumably arising from the diffuse neuroendocrine cell population found among the biliary epithelium and possibly within the hepatic parenchyma, have been reported in dogs, cats, and cattle. They may arise within the liver, in the extrahepatic bile ducts or within the gallbladder. The gross pathologic appearance of carcinoids is typically that of disseminated pale gray to tan small masses within the liver, but on some occasions, only a single mass is formed (eFig. 2-33). Like other neuroendocrine tumors, hepatic carcinoids typically form nests of cells separated by a fine fibrovascular stroma. The cells are oval to spindle shaped, and may form a rosette or pseudolobular pattern (Fig. 2-118 ). Mitotic figures are usually frequent. Argyrophilic cytoplasmic granules may be detected by silver impregnation stains; more precise identification of carcinoids may require immunohistochemical stains for neurosecretory products, such as neuron-specific enolase or serotonin. These are aggressive neoplasms, with frequent intrahepatic spread, and metastasis to local lymph nodes, peritoneum, and lung. Figure 2-118 Hepatic carcinoid from a cat with characteristic islands and rosettes separated by fine fibrovascular stroma and characteristic invasive behavior. eFigure 2-33 Hepatic carcinoid from a cat. Mesodermal tumors Primary mesenchymal tumors of the liver are quite uncommon. Of the mesenchymal tumors, primary hemangiosarcomas are likely the most frequent. Primary hepatic hemangiosarcomas occur in dogs, cats, cattle, and sheep. Hemangiosarcoma of the liver should always be considered metastatic until proven otherwise by diligent search. Some of these tumors are solitary, large, and gray-white, with scattered hemorrhagic areas, and others are ill defined and cavernous (Fig. 2-119 ). The latter may rupture into the peritoneal cavity to produce severe hemorrhage. Microscopically, hepatic hemangiosarcomas resemble those found in other sites; however, it may be impossible to find malignant cells in or lining cavernous areas. At the margins of the tumor, there is a distinctive pattern or growth in which small, solid nodules of malignant cells may be found, or these cells can be found forming capillary structures or invading along pre-existing sinusoids. The latter phenomenon is particularly characteristic. As the cells invade along the sinusoids, perhaps in single file, they initially produce little distortion of the hepatic cords. Behind them, the sinusoids are spread widely apart, and individual hepatocytes or portions of cords are isolated and appear to be floating freely, surrounded by a thin layer of connective tissue and neoplastic cells. Differential diagnoses other than metastatic lesions include the syndrome of telangiectasia in Pembroke Welsh Corgi dogs, telangiectatic lesions in older animals, and vascular hamartomas in cattle. Hepatic hemangiomas have been reported in dogs and a pig. Figure 2-119 Hepatic hemangiosarcoma in the liver of a dog. Careful examination is required to distinguish primary from metastatic disease. Leiomyomas are occasionally observed in the gallbladder of dogs and cattle. Leiomyosarcomas and fibrosarcomas have been reported in the liver of cats and dogs, hemangiopericytoma and fibrosarcoma in cattle, and a single case of botryoid-type embryonal rhabdomyosarcoma in a cat. Other rare mesenchymal tumors reported in either dogs or cats include lymphangioma, plasmacytoma, osteosarcoma, nerve sheath tumor, liposarcoma, and chondrosarcoma. The myelolipoma is an unusual tumor that develops in the livers of domestic cats and wild felids. The tumor develops as multiple growths, 0.5-5 cm diameter, in one or more lobes; if they project above the surface of the liver, they are irregularly nodular. Myelolipomas are friable, and yellow to orange because of their high fat content. The neoplasm is composed of normal-appearing, mature adipocytes with a variable admixture of myeloid cells, including both mature and immature cells of the granulocytic, erythrocytic, and megakaryocytic series (Fig. 2-120 ). In captive wild cats, similar lesions have been seen in the spleen. These were judged to be separate developments of the same process. Metastasis to other organs has not been reported. Figure 2-120 Myelolipoma of the liver of a dog. Metastatic neoplasms The liver is particularly “fertile soil,” as metastatic lesions markedly outnumber primary hepatic neoplasms. Metastatic solid neoplasms may be multiple, but are usually not numerous and usually do not elicit clinical or biochemical evidence of hepatic injury or dysfunction. Malignancies arriving via the portal vein, such as pancreatic or gastric carcinoma or hemangiosarcomas, may practically replace the liver before producing clinical signs, one of which may be icterus caused either by extrahepatic bile duct obstruction, or by intrahepatic cholestasis, or both. In dogs, the most common metastatic hematopoietic, mesenchymal, and epithelial neoplasms are lymphoma, hemangiosarcoma, and pancreatic carcinoma, respectively. Some of the carcinomas (e.g., thyroid, mammary), melanoma, and sarcomas from more remote sites also metastasize to the liver via the lungs and hepatic artery, and some (e.g., ovarian carcinoma, mesothelioma) implant on the capsular surface from within the peritoneal cavity. The hepatic perisinusoidal and periportal compartments are hospitable to various hematopoietic neoplastic cell types. Accordingly, malignant histiocytosis, myeloid and erythroid leukemias, and mast cell tumors often localize in the liver along hepatic sinusoids. Some lymphomas, histiocytic sarcomas (Fig. 2-121 ), plasmacytomas (Fig. 2-122 ), and mast cell tumors also produce solid sarcoma masses in the liver as primary or metastatic lesions. Lymphoma in the liver is common, especially when the spleen is involved, and occasionally, the liver appears to be the major or primary site affected. Typically, lymphoma is most apparent in the portal tracts, and the connective tissue surrounding the central veins can also be infiltrated, but late in the course of disease, the sinusoids are affected (eFig. 2-34). There are occasional exceptions to this pattern. Hepatosplenic γδ T-cell lymphomas (also CD3+ and CD11d+) in dogs demonstrate preferential involvement of the liver and spleen, and neoplastic lymphocytes are most common within the sinusoids. Hepatocytotrophic lymphoma is a T-cell lymphoma variant (CD3+, CD11d−) that invades hepatocytes. Diffuse infiltration of the liver in myeloproliferative disorders and mast cell leukemia may also cause extreme enlargement of the organ; the infiltrates localize preferentially in and around the sinusoids (Fig. 2-123 ). Hepatic infiltration may be in discrete nodules 2 cm or more in size, particularly lymphoma in horses, but it is usually diffuse in the connective tissues of the portal triads. Figure 2-121 Histiocytic sarcoma in the liver of a dog. Figure 2-122 Primary hepatic plasmacytoma in the liver of a dog. Figure 2-123 Monomyelocytic leukemia cells typically array along the hepatic sinusoids as seen in this dog. eFigure 2-34 Lymphoma typically infiltrates the stroma of portal tracts and hepatic venules as seen in this dog. Melanomas and hemangiosarcomas have characteristic gross features in the liver, but most metastatic tumors cannot be distinguished by their gross appearance. Sarcomas do tend to form a few large, smooth-surfaced nodules, and carcinomas do tend to form more nodules and to be umbilicate when in contact with Glisson's capsule. Hemangiosarcomas come from the spleen, usually, and may virtually replace the liver with small, blood-filled caverns. Their microscopic appearance is the same as that of the primary tumors. A non-neoplastic condition, hepatic splenosis, a rare condition in which normal splenic tissue becomes implanted into the liver, can be confused with metastatic disease. Normal spleen enters the liver, often via the portal vein after surgery or trauma to the spleen and multiple soft red masses up to ~4 cm in diameter can develop. Histologically, the masses resemble splenic tissue. Further reading Beeler-Marfisi J, et al. Equine primary liver tumors: a case series and review of the literature. J Vet Diagn Invest 2010;22:174-183. Bergman JR. Nodular hyperplasia in the liver of the dog: an association with changes in the Ito cell population. Vet Pathol 1985;22:427-438. Bettini G, Marcato PS. Primary hepatic tumours in cattle. A classification of 66 cases. J Comp Pathol 1992;107:19-34. Bosje JT, et al. Polycystic kidney and liver disease in cats. Vet Q 1998;20:136-139. Cantile C, et al. Hepatoblastoma in a foal. Equine Vet J 2001;33:214-216. Charles JA, et al. Morphological classification of neoplastic disorders of the canine and feline liver. In: Rothuizen J, et al., editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Philadelphia: Saunders Elsevier; 2006. p. 117-124. Desmet VJ. Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation.” Hepatol 1992;16:1069-1083. Fabry A, et al. Nodular hyperplasia of the liver in the beagle dog. Vet Pathol 1982;19:109-119. Fry MM, et al. Hepatosplenic lymphoma in a dog. Vet Pathol 2003;40:556-562. Gamlem H, et al. Canine vascular neoplasia—a population-based clinicopathologic study of 439 tumours and tumour-like lesions in 420 dogs. APMIS Suppl 2008;125:41-54. Haddad JL, Habecker PL. Hepatocellular carcinomas in Vietnamese pot-bellied pigs (Sus scrofa). J Vet Diagn Invest 2012;24:1047-1051. Haines VL, et al. Adenocarcinoma of the hepatopancreatic ampulla in a domestic cat. Vet Pathol 1996;33:439-441. Knostman KA, et al. Intrahepatic splenosis in a dog. Vet Pathol 2003;40:708-710. Lawrence HJ, et al. Nonlymphomatous hepatobiliary masses in cats: 41 cases (1972 to 1991). Vet Surg 1994;23:365-368. Minkus G, Hillemanns M. Botryoid-type embryonal rhabdomyosarcoma of liver in a young cat. Vet Pathol 1997;34:618-621. Moore FM, Thornton GW. Telangiectasia of Pembroke Welsh corgi dogs. Vet Pathol 1983;20:203-208. Mueller POE, et al. Antemortem diagnosis of cholangiocellular carcinoma in a horse. J Am Vet Med Assoc 1992;201:899-901. Neu SM. Hepatoblastoma in an equine fetus. J Vet Diagn Invest 1993;5:634-637. Patnaik AK, et al. Canine bile duct carcinoma. Vet Pathol 1981;18:439-444. Patnaik AK, et al. Canine hepatic carcinoids. Vet Pathol 1981;18:445-453. Patnaik AK, et al. Canine hepatocellular carcinoma. Vet Pathol 1981;18:427-438. Patnaik AK, et al. Hepatobiliary neuroendocrine carcinoma in cats: a clinicopathologic, immunohistochemical, and ultrastructural study of 17 cases. Vet Pathol 2005;42:331-337. Pollock S, Wagner BM. Hemangioma of the liver in a dog. Vet Med Small Anim Clin 1972;67:863-867. Post G, Patnaik AK. Nonhematopoietic hepatic neoplasms in cats: 21 cases (1983-1988). J Am Vet Med Assoc 1992;201:1080-1082. Rodríguez F, et al. Cholangiocarcinoma in a goat. Vet Rec 1996;139:143-144. Shiga A, et al. Hepatoblastoma in a dog. J Vet Med Sci 1997;59:1167-1170. Tanimoto T, Ohtsuki Y. Hepatic haemangioma in a pig. Vet Rec 1992;131:176-177. van den Ingh TS, Rothuizen J. Congenital cystic disease of the liver in seven dogs. J Comp Pathol 1985;95:405-414. van den Ingh TSGAM, et al. Morphological classification of biliary disorders of the canine and feline liver. In: Rothuizen J, et al., editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Philadelphia: Saunders Elsevier; 2006. p. 61-76. van Sprundel RG, et al. Classification of primary hepatic tumours in the dog. Vet J 2013;197:596-606. Zini E, et al. Paraneoplastic hypoglycemia due to an insulin-like growth factor type-II secreting hepatocellular carcinoma in a dog. J Vet Intern Med 2007;21:193-195.

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Journal ID (nlm-ta): Jubb, Kennedy & Palmer's Pathology of Domestic Animals: Volume 2

Title: Jubb, Kennedy & Palmer's Pathology of Domestic Animals: Volume 2

Publication date PMC-release: 5 February 2016

Publication date (Print): 2016

Publication date (Electronic): 5 February 2016

Pages: 258-352.e1

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Co-Executive Director, Laboratory Service Division, Director, Animal Health Laboratory, University of Guelph, Guelph, Ontario, Canada

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Publisher ID: B978-0-7020-5318-4.00008-5

DOI: 10.1016/B978-0-7020-5318-4.00008-5

PMC ID: 7149729

SO-VID: 0c24cefd-0636-4b22-9e8f-b1c85ff654ae

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