Wilson's disease is an autosomal recessive inherited disorder of copper metabolism 354546. Wilson's disease results in copper accumulation in the liver, cornea and brain 1. The worldwide incidence of Wilson's disease, independent of ethnic and geographic origin, is approximately 1 in 30,000 47. However, it has been noted that the disease may be more common than previously expected, because most incidence estimations are based on adolescent or adults presenting with neurologic symptoms, which occur only in about half of the patients 48. The Wilson's disease gene is localized on human chromosome 13 and codes for ATP7B, a copper transporting P-type ATPase 21. The Wilson's disease mutations occur throughout the whole gene and include missense and nonsense mutations, deletions and insertions 49. Most of the more than 80 mutations are present at a low frequency, and mutations differ between ethnic groups 50. Thus, diagnosis of Wilson's disease is challenging and requires a battery of tests, including morphologic evaluation and copper analysis of liver tissue 5152. Liver disease may mimic various forms of common liver conditions, ranging from fulminant hepatic failure, chronic hepatitis, and cirrhosis 1.

The LEC rat 36 and the toxic milk mouse are the only known valid animal models of Wilson's disease 1137.

The LEC rat with a hooded dilute agouti coat is a mutant inbred strain, which was established from a closed colony of randomly bred Long-Evans rats 36. Long Evans Cinnamon rats suffer from fulminant hepatitis and severe jaundice at about 4 months of age and show similarities to Wilson's disease in many clinical and biochemical features 1036. This mutant has a deletion in the copper transporting ATPase gene (Atp7b) homologous to the human Wilson's disease gene (ATP7B) 1053, and the mode of inheritance of hepatitis is also autosomal recessive 54.

Similar to the condition in Wilson's disease, LEC rats manifest elevated hepatic copper levels, defective incorporation of copper into ceruloplasmin, and reduced biliary excretion of copper 55. LEC rats develop intravascular haemolysis secondary to the release of large amounts of non-ceruloplasmin copper into the bloodstream 56, as it has been described in patients with Wilson's disease 14.

The hepatic copper concentration can rise to 2,126 ppm dry weight 57. It is also known that LEC rats may accumulate as much iron as copper in the liver as a result of hemolysis 56. This mutant strain also possesses reduced hepatic selenium 58. Both accumulation of iron and depletion of selenium in the liver may contribute to the development of fulminant hepatitis, hepatic fibrosis, and subsequent hepatocarcinogenesis in LEC rats by increasing the process of oxidative damage with copper, and a reduction in the antioxidant capacity against copper-induced free-radical damage 5658.

It has been suggested that an immune-mediated mechanism may play a role in the development of acute lethal hepatitis in LEC rats. Autoimmune antibodies to liver microsomal proteins have been demonstrated 3–7 weeks before death in these rats 59. Protein disulfide isomerase and calreticulin have been identified as antigens in liver microsomes of this mutant 60, and treatment with immunosuppressant drugs such as cyclosporin-A reduced the mortality in LEC rats 61. However, a recent study showed that the development of antimicrosomal antibodies does not precede the development of severe liver damage in the LEC rat model 62.

The clinical signs of hepatitis include severe jaundice, a bleeding tendency, oliguria, lethargy, and loss of body weight. During this period, activities of serum enzymes, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) 63, aspartate aminotransferase (AST), and γ-glutamyltransferase (GGT), as well as bilirubin levels, are increased significantly 365462. While the serum levels of ceruloplasmin remain reduced all the time 60, the copper concentration in serum increases mainly after the onset of jaundice 63. About half of the animals die within a week of the onset of jaundice 36.

Histological changes of acute hepatitis in LEC rats occur prior to 8 weeks of age, and the most drastic changes occur from 17 to 20 weeks of age 36. A recent study utilizing female LEC rats reports biochemical and morphological evidence of severe liver damage at 12 weeks of age 62. Histological changes are characterized by hepatocellular karyomegaly, large numbers of Councilman bodies, submassive necrosis 54, mitosis of hepatocytes 36, and apoptosis 56. After this stage, surviving rats develop chronic hepatitis, cholangiofibrosis, preneoplastic foci and nodules, and hepatocellular carcinomas 465456. Histochemical examination of LEC rat liver for copper reveals that copper accumulates preferentially in hepatocytes and distributes diffusely throughout the cytoplasm. Copper accumulates in virtually all hepatocytes throughout the entire liver lobule, but shows a tendency to localize in the periportal areas 64. LEC rats that survive the stage of fulminant hepatitis develop cirrhosis 65, and are highly susceptible to the development of hepatocellular carcinoma 64. This is an interesting feature of this particular animal model because hepatocellular carcinoma is rarely diagnosed in Wilson's disease patients 66.

Toxic milk is an autosomal recessive mutation which alters copper homeostasis in mice 37. Offspring of mutant females are born copper-deficient and since their mother's milk is also low in copper, babies die at 2 weeks of age. The progeny of affected dams fostered to lactating normal females survive but with age copper accumulate in their livers 67. By 6 months of age, liver changes are characterized by nodular fibrosis, bile duct hyperplasia and portal lymphocytic inflammatory cell infiltration 67. Toxic milk mice share some biochemical abnormalities with Wilson's disease for example concentration of serum copper and ceruloplasmin are decreased. The genetic defect in toxic milk mice is similar to that of Wilson's disease 11. Although gross and histologic changes in the liver in both rodent models (i.e. LEC rat and toxic milk mice) resemble Wilson's disease 67, same differences have been noted. Neither of the rodent models has neurologic symptoms, and in the toxic milk mice model affected dams produce Cu-deficient milk, whereas there are no reports of Cu-deficient milk in humans mothers with Wilson's disease 68.

Indian childhood cirrhosis and its analogues endemic Tyrolean infantile cirrhosis, and idiopathic copper toxicosis, are fatal liver diseases seen in young children due to genetic susceptibility to minimal excess in dietary copper 2347. The gene for Indian childhood cirrhosis diagnosed in North America has recently been identified 69.

This primitive breed has adapted to copper impoverished environment and display an abnormal sensitivity to copper toxicity when transferred to copper adequate location 70. A recent study reports the remarkable similarities between the condition in North Ronaldsay sheep and Indian childhood cirrhosis, whereby affected sheep exhibited liver changes varying from active hepatitis to panlobular pericellular fibrosis, and cirrhosis. Histochemical stains demonstrated periportal to panlobular histochemical copper retention 6.

Primary biliary cirrhosis is a chronic progressive, often fatal liver disease, characterized by the eventual development of cirrhosis and liver failure 3141. Middle-aged females are predisposed to the condition 71. The immunological abnormalities and morphologic features observed in primary biliary cirrhosis, favour the hypothesis of an immune-mediated mechanism 7273. However, the nature of the initiating factor(s) and of the sensitizing antigen is unknown. The only effective treatment for this disease is liver transplantation 74. Indeed, primary biliary cirrhosis is one of the five most frequent causes of liver transplantation 75. Although copper accumulation is a secondary event 76, therapeutic approaches have included attempts to remove excess hepatic copper in order to avoid possible synergism between the initiating factor(s) and release of copper into the liver 77.

Certain disorders, such as chronic active hepatitis in Doberman pinschers and Skye terrier hepatitis are characterized by copper retention secondary to the underlying disease, thus resembling primary biliary cirrhosis in humans 44. Copper values are never so elevated as in the familial storage diseases.

Doberman hepatitis is a disorder associated with histologic features of chronic active hepatitis, cholestasis, and cirrhosis. Middle aged, spayed female dogs are predisposed to the disease 94378. The cause of the disease remains undetermined, but histopathological changes support the idea of immune-mediated disorder 79. Copper accumulates in centroacinar (portal) areas 980. The present knowledge on the role of copper in this disorder is incomplete; however, the presence of copper in liver biopsies constitutes one of the essential diagnostic criteria for subclinical Doberman hepatitis 80.

This condition appears to be an unusual lesion characterized by intracanalicular cholestasis, with copper accumulation and hepatocellular degeneration culminating in cirrhosis 44. Copper accumulation occurs primarily in the periacinar area, which is inconsistent with other disorders associated with cholestasis and subsequent tissue copper retention 81. The cause is unknown, but an inheritable metabolic defect involving membrane transfer and transport systems in the periacinar zone, resulting in disturbed bile secretion and excessive copper accumulation have been suggested 44.

This condition has three well-characterized histopathologic lesions, but the etiology and pathogenesis of the condition is poorly understood 82. The non-suppurative type has been compared to primary biliary cirrhosis in humans 8384. Non-suppurative cholangitis in cats is characterized by lymphocytic and plasmacytic portal infiltration, bile duct hyperplasia, and portal fibrosis. Histochemical stains demonstrate copper positive granules within portal hepatocytes (ICF personal observation). The value of this model in the study of primary biliary cirrhosis has not been thoroughly assessed.

In general, naturally occurring canine genetic diseases resemble human diseases more faithfully than their rodent counterpart, as there is a higher degree of DNA sequence identity between humans and dogs than between humans and rodents 85. Familial copper storage disorders in Bedlington and West Highland white terriers are usually associated with early subclinical disease during which copper accumulates with subsequent liver injury culminating in cirrhosis 4086. Occasionally however, there may be a copper-induced hemolytic crisis in Bedlington terriers 8687. Bedlington Terrier copper toxicosis has generated much interest as a possible animal model for Wilson's disease. However, it has been proven that Wilson's disease and Bedlington Terrier copper toxicosis do not share the same genetic defect 88. The canine copper toxicosis locus in Bedlington terriers has been mapped to canine chromosome region CFA 10q26 88 and in addition to ATPB, ATOX 1 8990, ATP6H 91 have been excluded as candidate genes underlying copper toxicosis in Bedlington terriers 8990. Recently, a mutated MURR1 gene was discovered in Bedlington terriers affected with the disease 85.

Some similarities have been noted between Bedlington terriers and idiopathic childhood cirrhosis. Idiopathic childhood cirrhosis is biochemically similar to copper toxicosis in Bedlington terriers 85, but clinically much more severe 7. Both conditions are characterized by the absence of neurologic damage and Kayser-Fleisher rings 9293, and normal ceruloplasmin levels 47.

Liver copper values can be as high as 12,000 ppm dry weight in Bedlington terriers 94 whereas the highest copper value recorded in West Highland white terriers is 6,800 ppm dry weight 95. In Bedlington terriers older than 1 year of age, there is a progressive increase in the accumulation of tissue copper until 8 years of age 81. There is no relationship between age, histomorphological changes and hepatic copper concentration in West Highland white terriers. The clinical signs vary widely, depending on the stage of the disease. Affected animals in the early stages usually are asymptomatic. Depression, anorexia, lethargy, vomiting, and increased ALT are usually associated with an acute onset of hepatic necrosis. Microscopically, there is a spectrum of recognizable changes. In the least affected or subclinical cases, accumulation of intracytoplasmic refractile, light-brown granules in vacuolated parenchymal cells of the periacinar zones are observed which stain histochemically positive for copper. This is followed by the development of foci of hepatocellular degeneration and necrosis with scattered inflammatory response of neutrophils, lymphocytes and plasma cells. Fine fibrous septa extend from the portal areas into the lobules. More advanced stages are characterized by periportal infiltrates of inflammatory cells and prominent piecemeal necrosis (resembling chronic active hepatitis). Piecemeal necrosis has not been described in West Highland white terriers toxicosis. Finally, there is a complete architectural disorganization of the liver with variable sized nodules of hepatocytes (with focal degeneration and aggregates of neutrophils) separated by bands of fibrous connective tissue with portal-central bridging 9496. Contrary to Wilson's disease, hepatocytes with Mallory bodies have not been identified 97.

Copper-associated liver disease has increasingly being recognized in Dalmatians 9899 (Figs. 1,2). Fulminant hepatic failure characterized by high serum activities of ALT and AST. Histologically, there is severe necrosis of hepatocytes involving the centrilobular areas. Macrophages and surviving hepatocytes contain copper-positive material 98. Primary copper storage disease was suspected on the basis of histologic findings and high copper concentration in the liver 99. Morphologic changes observed in Dalmatians are strikingly similar to those observed in LEC rats (ICF personal observation) suggesting that copper toxicosis in Dalmatians is a promising spontaneous animal model of Wilson's disease.

Excess copper accumulation occurs as a consequence of chronic liver disease in other canine breeds dogs 594344, particularly Cocker Spaniels and Poodles 9. Similarly, secondary copper accumulation has been described in chronic hepatitis in humans 42.

Sheep are particularly susceptible to copper poisoning 100101. The condition occurs when sheep are accidentally fed rations prepared for other species (i.e. bovine or swine) 100102. Copper poisoning in sheep is commonly diagnosed. In contrast, copper toxicosis in other farm animals is rarely reported 103.

Chronic copper poisoning in sheep results from the accumulation of copper in hepatic tissue over a period of a few weeks to more than a year 100101, and is considered to have two distinct phases 104. During the accumulation or pre-hemolytic phase, animals may be clinically normal, even with liver copper concentrations of 1,000 ppm, so long as increasing mitotic rate produces enough new hepatocytes to take up the copper released by dying cells 94. However, liver damage does occur during this period as indicated by increased levels of lactic dehydrogenase and AST 100. The second phase, or hemolytic crisis, lasts from hours to days and is characterized by the sudden onset of severe intravascular hemolysis and hemoglobinemia associated with increased blood copper levels, with resulting liver 104105, kidney, and brain damage 106.

During the hemolytic phase, elevated serum values of AST, GGT, ALP, copper, urea nitrogen (BUN), bilirubin 100107, and ceruloplasmin 108 have been observed. Elevation of blood copper occurs prior to and during the hemolysis; the ceruloplasmin levels tend to fluctuate but in most instances there is a two-fold or more increase immediately before and during the hemolytic crisis 108. The histologic changes are present in the liver in the preclinical stages and can be somewhat obscured by the periacinar (centrilobular) necrosis of hypoxemia and the bile accumulations of hemolytic disease 94. Copper can be demonstrated histochemically with rubeanic acid or rhodanine as fine granules within the cytoplasm of parenchymal and Kupffer cells 104108. Copper in hepatocytes is mostly located within lysosomes 109. It has been demonstrated that copper deposition begins in the periacinar areas extending to the mid and periportal zones with progressive copper-loading 109.

Liver disease associated with centrilobular (periacinar, zone 3) copper accumulation was described in a Siamese cat 110. One of the authors (ICF) has examined cases of copper-associated liver disease in cats, with morphologic characteristics similar to those reported in Siamese cats (Figs. 3,4), suggesting that metabolic defects of copper metabolism occur with relative frequency in the feline specie and may have been overlooked in the past.

Two cases have been described in ferrets 111. One case was characterized by chronic hepatopathy, with diffuse hepatocellular vacuolation, and the second case had centrilobular degeneration and necrosis. In both ferrets, liver copper concentration was markedly elevated and special staining revealed copper pigment in hepatocytes and macrophages. An inherited defect of copper metabolism was suspected in these ferrets based on the lack of related illness in 11 other ferrets housed in the same environment and receiving the same diet.

Liver sections are dewaxed and hydrated to distilled water. Then, sections are placed in rhodanine working solution at 37°C for 18 hours. Rhodanine working solution is prepared using 3.0 ml of 0.2% Rhodanine stock solution (0.2 g rhodanine in 100 ml 100% ethanol) and 50 ml buffer acetate (5.0 ml 40% formalin, 20 g sodium acetate in 1000 ml distilled water).

Slides are rinsed in 3 changes of acetate buffer solution, counterstained in Mayer's hematoxylin solution, rinsed in acetate buffer, dehydrated, cleared and mounted.

Liver samples are placed in plastic bags, frozen at -80°C and stored for 24 hours. Tissues are then processed for atomic absorption spectrophotometry. Briefly, approximately 1 gram of tissue from each sample is weighed and placed in a Teflon container for microwave digestion. All samples are weighed in duplicate. Control samples with known amounts of copper are also included. To each vessel, including a blank containing no tissue, 1.0 ml of deionized, distilled water and 2.5 ml full strength, trace metal free nitric acid (10.95 M HNO₃) is added. Vessels are capped and placed in a MDS-200 microwave oven and digested. The digested contents are poured off and diluted to 10 ml with distilled water and thoroughly mixed. Copper concentrations are measured using a spectrophometer equipped with a copper hollow cathode lamp, with the following instrument settings: wavelength 324.8 nm, slit width 0.7 nm, lamp current 17 mA. Standards used are 3, 6 and 12 ppm prepared from 1000 ppm stock standard in 0.2 M HNO₃. Peak area is read with a read delay of 0 s, and a read time of 5 s. Standard reference materials are used as quality control substances, and are processed with each batch of liver samples. Samples are not corrected for recovery if the he recovery of the quality control samples is 95% or more. Cu concentrations are recorded in μg/g wet weight tissue.