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Chronic hepatitis: A retrospective study in 34 dogs. Carmen Fuentealba, Shirleen .... such as, autoimmune hemolytic anemia and systemic lupus erythematosus.

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Chronic hepatitis: A retrospective study in 34 dogs Carmen Fuentealba, Shirleen Guest, Susan Haywood, Barbara Horney Abstract The aims of this study were to characterize the histological changes observed in 34 accessioned cases of canine chronic hepatitis and to correlate these changes with the clinical pathological data. Cases of chronic hepatitis were subdivided into 6 categories: chronic active hepatitis (10/34), chronic persistent hepatitis (7/32), chronic cholestatic hepatitis (6/34), fibrosing hepatitis with cirrhosis (3/34), chronic cholangiohepatitis (3/34), and miscellaneous secondary hepatitis (5/34). Iron accumulation was a consistent finding in all livers examined. Although all cases of chronic hepatitis had elevated liver enzymes, no correlation was detected between biochemical parameters and the severity of morphologic changes. Similarly, no correlation was detected between rhodanine staining for copper and morphologic or biochemical indicators of cholestasis. However, presence of copper correlated well with reticulo-fibrosis (r = 0.8) and bile duct hyperplasia, suggesting that changes in the hemodynamics of the hepatic acini due to fibrosis could influence storage of copper. Resume Hepatite chronique: etude retrospective chez 34 chiens. L'objectif de cette etude etait de caracteriser les modifications histologiques observees chez 34 cas etablis d'hepatite canine chronique et de faire des correlations entre ces modifications et les donnees de pathologie clinique. Les cas d'hepatite chronique ont ete subdivises en 6 categories : hepatite chronique active (10/34), hepatite chronique persistante (7/34), hepatite cholestatique chronique (6/34), hepatite fibrosante avec cirrhose (3/34), cholangiohepatite chronique (3/34) et hepatites secondaires diverses (5/34). L'accumulation de fer etait un fait constant dans tous les foies observes. Bien que tous les cas d'hepatite chronique montraient un taux d'enzymes hepatiques eleve, aucune correlation n'a ete etablie entre les parametres biochimiques et la severite des modifications morphologiques. Une similarite sans correlation a pu 'tre detectee entre la coloration 'a la rhodanine pour le cuivre et les indicateurs morphologiques ou biochimiques de cholestase. Cependant, la presence de cuivre avait une bonne correlation avec la reticulofibrose (r = 0,8) et l'hyperplasie des canaux biliaires, suggerant que les changements hemodynamiques relies 'a la fibrose qui affectent les acini hepatiques puissent influencer l'entreposage du cuivre. (Tr-aduit par docteur Andre Blouin) Can Vet J 1997: 38: 365-373

Introduction Chronic hepatic disease comprises a variety of pathologic processes that can affect dogs of any age (1). The liver's response to injury by a variety of etiologic agents results in similar microscopic patterns, making it difficult to distinguish specific disease entities (2,3). Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenuce, Charlottetown, Prince Edward Island CIA 4P3 (Fuentealba, Guest, Horney); Department of Veterinary Pathology, Faculty of Veterinary Sciences, University of Liverpool, Liverpool, England (Haywood). Susan Haywood was the recipient of a Wellcome Trust Veterinary Research Travel Grant held at the Atlantic Veterinary College. Shirleen Guest was supported by an Animal Health Trust of Canada Student Summer Research Award. This study was funded by a Companion Animal Trust Fund Award. Can Vet J Volume 38, June 1997 1997365

Adding to these obstacles, the terminology used to classify chronic hepatitis is often confusing. In the human medical literature, chronic hepatitis is defined as mononuclear or mixed inflammatory infiltration associated with clinical or biochemical evidence of hepatocellular dysfunction continuing without improvement for at least 6 mo (4), whereas in the veterinary literature, it has been suggested that a diagnosis of chronic hepatitis should be made after a documented rise of alanine aminotransferase (ALT) for a minimum of 4 mo, with concomitant histologic evidence of liver damage during that 4-month period (3). In humans, chronic hepatitis has been divided into 2 groups, chronic persistent hepatitis and chronic active hepatitis, based on the predominant microscopic pattern and relationship of the inflammatory reaction to the liver parenchyma (5). No such categorization has been achieved in veterinary medicine, and a somewhat arbitrary classification has been ascribed to canine diseases of a chronic nature, such as, chronic 365

active hepatitis (1), lobular dissecting hepatitis (6), and postnecrotic cirrhosis (3,7-9). The cause and pathogenesis of chronic hepatic disease is usually unknown. In humans, chronic active hepatitis most often occurs as a sequel to viral hepatitis or immune-mediated diseases, such as, autoimmune hemolytic anemia and systemic lupus erythematosus (10). In dogs, chronic hepatitis with fibrosis has been induced experimentally with infectious canine hepatitis virus (11). Additionally, inflammatory conditions of the pancreas and gastrointestinal tract (12), and prolonged use of corticosteroids (13), anticonvulsants (14), and Corynebacterium parvum immunotherapy (15) have been implicated as causes of chronic hepatic injury in dogs. Finally, copper-induced chronic hepatitis culminating in cirrhosis occurs in certain breeds of dogs (Bedlington terrier) as a genetic defect (16). The aims of this study were to (1) characterize the histological changes observed in accessioned cases of canine chronic hepatitis; (2) correlate the histopathologic changes with the clinical pathology data; (3) correlate the presence of copper, iron, fibrous connective tissue, and reticulin fibers with indicators of cholestasis; and (4) compare the morphologic with the clinical pathologic indicators of cholestasis.

Materials and methods A case search was performed on the pathology records held at the Atlantic Veterinary College. The number of canine cases submitted between January 1988 and May 1994 was recorded, and all cases of "chronic hepatitis" diagnosed by histologic examination were selected. All clinical pathology and morphologic pathology records relevant to the cases studied were located and the clinical pathology data were recorded for statistical analysis. In cases where there were more than 1 set of biochemical and hematologic results, the one closest to the date of biopsy or postmortem examination was chosen.

Histopathology The paraffin blocks of liver tissue were retrieved, and sections, 6 pm thick, were cut and processed for routine hematoxylin and eosin (HE) staining. Additionally, silver staining for reticulin fibers, Masson's trichrome stain for fibrous connective tissue, Perl's Prussian blue method for iron (17), and rhodanine stain for copper (18) were used. Both the HE and histochemically stained sections were examined by light microscopy. The HE stain was used to characterize the inflammatory infiltrate and to evaluate the presence and distribution of fibrosis, necrosis, bile duct hyperplasia (BDH), and pigment. Evaluation was done using the following grading system: - = absent, + = mild, ++ = moderate, and +++ = severe. The nomenclature employed for the designation of the liver lobule into zones was based on the microcirculatory unit described by Rappaport (19). Zone 1, the periportal zone, refers to the cells closest to the afferent vessels receiving enriched blood; zone 2, also known as the midzone, corresponds to the parenchyma between zones 1 and 3; and zone 3, the former centrilobular 366

area, includes cells at the microcirculatory periphery of the acinar unit receiving blood low in 02 and nutrients. The zonal hepatic distribution of histochemical stains was noted and the histochemical staining in the 3 hepatic zones was evaluated, as previously described (18). Following histological examination, chronic hepatitis was subdivided into 6 general categories: chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), chronic cholestatic hepatitis CCH), fibrosing hepatitis with cirrhosis (FH/C), chronic cholangiohepatitis (CH), and miscellaneous secondary hepatitis (MSH). The standard used for this classification was a modification of that described by Anderson and Sevelius (20).

Statistical analysis Analysis of variance and Duncan's multiple range test were used to compare the histopathological, hematological, and biochemical data. Pearson's correlation coefficient was used to assess the relationship between the presence of copper, iron, reticulin fibers, and fibrous connective tissue and the clinical pathological indicators of cholestasis, namely, bilirubin, alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and cholesterol. The level of significance was set at P < 0.05.

Results Forty-six hundred and eighty-seven canine cases were submitted to the Pathology Diagnostic Services at the Atlantic Veterinary College between January 1988 and May 1994. Hepatitis was diagnosed in 47 cases (1.0%). Chronic hepatitis represented 72.3% (34/47) of the total number of hepatitis cases. The group of dogs with chronic hepatitis consisted of 23 females (70.6%) and 11 males (29.4%), with an age range of 3 to 17 y and a mean of 7.6 y (Table 1). Of the breeds represented, Doberman pinscher (6/34), cocker spaniel (3/34), and poodles (3/34) appeared more than once (Table 1). Doberman pinschers accounted for 17.6% of the cases of chronic hepatitis, but only 2.6% (122/4687) of the total canine submissions. All affected Doberman pinschers were female and 4 of the 6 had been spayed. Clinical signs included anorexia, ascites, vomiting, diarrhea, depression, polyuria, and polydipsia. Gross findings The cases of chronic hepatitis comprised 19 necropsy and 15 biopsy submissions. Postmortem examination in all cases studied revealed a firm, yellowish liver, which was generally reduced in size and had a coarsely nodular surface.

Histopathological findings Microscopic changes are presented in Tables 2 and 3. The most common forms of chronic hepatitis detected in this study were CAH, CPH, and CCH. Chronic active hepatitis was diagnosed in 10 of the 34 cases (29.4%), 8 of which were females, ranging in age from 5 to 11 y. Six cases were diagnosed in Doberman pinschers. The condition was characterized by moderate to severe inflammatory infiltration, composed principally of lymphocytes and plasma cells in the portal areas and extending into the midzone. Piecemeal

-ft . I

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Table 1. Breed, age, sex, and clinical signs for 34 dogs with chronic hepatitis Clinical

Dog

Breed

Age (y)

Sex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Labrador Cocker spaniel Lhasa apso Dalmatian Old English sheepdog Doberman pinscher Sheltie Terrier Scottish terrier Doberman pinscher Mixed Terrier cross Doberman pinscher Cocker/poodle Golden retriever Doberman pinscher Poodle Doberman pinscher Mixed German shepherd cross Doberman pinscher Cocker spaniel Cocker spaniel West Highland white terrier English springer spaniel X corgi Corgi Mixed Border collie Irish wolfhound Pug Poodle Mixed Poodle Basenji

4 5 5 9 10 6 6 17 10 7 6 7 11 8 4 7 7 7 12 4 6 7 3 5 5 4 9 6 4 7 10 4 5 5

FS F MC FS MC F MC M MC F M FS FS FS MC FS FS F FS M FS F F F FS M FS F M M FS FS F F

history Ascites Ascites Vomiting, diarrhea Vomiting, diarrhea Ascites Anorexia, dewormed Anorexia, weight loss Weight loss

Vomiting No history

Vomiting Vomiting Ascites, vomiting, icterus Anorexia, icterus Anorexia, icterus Elevated liver enzymes Icterus Weight loss, PD/PU, vomiting Increased liver enzymes Ascites, icterus Increased liver enzymes Anorexia Increased liver enzymes Vomiting, diarrhea Chronic liver disease Icterus, vomiting, diarrhea Diarrhea, anorexia Vomiting, depression Weight loss Ascites Diarrhea Icterus Increased liver enzymes Diarrhea

FS = female spayed; F = female; MC = male castrated; M = male; PD = polydipsia; PU = polyuria

Table 2. Canine chronic liver disease. Histopathologic and histochemical findings in cases of chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and chronic cholestatic hepatitis (CCH) Hepatitis type and case

number

CAH (5) CAH (6) CAH

(10)

CAH (13) CAH (14) CAH (16) CAH (18) CAH (21) CAH (25) CAH

(26)

CPH4) CPH (22) CPH (23 CPH (24) CPH (29) CPH (30) CPH (31) CCH (2) CCH (8) CCH (17) CCH (27) CCH (28) CCH (32)

Inflammation grade and zone +++ +++ I +++ 1 +++ I

Bile duct

hyperplasia + +

-

+++ I ++ I ++ I +1 +++ I ++ I ++ I +1 +++ 1 ++ I +1 +1 +3 ++ 3

++ ++ ++

++3

Can Vet J Volume 38, June 1997

++

+++ + + ++

++ ++ ++ ++ ++ ++ ++ ++ +

-

+++ ++ ++ + + + ++

+++ ++ ++

Reticulin silver stain

++++++

+++1

+3 ++ 3 ++ 3

Necrosis

++ ++ + + + +

Masson trichrome

Copper (rhodanine stain) and zone

++

-

+++

++ I +++ 1 ++ I +1 +++ I +++ +1

++ +++ ++ ++ +++ ++ ++ + +++ ++ ++ ++ +++ + + + ++ + +++ +++

+++ ++ ++ ++ +++ +++

+ +

++ ++

+++ +++

+++ +++ +++ ++ +++

+++ ++ ++ ++ +++

+++1 +++ 1

+1 ++

Iron stain + + ++ +++ +++

+++ ++ ++ ++ ++ + +

+1 ++

+ + ++ +++ -

+++ + 1

+ ++ +++ ++ ++

++l

++ -

+1

+

Bile + + + + + + -

+ -

-

++

+ ++ + + +

367

Table 3. Canine chronic liver disease. Histopathologic and histochemical findings in cases of fibrosing hepatitis (FH), chronic cholangiohepatitis (CH), and miscellaneous secondary hepatitis (MSH) Copper Hepatitis type and case number

FH/C (1) FH/C (I1) FH/C (12) CH (15) CH (20) CH (34) MSH (3) MSH (7) MSH (9) MSH (19) MSH (33)

(rhodanine

Inflammation grade and zone +1 +1 ++ I +++ l +++ 1 ++ I ++ 1,2 + 1,2 +++ 1,2 + 1,2 ++ 1,2

Bile duct

hyperplasia

Necrosis

Reticulin stain

Masson trichrome

+

+

-

-

+++ ++

+ ++ +

+ + ++

++ + +

+

-

+

++ +

+++

++ + + + + ++ +

+++ +++ +++

++ + ++ ++ + +

stain) and zone +3

-

+++

+++1

+1 +1

+1

Iron stain

Bile

++ ++ +

+

+

++

++ -

+ ++

+ + +

+ + +

+ -

Figure 1. Canine chronic active hepatitis. Severe lymphoplasmacytic infiltration and erosion of liver parenchyma in portal areas (zone 1). Hematoxylin and eosin. Bar = 50 pm.

Figure 2. Canine chronic active hepatitis. Intracytoplasmic copper granules in portal areas (zone 1). Rhodanine stain. Bar = 25 pm.

necrosis was characterized by the erosion of the liver parenchyma by areas of coagulative necrosis at its junction with the portal tract and extending into the midzone (Figure 1). Single cell necrosis was represented by scattered hepatocytes with rounded, condensed, dark eosinophilic cytoplasm, containing a lysed or shrunken, pyknotic nucleus. Reticulo-fibrosis, as evidenced by an increased number of reticulin fibers and fibrous connective tissue, was a prominent feature of this condition. Bile duct hyperplasia was found in 7 of 10 cases and was often moderate. Copper was detected in 9 of 10 cases and consisted of intracytoplasmic granules in zone 1 (Figure 2). Cells containing golden-brown intracytoplasmic pigment, identified as iron using the Prussian blue stain, were a consistent finding in all dogs with CAH. Iron pigment was present in zone 1 and also scattered in small clusters in the midzone (Figure 3). Bile was often seen within the canaliculi. Chronic persistent hepatitis was diagnosed in 7 cases (20.6%), 5 of which were females, ranging in age from 3 to 19 y. Cases compatible with CPH had mild to moderate lymphoplasmacytic inflammatory infiltration in zone 1, with moderate bile duct hyperplasia and moderate to severe reticulofibrosis. Necrosis was not a feature in cases of CPH. Fibrous connective tissue was often prominent in the portal areas and thin bands

tended to encircle small groups of hepatocytes, resulting in the formation of sublobules and coarse pseudolobules (Figure 4). In addition, bands of fibrous connective tissue often extended from one central vein to another. Copper was found in zone 1 in 3 of 7 cases, and iron was found in all 7 cases. The iron was generally localized in the portal areas, at the periphery of the pseudolobules, and, occasionally, in clusters within the midzone

368

(Figure 4).

Chronic cholestatic hepatitis was diagnosed in 5 females and 1 male (17.6%), ranging in age from 4 to 17 y. The main histological changes were confined to zone 3 (perivenular) and consisted of infiltration by lymphocytes and plasma cells, zonal coagulative necrosis, and prominent single cell necrosis. Marked intracanalicular bile plugs or thrombi were seen in perivenular areas (Figure 5), associated with zonal coagulative necrosis (also known as biliary piecemeal necrosis). Additionally, mild to moderate lymphoplasmacytic inflammatory infiltration of zone 1, bile duct hyperplasia, and reticulofibrosis were prominent in these cases. Copper was found in zone 1 hepatocytes in 4 of 6 cases and iron in 5 of 6. Fibrosing hepatitis with cirrhosis was observed in 3 of the 34 cases (8.8%), 2 females and 1 male, between 5- and 7-years-old, and was characterized by mild to

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-4:i -.A

--

Figure 3. Canine chronic active hepatitis. Iron pigment (arrows) in portal areas (zone l). Perl's Prussian blue stain. Bar = 50 pm.

er5~ ~~~~~~~- 5*S

m

i

"; -.3M¢

1-iw-vi

Figure 4. Chronic persistent hepatitis. Dissection of liver parenchyma by fibrous connective tissue with formation of sublobules and pseudolobules. Note iron pigment at the periphery of pseudolobules (arrows). Perl's Prussian blue stain. Bar = 100 pm.

moderate reticulofibrosis with very mild lymphoplasmacytic infiltration in zone and mild bile duct hyperplasia. Ito cell (lipocyte) hyperplasia was often prominent. Mild copper deposition was seen in zone 3 in 1 case, and scattered iron granules within Kupffer cells were detected in all 3 cases. The histological features of CH and MSH were quite distinct from those of the other conditions. Chronic cholangiohepatitis was diagnosed in 3 cases (8.8%) and consisted of moderate to severe lymphoplasmacytic inflammatory infiltration of zone 1. Inflammatory cells were often seen in the lumen of the bile ducts and migrating into the bile duct walls (Figure 6). Copper accumulation was detected in zone in all 3 cases, and iron pigment was seen in zone 3 in 2 of the 3 cases. Miscellaneous secondary hepatitis was diagnosed in 5 of the 34 cases (14.7%), 3 males and 2 females, varying from 5 to 15 y of age. It varied from a multifocal to coalescent lymphoplasmacytic to a pyogranulomatous inflammatory reaction, affecting mainly zones 1 and 2. Reticulofibrosis was mild. Copper was found in 2 cases and iron pigment was seen scattered throughout all liver zones. Can Vet J Volume 38, June 1997

Figure 5. Chronic cholestatic hepatitis. Intracanalicular bile plugs (arrows) in centrolobular area (zone 3) with zonal coagulative necrosis. Hematoxylin and eosin. Bar = 50 pm.

t

Figure 6. Chronic cholangiohepatitis. Severe lymphoplasmacytic inflammatory infiltration, with fewer numbers of neutrophils in zone 1. Hematoxylin and eosin stain. Bar = 25 pm.

In general, a statistically significant correlation was found between the recognition of reticulofibrosis by routine HE and histochemical demonstration of reticulin fibers and fibrous connective tissue (r = 0.9 and 0.6, respectively). Rhodanine staining for copper correlated well with Masson's trichrome stain for fibrous connective tissue (r = 0.8); a slightly lower correlation coefficient was obtained when it was compared with silver staining for reticulin fibers (r = 0.6). A significant correlation was also detected between bile duct hyperplasia and copper staining.

Clinical pathology Clinical pathologic data were available in 22 of the 34 dogs, and partial laboratory results are presented in Table 4. Hematological values were unremarkable. Alanine aminotransferase and ALP activities were elevated in 20 of the 22 dogs. Aspartate aminotransferase was elevated in 14 of 21 dogs; GGT in 14 of the 22, and sorbitol dehydrogenase (SDH) in 14 of 21. Cholesterol was elevated in 13 of the 22 dogs and total bilirubin in 7 of the 22. Bile acids were elevated in 5 of 6 dogs tested, while protein concentration was variable and 369

369

Table 4. Partial laboratory findings in 21 dogs with chronic liver disease Hepatitis type and case # CAH (5) CAH (10) CAH (13) CAH (14) CAH (16) CAH (18) CAH (21) CAH (25) CPH (4) CPH (29) CPH (30) CPH (31) CCH (17) CCH (27) CCH (28) CCH (32) FH/C (I1) CH (15) CH (20) MSH (3) MSH (7)

MSH(19)

Total bilirubin

ALP U/L (Normal 23-87)

ALT U/L (Normal 5-69)

AST U/L (Normal 20-50)

GGT U/L (Normal 0-8)

SDH U/L (Normal 2-20)

Chol mmol/L (Normal 2.4-7)

(Normal 0-17)

390 452 872 2834 1690 415 58 274 949 230 5886 2260 2967 77 910 1785 727 218 485 3278 224 424

170 796 819 1569 293 459 163 532 495 204 1480 338 1629 40 1809 291 505 371 117 1066 18 124

87 313 256 142 77 NA 42 101 33 26 170 174 165 34 218 646 45 260 90 92 37 21

10 30 13 74 7 0 8 32 2 14 63 106 48 2 23 44 30 4 27 406 0 3

39 32 38 9 19 9 24 34.5 25 16.9 31 NA 29 22.7 71 26.5 43 19 47 169 17 14

3.28 7.28 7.76 11.03 8.98 3.1 13.14 6.10 9.21 4.89 20.43 15.93 21.43 3.21 4.15 4.15 9.45 3.01 5.74 9.67 12.9 8.5

2 62 14 143 13 218 4 4 7 2 11 17 166 3 122 184 2 166 15 2 10 0

umol/L

ALT = alanine amine transferase; ALP = alkaline phosphatase; AST = aspartate amino transferase; GGT gamma glutamyl transferase; SDH = sorbitol dehydrogenase; Chol = cholesterol; CAH = chronic active hepatitis; CPH = chronic persitent hepatitis; CCH = chronic cholestatic hepatitis; FH/C = fibrosing hepatitis with cirrhosis; CH = chronic cholangiohepatitis; MSH = miscellaneous secondary hepatitis

albumin

was low in 7 of the 22 dogs tested (data not shown). A slight positive correlation was found between

different indicators of liver function. Alanine aminotransferase correlated with ALP and GGT (r = 0.6), ALP correlated with GGT and cholesterol (r = 0.6 and 0.7, respectively), and a positive correlation was detected between AST and total bilirubin (r = 0.7). In general, dogs with CAH were characterized by increased ALP, ALT, and AST activities. Gamma glutamyl transferase, SDH, and cholesterol were elevated in over 50% of the cases (5/8), whereas total bilirubin was elevated in only 3 of the 8 cases. Chronic persistent hepatitis was characterized by remarkably high ALP and increased ALT activities, with frequent increases in AST (2/4), GGT (3/4), SDH (2/3) activity, and cholesterol values (3/4). Total bilirubin was within normal limits. Chronic cholestatic hepatitis was characterized by increased ALP, ALT, AST, and GGT activities, and marked increases in total bilirubin. However, 1 case histologically compatible with CCH was characterized by an increase in SDH activity only. Cases of fibrosing hepatitis with cirrhosis and miscellaneous secondary hepatitis had elevated ALP and ALT activity and variable elevation of AST, GGT, and SDH activity. Cholesterol was high in all cases; however, total bilirubin was within normal limits. Two cases of cholangiohepatitis had an increase in ALP, ALT, AST, and SDH activity, with variable values of GGT and total bilirubin. Cholesterol was within normal values. Sorbitol dehydrogenase was high in 1 case and below the upper limit in another case. Despite obvious contrasts among the 6 groups, small numbers and wide standard deviations contributed to a lack of statistical significance. Similarly, no correlation was 370

detected between reticulofibrosis and the clinicopathologic indicators of cholestasis.

Discussion Chronic hepatitis is insidious, in that the primary clinical signs are not readily evident until a well-established pathologic process has developed. Ascites, vomiting, weight loss, anorexia, and jaundice are consistent clinical signs; polydipsia, depression, and weakness have also been reported (1-8). The primary signs of chronic liver disease (aundice, depression, anorexia) can be explained on the basis of the main biochemical and morphologic changes. The age and sex distribution of cases of chronic active hepatitis in this study is comparable with that reported in the literature (1,21,22). Similarly, as previously reported by other authors (6,7,21-23), chronic hepatitis was diagnosed with more frequency in Doberman pinschers, cocker spaniels, and poodles. The results of the serum biochemical assays were consistent with hepatic disease but were not characteristic of any form of chronic hepatitis. Increased ALT activity was a consistent finding in 20 of the 22 for which data were available. However, normal ALT activity does not rule out the possibility of chronic hepatitis, and ALT values within normal limits have been reported in dogs affected with chronic active hepatitis and chronic lobular hepatitis (1,21). The half-life of the enzyme is short (2.5 h), so high concentrations that return to normal within 24 h are observed (24). A single insult to the liver can result in a high ALT that returns to normal before the morphologic evidence of necrosis disappears (24). Thus, it is possible to have near normal ALT activity with histologic findings of necrosis.

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Increased ALP activity was also found in 20 of the 22 dogs. An elevation in ALT and ALP activities concurrent with increased bromosulphalein retention and a low albumin concentration suggest a progressive hepatic disease (21). However, an increase in ALP alone is nonspecific and is said to reflect the proliferation of bile ductule epithelium that is seen with biliary obstruction and hepatic regeneration (25). Large amounts of ALP are normally found in bile duct epithelium (1). On the other hand, an association between increased ALP activity and the presence of bile duct hyperplasia was not found in the present study. Other sources of increased serum ALP activity (bone or steroid induced isoenzyme) cannot be differentiated without investigation of isoenzyme patterns.

Biochemical alterations associated with chronic liver disease also included increases in AST, GGT, and SDH activity, and cholesterol and total bilirubin concentrations. Furthermore, some correlation was found between ALT and ALP, ALT and GGT, ALP and GGT, ALP and cholesterol, GGT and cholesterol, and AST and total bilirubin. Conversely, no correlation could be identified between biochemical parameters and the severity of morphologic changes in any of the cases of chronic hepatitis studied. It is not uncommon to find a lack of correlation between clinicopathologic manifestations of liver disease and the severity of the clinical signs (1). The gross appearance of the livers was similar to that of other reported cases of chronic liver disease (1-7,14). In dogs, the diagnosis of chronic active hepatitis has been used to include various forms of chronic liver disease, in contrast to the situation in humans in which CAH implies a immune-mediated pathogenesis (26). It is not unusual to find discrepancies in the terminology used in human versus veterinary medicine, and the demonstration of an immune-mediated mechanism identical to that occurring in people is not absolutely necessary to make a histopathologic diagnosis of CAH (20). Indeed, the morphologic characteristics that distinguish canine chronic active hepatitis from other types of hepatic disease are very distinct: foci of hepatocellular necrosis, a cellular response consisting primarily of lymphocytes, and diffuse fibrosis (1,2,20,21). In humans, the destruction of normal hepatocytes is mediated by lymphocytes and plasma cells, which are the predominant cell types that infiltrate the liver. Antibody directed against a liver cell membrane lipoprotein in cooperation with cytotoxic lymphocytes may be responsible for ongoing liver cell injury in chronic active hepatitis and primary biliary cirrhosis (27,28). These immunocytes are capable of transformation into cells that are directly cytotoxic for hepatocytes by an antibodymediated response (27). In humans and rabbits, lymphocytes produce complement-fixing antibodies directed against other tissue-specific antigens, such as, smooth muscle, nuclear material, and mitochondria, which can cause tissue damage without an effect from cytotoxic cells (29,30). In humans and dogs, limiting plate necrosis is an important feature of chronic active hepatitis. Regardless of the tissue-specific antigens, the immunocyte-mediated inflammatory process causes necrosis of the hepatocytes that form a plate adjacent to the portal triads (31,32), and the hepatic necrosis can extend into Can Vet J Volume 38, 38, June 1997

the lobule when the limiting plate is damaged (32). Furthermore, with the development of massive or bridging necrosis, a path is formed through which immunocytes can advance into the lobule (32). Middle-aged, female Doberman pinschers represented 60% of all cases diagnosed as CAH. A similar breed and sex predisposition has been extensively described in the literature (21,22,33,34). Chronic persistent hepatitis was diagnosed in 20.6% of all the cases of chronic hepatitis examined. In humans, the essential diagnostic feature of CPH is portal inflammation with absence of piecemeal necrosis (31). Although chronic persistent hepatitis has not been described in detail in the veterinary literature, a few cases have been diagnosed in dogs (20,35). In general, causes of zone 1 (portal) inflammation include resolving acute hepatitis and nonspecific reactive inflammation near a focal lesion (31). In humans, the prognosis of CPH is uncertain, as treatment with immunosuppressive drugs may temporarily change the lesion of CAH to CPH by reducing inflammation and piecemeal necrosis, or some patients with mild CAH have portal tracts with only changes of CPH (31). The term chronic cholestatic hepatitis was originally used to describe Skye terrier hepatitis. This seems to be a distinct disease entity, possibly derived from a disorder of intracanalicular bile metabolism culminating in disturbed bile secretion, accumulation of copper, and terminal cirrhosis (36). In humans, biliary piecemeal necrosis results from retention of bile acids in precholestasis or cholestasis in primary biliary cirrhosis, sclerosing cholangitis, and other conditions with prolonged interference with biliary secretion (37). In dogs, zone 3 cholestasis with secondary hepatocellular damage has been observed in a condition known as idiopathic canine intrahepatic fibrosis (38). Fibrosing hepatitis was diagnosed in 8.8% of cases of chronic hepatitis. A progression from piecemeal necrosis to fibrosing hepatitis has been described previously (7) and dissection of the lobular parenchyma by reticulin and fine collagen fibres with minimal portal inflammation has been identified in standard poodles (6). Recently, 2 conditions characterized histologically by a noninflammatory fibrosis, which varied from diffuse to zonal reticulofibrosis, have been reported (38,39) and named noninflammatory primary hepatic fibrosis (39) and idiopathic hepatic fibrosis (38). In the latter condition, the young age of the patients and overrepresentation of German shepherds suggested a genetic component (38). Fibrosis commonly occurs as a sequel to hepatic parenchymal damage and inflammation, and often progresses to cirrhosis (21,31,40,41). Fibrosis is associated with increases in interstitial collagen, basement membrane collagen (type IV), and proteoglycans (42). Disse's spaces contain increased amounts of laminin and fibronectin, as well as collagen (43,44). In the present study, a morphologic association between Ito cell hyperplasia and fibrosis was observed in cases of fibrosing hepatitis. The role of Ito cells in hepatic fibrosis has been the subject of interest in the last decade. One of the likely initiating factors of fibrosis is changes involving cellular fibronectin, which seems to arise from activated lipocytes (45). 371

A positive correlation was found between hepatic reticulofibrosis and copper accumulation, and bile duct hyperplasia and copper accumulation. Copper and iron accumulations were seen in the portal regions (zone 1) in 9 of 10 cases of CAH. Copper was found in zone 1 in 3 of 7 cases of CPH, whereas iron was found in all 7 cases. The presence of copper in cases of CAH has been a controversial issue; it is unknown whether increases in liver copper content are secondary to cholestasis (33) or portray a manifestation of a primary copper storage disease (8,23). Franklin and Saunders (46) interpreted the lack of copper in some cases of CAH as being consistent with the concept that hepatic copper is not the primary cause of chronic active hepatitis in Doberman pinschers. Centrilobular (zone 3) localization of copper, as in Bedlington (16,47) and West Highland white terriers (48), has been associated with a hereditary copper metabolism defect, whereas portal (zone 1) localization of copper has been interpreted as being secondary to cholestasis (7). However, in the present study, no correlation was detected between the presence of copper and the morphologic and biochemical indicators of cholestasis. It is possible that cholestasis is not the only mechanism responsible for copper accumulation, and the fact that intracytoplasmic copper has been identified in dogs with idiopathic hepatic fibrosis (38) suggests that changes in the hemodynamics of the hepatic acini due to fibrosis could influence the centroacinar (zone 1) storage of copper. Iron accumulation within the cytoplasm of Kupffer's cells was a consistent finding in this study. Accumulation of iron has also been described in cases of chronic active hepatitis (1,21), copper-associated liver disease in dogs (49), idiopathic hepatic fibrosis (38), and copperassociated disease in Skye terriers (36). Demonstrable presence of iron in tissues is known as siderosis, which differs from hepatic hemochromatosis only in that there is no hepatocellular damage or fibrosis attributable to the iron overload at the time of examination (31). Causes of Kupffer's cell siderosis in humans include acute hepatitis, blood transfusion, hemolysis, and thalassemia (31). The cause and pathogenesis of chronic hepatic disease is usually unknown in most species. Recognized causes of chronic hepatitis in dogs include infectious canine hepatitis virus (1 1), leptospirosis (2), and prolonged use of corticosteroids (13) and anticonvulsants (14). Immune-mediated diseases and inflammatory conditions of the pancreas and gastrointestinal tract have also been associated with chronic hepatitis (1 1). Inherited hepatic copper storage disease in Bedlington (16,47) and West Highland white terriers (48) and copper-associated disease in Skye terriers (36) manifest as chronic hepatitis culminating with end-stage hepatic fibrosis. Additionally, hepatitis varying from acute to chronic has been observed in Scotland in canine acidophilic cell hepatitis (50). The pathogenesis of this putative infectious condition is intriguing, as a progression from chronic hepatitis to hepatocellular carcinoma has been implied (50). A similar relationship between a viral disease and cancer has been found in hepatitis B virus and hepatocellular carcinoma in humans (51 ) and hepadnavirus infection and hepatocellular carcinoma in woodchucks (52). 372

Unfortunately, clinical signs associated with liver disease are often detected only when the hepatic damage is severe enough to cause dysfunction. Detection of increased serum activity of liver enzymes provide a reliable indicator of liver disease, but clinicopathologic data are insufficient indicators of the particular character of the liver injury. Histological classification, whilst not resolving the etiology of the disease, may be a useful indicator for symptomatic treatment and provides an indication of the severity of the lesion. More importantly, it provides a basis for a more detailed enquiry into the cause of different types of chronic hepatitis, be it infectious or immune-mediated, genetic or metabolic, toxic, etc. Finally, the relationship of copper and iron retention to bile secretion or retention and possible hemodynamic changes associated with reticulofibrosis may provide a promising area of future study. c

References 1. Strombeck DP, Gribble D. Chronic active hepatitis in the dog. J Am Vet Med Assoc 1978; 173: 380-386. 2. Bishop L, Strandberg JD, Adams RJ, et al. Chronic active hepatitis in dogs associated with leptospires. Am J Vet Res 1979; 40: 839-844. 3. Thornburg LP. A study of canine hepatobiliary diseases. Part 3: Hepatitis and Cirrhosis. Companion Anim Pract 1988; 2: 12-17. 4. Hazzi CH. Diagnosis and management of chronic active hepatitis. Am J Gastrenterol 1986; 81: 85-90. 5. Popper H, Schaffner F. The vocabulary of chronic hepatitis. N Engl J Med 1971; 284: 1154-1156. 6. Bennett AM, Davies JD, Gaskell CJ, Lucke VM. Lobular dissecting hepatitis in the dog. Vet Pathol 1983; 20: 179-188. 7. Thornburg LP, Childs A, Toomey AA, Roudebush P. Postnecrotic canine cirrhosis I. Clinicopathologic features. Vet Med Small Anim Clin 1983; 78: 43-50. 8. Thornburg LP, Rottinghaus G, Gage H. Chronic liver disease associated with high hepatic copper concentration in a dog. J Am Vet Med Assoc 1986; 188: 1190-1191. 9. Thornburg LP. A study of canine hepatobiliary diseases 4: Copper and liver disease. Companion Anim Pract 1988; 2: 3-6. 10. Wright R, Rassam S. The immunology of acute and chronic hepatitis. J Clin Gastroenterol 1976; 5: 387-417. 11. Gocker DJ, Priesig R, Morris TW, et al. Experimental viral hepatitis in the dog: Production of persistent disease in partially immune animals. J Clin Invest 1967; 46: 1506-1517. 12. Strombeck DR. Small Animal Gastroenterology. Davis, Califomia:

Stonegate Publ, 1979: 425-449 13. Rogers WA, Ruebner BH. A retrospective study of probably glucocorticoid-induced hepatopathy in dogs. J Am Vet Med Assoc 1977; 170: 603-606. 14. Bunch SE, Castleman WL, Hombuckle WE, Tennant BC. Hepatic cirrhosis associated with long-term anticonvulsant drug therapy in dogs. J Am Vet Med Assoc 1982; 181: 357-362. 15. Leifer CE, Page RL, Matus RE, et al. Proliferative glomerulonephritis and chronic active hepatitis with cirrhosis associated with Corynebacterium parvum immunotherapy in a dog. J Am Vet Med Assoc 1987; 190: 78-80. 16. Ludwig J, Owens CA, Barham SS, et al. The liver in the inherited copper disease of Bedlington Terriers. Lab Invest 1980; 43: 82-87. 17. Luna LG, ed. Manual of Histological Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. Toronto: McGrawHill, 1968: 184. 18. Fuentealba IC, Haywood S, Trafford J. Evaluation of histochemical methods for the detection of copper overload in rat liver. Liver 1987; 7: 277-282. 19. Rappaport AM, Borowy ZJ, Longhead WM, Lolto WN. Subdivision of hexagonal liver lobules into a structural and functional unit, role in hepatic physiology and pathology. Anat Rec 1954; 119: 11-33. 20. Anderson M, Sevelius E. Circulating autoantibodies in dogs with chronic liver disease. J Small Anim Pract 1992; 33: 389-394. 21. Doige CE, Lester S. Chronic active hepatitis in dogs -A review of fourteen cases. J Am Anim Hosp Assoc 1981; 17: 725-730. Can Vet J Volume 38, June 1997

22. Crawford MA, Schall WD, Jensen RK, Tasker JB. Chronic active hepatitis in 26 Doberman pinschers. J Am Vet Med Assoc 1985; 187: 1343-1350. 23. Thornburg LP, Rottinghaus. What is the significance of hepatic copper values in dogs with cirrhosis? Vet Med 1985; 80: 50-54. 24. Dixon MF, Fulker MJ, Walker BE, et al. Serum transaminase levels after experimental paracetamol-induced hepatic necrosis. Gut 1975; 16: 800-807. 25. Pekarthy JM, Short, Lansing AT, et al. Function and control of liver alkaline phosphatase. J Biol Chem 1972; 247: 1767-1774. 26. Rutgers HC, Haywood S. Chronic hepatitis in the dog. J Small Anim Pract 1988; 29: 679-690. 27. Geubel AP, Keller RH, Summerskill WHJ, et al. Lymphocyte cytotoxicity and inhibition studied with autologos liver cells: Observations in chronic active liver disease and the primary biliary cirrhosis syndrome. Gastroenterol 1976; 71: 450-456. 28. Jensen DM, McFarlane IG, Portman BS. Detection of antibodies directed against a liver-specific membrane lipoprotein in a patient with acute and chronic active hepatitis. N Eng J Med 1978; 299: 1-7. 29. Kawanishi H, MacDermott RP. Induction of antibody-dependent hepatocyte cytotoxicity by chronic active liver disease serum, mediated by normal null cells. Gastroenterol 1977; 72: 1078. 30. Hopf U, Meyer zum Buschenfelde KH. Studies on the pathogenesis of experimental chronic active hepatitis in rabbits II. Demonstration of immunoglobulin on isolated hepatocytes. BrJ Exp Pathol 1974; 55: 509-513. 31. Scheuer PJ. Liver Biopsy Interpretation, 4th ed. London: Bailliere Tindall, 1988. 32. Boyer JL. Chronic hepatitis - A perspective on classification and determinants of prognosis. Gastroenterol 1976; 70: 1161-1171. 33. Johnson GF, Zawie DA, Gilbertson SR, Sternlieb I. Chronic active hepatitis in Doberman pinschers. J Am Vet Med Assoc 1982; 180: 1438-1442. 34. Meyer DJ. Obstructive jaundice associated with chronic active hepatitis in a dog. J Am Vet Med Assoc. 1980; 176: 41-44. 35. Hardy RM. Chronic active hepatitis in dogs: A syndrome. Compend Contin Educ Pract Vet 1986; 8: 904-921. 36. Haywood S, Rutgers HC, Christian MK. Hepatitis and copper accumulation in Skye Terriers. Vet Pathol 1988; 25: 408-414. 37. Popper H. The problem of histologic evaluation of primary biliary cirrhosis. Virchows Arch A Path Anat Histol 1978; 379: 99-102.

38. Rutgers HC, Haywood S. Kelly DF. Idiopathic hepatic fibrosis in 15 dogs. Vet Rec 1993: 115-118. 39. Van den Ingh TSG, Rothuizen J. Hepatoportal fibrosis in three young dogs. Vet Rec 1982; 110: 575-577. 40. Kelly WR. The liver and biliary system. In: Jubb KVF, Kennedy PC, Palmer N, eds. Pathology of Domestic Animals. 3rd ed. vol 2. Toronto: Academic Pr, 1985: 319-402. 41. Rojkind M, Kershenobich D. Hepatic fibrosis. In: Popper H, Schaffner F, eds. Progress in Liver Diseases. vol 5. New York: Grune and Stratton, 1976: 363-397. 42. Bissell DM. Cell-matrix interaction and hepatic fibrosis. In: Popper H, Schaffner F, eds. Progress in Liver Diseases. vol 9. Philadelphia: WB Saunders, 1990: 143-155. 43. Hahn E, Wick G, Pencev D, et al. Distribution of basement membrane proteins in normal and fibrotic human liver: Collagen type IV, laminin, and fibronectin. Gut 1980; 21: 63-71. 44. Clement B, Rescan P-Y, Baffet G, et al. Hepatocytes may produce laminin in fibrotic liver and in primary culture. Hepatology 1988; 8: 794-803. 45. Ramadori G, Rieder H, Knittel TH, et al. Fat storing cells (FSC) of rat liver synthesize and secrete fibronectin. J Hepatol 1987; 4: 190-197. 46. Franklin JE, Saunders GK. Chronic active hepatitis in Doberman pinschers. Compend Contin Educ Pract Vet 1988; 10: 1247-1988. 47. Twedt DC, Sternlieb I, Gilbertson SR. Clinical, morphologic and chemical studies on copper toxicosis of Bedlington terriers. J Am Vet Med Assoc 1979; 175: 269-275. 48. Thomburg LP, Shaw D, Dolan M, et al. Hereditary copper toxicosis in West Highland white terriers. Vet Pathol 1986; 23: 148-154. 49. Thornburg LP, Rottinghaus G, McGowan M, et al. Hepatic copper concentrations in purebred and mixed-breed dogs. Vet Pathol 1980; 27: 81-88. 50. Jarrett WFH, O'Neil PW. A new transmissible agent causing acute hepatitis, chronic hepatitis and cirrhosis in dogs. Vet Rec 1985; 116: 529-635. 51. Karayiannis P, Fowler MSF, Lik ASF, et al. Detection of serum HBV-DNA by molecular hybridization. Correlation with HBeAg/ anti HBe status, racial origin, liver histology and hepatocellular carcinoma. J Hepatol 1985; 1: 99-106. 52. Summers J, Smolec JM, Snyder R. A virus similar to hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci 1978; 75: 533-4537.

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