CLINICO-PATHOLOGICAL AND THERAPEUTIC STUDIES ON … · CERTFICATE – II This is to certify that...

CLINICO-PATHOLOGICAL AND THERAPEUTIC

STUDIES ON HEPATIC INSUFFICIENCY

IN DOGS

Dissertation

Submitted to the Guru Angad Dev Veterinary and Animal Sciences

University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in

VETERINARY MEDICINE

(Minor Subject: Veterinary Pharmacology and Toxicology)

By

Murad A. M. Hiblu

(L-2010-V-06-D)

Department of Veterinary Medicine

College of Veterinary Science

GURU ANGAD DEV VETERINARY AND ANIMAL SCIENCES

UNIVERSITY

LUDHIANA -141 004

2015

CERTIFICATE – I

This is to certify that the dissertation entitled, “Clinico-pathological and

therapeutic studies on hepatic insufficiency in dogs” submitted for the degree of

PhD, in the subject of Veterinary Medicine (Minor Subject: Veterinary

Pharmacology and Toxicology) of the Guru Angad Dev Veterinary and Animal

Sciences University, Ludhiana, is a bonafide research work carried out by Murad A.

M. Hiblu (L-2010-V-06-D) under my supervision and that no part of this dissertation

has been submitted for any other degree.

The assistance and help received during the course of investigation have been

fully acknowledged.

(Dr. Kirti Dua)

Major Advisor

Sr. Scientist and Incharge Centre for

Wildlife Studies and Research

Department of Veterinary Medicine

Guru Angad Dev Veterinary and

Animal Sciences University

Ludhiana-141004, India

CERTFICATE – II

This is to certify that the dissertation entitled, “Clinico-pathological and

therapeutic studies on hepatic insufficiency in dogs” submitted by Murad A. M.

Hiblu (L-2010-V-06-D) to the Guru Angad Dev Veterinary and Animal Sciences

University, Ludhiana, in the partial fulfillment of the requirements for the degree of

PhD. in the subject of Veterinary Medicine (Minor Subject: Veterinary

Pharmacology and Toxicology) has been approved by the Student‟s Advisory

Committee after an oral examination on the same, in collaboration with an external

examiner.

_____________________ _____________________

(Dr. Kirti Dua) (Dr. D.S. Nauriyal)

Major Advisor External Examiner

Professor-cum-Head

Deptt. of Veterinary Medicine

COVSc & AH, AAU,

Anand, Gujrat

_____________________

(Dr. B.K. Bansal)

Head of the Department

_____________________

(Dr. Simrat Sagar Singh)

Dean, Postgraduate Studies

ACKNOWLEDGEMENT

“Foremostly with folded hands, I bow my head and kneel with reverence and dedicatedly

award my gratitude to the “Almighty Allah” the most merciful, the most gracious and

compassionate whose grace, glory and blessings in crunch situation made me able to float smoothly

up-till this chapter of my life.

Whenever a journey reaches its climax, it is always a pleasure to look back at all the noble

characters that had come in the way and made the expedition a remarkable one. I would like to

express my deepest sense of gratitude to many known and unknown hands, which trusted me

forward, learned souls who put me on the right path and enlightened me with their knowledge,

experience and skills in my humble acknowledgement.

I would like to dedicate my utmost gratitude to my esteemed advisor Dr. Kirti Dua, Sr.

Scientist and Incharge of Wildlife, Dept. of Veterinary Medicine for his crucial contribution,

meticulous guidance, constant supervision and persistent encouragement from the very beginning

till date. His involvement and originality has triggered and nourished my intellectual maturity

that I will benefit from, for a long time to come. I whole heartedly thank him for the time she

spent with me during which I had free access for formal and informal discussion, which helped me

immensely in my research work. During this period of association with him, I have not only

received knowledge but immense faith, determination, confidence and affection, which I will

always pursue and cherish in my life.

“The debt of gratitude we owe our mother and father goes forward, not backward. What

we owe our parents is the bill presented to us by our children”- Nancy Friday

Where would I be without my family? My mother and my wife deserve the most special

mention for their inseparable support and silent prayers besides their noble sacrifice in bringing

mere joy and happiness in my life. My Mother, Ms. Hawa Mohammed, in the first place is the

person who inspired me with her optimistic thoughts ever since I was a child. It’s all because of

her I’ve had the best of everything and never a no for anything. My wife, Ms. Ebtesam Omran, is

the wind beneath my wings who motivated and encouraged me in the intellectual pursuit in every

part of my PhD. Words cannot express my feelings for my beloved brothers and sisters. There is no

better friend than brothers and sisters, no better friends than you. For all the years of adoration,

emotional support, trust, firm belief and unconditional affection I love you more than I love

myself. I owe all my achievements to my family.

My advisory committee members, Dr. C.S. Randhawa, Professor, Department of

Veterinary Medicine, Dr. P. S. Dhaliwal, Dean PGS Nominee, Professor, Department of

Veterinary Medicine, Dr. N. K. Sood, Professor, Department of TVCC and Dr. V.K. Dumka,

Professor, Department of Veterinary Pharmacology and Toxicology have extended their complete

help and in all kinds of situations that arose during the course of the study. I would like to thank

them for their constructive criticism, healthy advice and suggestions. I owe my sincere thanks to

Dr. B.K. Bansal, Senior Scientist cum Head, Department of Veterinary Medicine, for his

cooperation, providing required facilities and inspirational guidance. I feel great elation in

expressing sincere thanks to Dean PG’s Dr. S. N. S. Randhawa for his support and sharing his

knowledge and experience.

I would like to express my sincere gratitude to Dr. S.K. Uppal, Dr. C.S. Randhawa and

Dr. P.S. Dhaliwal, Professors, Department of Veterinary Medicine, for their timely help and

meticulously scrutinizing this manuscript.

I would like to express my heartfelt thanks to the members of department of Surgery &

Radiology, for their moral support and encouragement from day one.

I further extend my cordial thanks to the professors of the my department, Dr. Ashwani

Kumar, Dr. S.S. Randhawa, Dr. D.K. Gupta, Dr. Sujatha, Dr. Rakesh Ranjan, Dr. Raj Sukhbir,

Dr. Sikh Tejinder, Dr. Naimi Chand, Sukrithi Sharma and Dr. Neetu Saini for being co-operative

and rendering guidance throughout.

I also thank the Head of TVCC, Dr. P.S Mavi for letting me avail all the facilities of the

TVCC and the TVCC laboratory.

Here is my special thanks to Dr. J. Mohindroo and Dr. S.K Mahajan, Department of

Veterinary Surgery & Radiology for their immense support and guidance during my research work

which have not only helped me get through this endeavor, but also helped me construct the

fundamentals of this subject for likely future applications. I express my sincere thanks to Dr. H.

Aashiq for his statistical analysis assistance.

I look back with fondness and nostalgia at the cherished moments I enjoyed in the

company of Dr’s. Arun Kumar Anand, Malik Rayess, H. Aashiq, Tawheed Shaffi, Riyaz Batt

and Abdulrahman Muhammed. The road to success would not have been smooth without their

great company, moral support and encouragement.

I reflect on the loveliest times I have spent in the large and small animal Medicine clinics

learning from my beloved teachers for their friendly approach and assistance which has been

fundamental in shaping up the whole saga of this task.

I’m extremely happy for the valuable support and kind cooperation from the small animal

clinics staff Bajaj and Suresh, department staff Mr. Kewal, Mr. Sachin, Mr. Parshotam, Mrs.

Anita, and Mrs. Sashi Bhatia & laboratory staff Mr. Pritpal Singh, Ms. Harpreet Kour and Mr.

Gurpreet Singh and the chemist Mr. Prabhdeep Singh.

For any errors or inadequacies that may remain in this work, of course, the responsibility

is entirely my own.

Place: Ludhiana

Date: Murad A. M. Hiblu

Title of the dissertation : Clinico-Pathological and Therapeutic Studies on

Hepatic Insufficiency in dogs

Name of the Student : Murad A. M. Hiblu

Admission No. L-2010-V-06-D

Major Subject : Veterinary Medicine

Minor Subject : Veterinary Pharmacology

Name and Designation of

Major Advisor : Dr. Kirti Dua, Sr. Scientist and Incharge Centre for

Wildlife Studies and Research

Degree to be awarded : Doctor of philosophy in Veterinary Medicine

Year of award the Degree : 2015

Total pages of Dissertation : 196 + ANNEXURE + VITA

Name of University : Guru Angad Dev Veterinary and Animal Sciences

University, Ludhiana – 141004 (Punjab), India

ABSTRACT

The present study on “Clinico-Pathological and Therapeutic Studies on Hepatic

Insufficiency in Dogs” was undertaken with the objectives of diagnosing and categorizing

various types of hepatopathies and monitoring the therapeutic response in the clinical

conditions. For this, a comprehensive study was undertaken on 140 dogs suffering from

hepatic insufficiency and it was observed that seventy per cent of cases (98) were

suffering from primary hepatopathies whereas thirty percent (42) constituted reactive

hepatopathies. Out of all the cases of hepatic dysfunction, chronic hepatitis/hepatosis

formed the largest group (30%) followed by acute hepatitis/hepatosis (26.43%). It was

followed by cholecystitis (11.43%), hepatic neoplasias (10.71%), cholangiohepatitis and

liver abscess (6.43%) each, liver cirrhosis (4.29%), obscured hepatopathy (2.86%) and

cholelithiasis (1.43%). Hepatic diseases were maximum (44.29%) in the young age group

(0-4 years), followed by middle age (4-8 years) group (35%), and minimum in geriatric (>

8 years) dogs (20.71%). The overall haemato-biochemical changes of dogs with hepatic

dysfunction revealed anaemia, neutrophilic leukocytosis, prolonged clotting time,

azotemia, hypoproteinemia, hyperbilirubinemia and rise of serum liver enzymes (ALT,

AST, ALP and GGT). The acute hepatic insufficiency had higher albumin level than

globulins level as compared to the chronic insufficiency. Hepatic radiography and

ultrasonography were very useful in diagnosing various hepatopathies; however, with

ultrasonography, detailed information pertaining to the liver dysfunction can be obtained.

Ultrasonographic guided fine needle aspiration cytology/biopsy of liver is useful in

approaching an accurate diagnosis provided that aspiration is taken from the right location

of the lesion. Peritoneal fluid analysis was useful for diagnosis of metastatic tumours,

peritonitis and sepsis. The presence of bilirubinuria and bilirubin crystals in the urine was

suggestive of canine hepatopathies. In the therapeutic management of hepatic

insufficiency, incorporation of N-acetylcysteine to conventional therapy enhances clinical

improvement during the early stages of hepatic disease and helps restoring normal

haemato-biochemical values. Regular screening of apparently healthy dogs will help in

early detection of hepatobiliary diseases.

Key words: Hepatic insufficiency, dogs, haemato-biochemical, urinalysis, imaging,

USG-guided, fine needle aspiration cytology/biopsy, N-acetylcysteine

________________________ ____________________

Signature of Major Advisor Signature of the Student

CONTENTS

CHAPTER TOPIC PAGE NO.

I. INTRODUCTION 1-4

II. REVIEW OF LITERATURE 5-35

III. MATERIALS AND METHODS 36-53

IV. RESULTS AND DISCUSSION 54-168

V. SUMMARY AND CONCLUSIONS 169-174

REFERENCES 175-196

ANNEXURE I i – vi

VITA

LIST OF TABLES

Table No. Title Page No.

1 Clinical signs associated with canine hepatic insufficiency 8

2 Breed wise distribution of cases with hepatic insufficiency 57

3 Sex wise distribution of various hepatic diseases (n=140) 59

4 Previous history of dogs with hepatic insufficiency (n=140) 65

5 Clinical manifestations of dogs with hepatic insufficiency

(n=140)

73

6 Vital body parameters of dogs in various hepatic disease

(n=140)

79

7 Morphological classification of anemia in dogs with hepatic

insufficiency (n=128)

83

8 Fibrinogen and clotting times in dogs with different hepatic

diseases (n=49)

89

9 Hemato-biochemical parameters in healthy and hepatic

insufficiency dogs on the day of presentation (Mean±SE)

92



93



97

12 Total serum bile acids concentrations in dogs with hepatic


103

13 Gross examination of urine in dogs with hepatic


109

14 Chemical analysis of urine in dogs with hepatic


110

15 Microscopic findings of urine in dogs with hepatic

dysfunction (n=76)

112

16 Electrolyte alterations (n=129) 113

17 Peritoneal fluid analysis of dogs with hepatic insufficiency

(n=50)

118

18 Ultrasonographic features in acute versus chronic

hepatitis/hepatosis

126

19 Fine needle aspiration Cytology/Biopsy of the liver (n=31) 137

20 Causes of hepatic insufficiency (n=140) 140

Table No. Title Page No.

21 Haematological changes in dogs with acute hepatitis/hepatosis

following treatment 143

22 Biochemical changes in dogs with acute hepatitis/hepatosis


23 Haematological changes in dogs with chronic hepatitis/hepatosis


24 Biochemical changes in dogs with chronic hepatitis/hepatosis


25 Haemato-biochemical changes in dogs with cholangiohepatitis

following conventional treatment 150

26 Haemato-biochemical changes in dogs with cholecystitis

following conventional treatment 151

27 Haematological changes in dogs with cholecystitis

following treatment with conventional treatment + NAC

153

28 Biochemical changes in dogs with cholecystitis following

treatment with conventional treatment + NAC

154

29 Haematological changes in dogs with primary

hepatopathies following treatment

156

30 Biochemical changes in dogs with primary hepatopathies

following treatment

157

31 Haematological changes in dogs with reactive

hepatopathies following treatment

159

32 Biochemical changes in dogs with reactive hepatopathies

following treatment

160

33A Post mortem changes in dogs with hepatic failure (n=6)

plus one case with core needle biopsy

167

33B Post mortem changes in dogs with hepatic failure 168

LIST OF FIGURES

Fig. No. Title

1 A 20 G hypodermic needle connected to a syringe (a) and spinal needle

(b) used for aspiration biopsies.

Ultrasound-guided fine needle aspiration cytology/ biopsy

2 Age wise distribution of cases with hepatic insufficiency (n=140)

3 Breed wise distribution of dogs with hepatic insufficiency

4 Sex wise distribution of cases with hepatic insufficiency irrespective of

classification

5 Sex wise distribution of various hepatic diseases

6 Distribution of acute and chronic hepatitis/hepatosis versus

primary/reactive hepatopathies

7 Distribution of primary versus metastatic neoplasia

8 Previous history of dogs with hepatic insufficiency

9 History of vaccination status

10 History of deworming status

11 History of feeding regimen

12 Veg versus non-veg diet

13 Clinical manifestations of dogs with hepatic insufficiency

14 Acholic faeces from a dog with complete bile outflow obstruction

15 Severe abdominal pain (hepatodynia) - position of relief

16 Skin bruising and jaundice

17 Bilateral corneal opacity (blue eye syndrome) compared to normal eyes

after recovery

18 Petechial hemorrhages and ecchymosis

19 Severe abdominal enlargement due to ascites/haemoperitoneum

20 Severe jaundice of sclera (a), gum and skin (b).

21 Petechial hemorrhages and ecchymoses on oral mucus membranes

22 Hepatomegaly- moderate abdominal enlargement

23 Vital body parameters of dogs in various hepatic disease (n=140)

24 Morphological classification of anaemia in dogs with hepatic insufficiency

25 Blood smear from a dog with immune mediated hemolytic anaemia

26 Dark yellowish (icteric) urine (A); green colored urine (B)

Fig. No. Title

27 Urine sediment of icteric dog reveals bilirubin casts

28 Peritoneal fluids colour

29 Cytology of peritoneal fluids reveals an overwhelming infection with bacteria,

fibrinopurulent exudate with less immune response suggestive of severe sepsis

30 Cytology of peritoneal fluids reveals chronic active peritonitis. Many markedly

degenerated neutrophils, a few activated macrophages and mesothelial cells with

small clumped bacteria

31 Peritoneal fluid cytology of dog with hepatocellular carcinoma reveals the

metastatic neoplastic cells

32 Peritoneal fluid cytology reveals metastatic hepatocellular carcinoma

33 Peritoneal fluid cytology reveals metastatic adenocarcinoma

34 Cytology of peritoneal fluids reveals hemangiosarcoma

35 Peritoneal fluid examination reveals numerous intact to moderately degenerated

neutrophils along with few abnormal and pleomorphic cells resembling neoplastic

hepatocytes suspected for primary/metastatic carcinoma, high protein

concentration, numerous RBCs and few mesothelial cells.

36 Right lateral recumbency abdominal radiograph of a dog revealing hepatomegaly

with sharp liver margins

37 Right lateral recumbency abdominal radiograph of a dog reveals

hepatosplenomegaly

38 Right lateral recumbency abdominal radiograph of a dog with chronic hepatitis

reveals hepatomegaly with rounded liver margins

39 Right lateral recumbency abdominal radiograph of a dog reveals

hepatomegaly with rounded liver margins and pushing the stomach

caudally.

40 Right lateral recumbency abdominal radiograph of ascitic dog with classic

“ground glass appearance” of abdomen masking abdominal cavity details

41 Right lateral recumbency abdominal radiograph of a dog shows radiopaque non

uniform opacity with irregular margins in cranial ventral region caudal to the liver

and ventral to the pylorus (liposarcoma).

42 Right lateral recumbency abdominal and chest radiograph of a dog with

hepatocellular carcinoma demonstrating generalized hepatomegaly with rounding

of liver margins and metastasis to the lung

43 Hepatic congestion

44 USG examination reveals hypoechoic liver with hypoechoic liver margins,

multiple focal hypoechoic areas suggestive of hepatic congestion

45 An ultrasonogram shows mildly congested liver with sharp liver margins

46 USG examination shows the gall bladder distended and contains cellular

debris (sludge)

Fig. No. Title

47 An ultrasonogram reveals liver is hyperechoic in general as compared to

spleen with slightly rounded margins and lots of anechoic fluids separating

the hepatic lobes suggestive of chronic liver disease

48 An ultrasonogram shows grossly enlarged hyperechoic liver (A) and

congested with slightly rounded and irregular margins and few hyperechoic

and lots of free anechoic fluids suggestive of chronicity (B).

49 Ultrasonographic features in acute versus chronic hepatitis/hepatosis

50 An ultrasonogram show double walled bladder suggestive of gall bladder

edema

51 An ultrasonogram shows thickening of gall bladder wall in a dog with

cholecystitis

52 An ultrasonogram of a dog with cholelithiasis reveals distended gall

bladder with thickened wall and contains multiple concretions and sludge

without acoustic shadow.

53 An ultrasonogram of gall bladder shows cholelithiasis with acoustic

shadow

54 An ultrasonogram of a dog with suppurative hepatitis/liver abscess shows

hyperechoic liver as compared to spleen.

55 An ultrasonogram of a dog with liver abscess shows multiple hyperechoic

areas in the right liver lobe with a lot of free anechoic fluid present in

abdomen suggestive of ascites.

56 An ultrasonogram of a dog with liver abscess shows a large anechoic pocket

measuring about 10 cm in the left liver lobe (A) and ~ 400 ml of

sanguinopurulent fluids (B) was drained under USG guidance (C).

57 An ultrasonogram of a dog with liver abscess shows multiple hypoechoic

nodules measuring about 1.5x1.9 cm in the caudal and right lobes of the

liver

58 USG examination of a dog with liver cirrhosis shows rounded and slightly

irregular liver margins and generally hyperechoic hepatic parenchyma

(Bright liver).

59 USG examination of a dog with liver cirrhosis shows generalized

hyperechoic liver with multiple hypoechoic cavitations/lesions (A) and

irregular margins with multiple small nodules on the surface with

suppuration. Gall bladder was distended with the wall thickened and lots of

free anechoic fluids in the abdominal cavity (ascites)

60 An ultrasonogram shows gall bladder wall thickening and contains some

debris.

61 An ultrasonogram of a dog with hepatic adenocarcinoma shows enlarged

liver with heterogeneous echotexture and few anechoic cavitations

Fig. No. Title

62 An ultrasonogram of a dog with hepatic adenocarcinoma shows a tumor

mass involving both liver and spleen.

63 An ultrasonogram of a dog with hepatic adenocarcinoma shows mixed

hepatic echotexture with irregular mass originating from spleen suggestive

of nodular hyperplasia

64 An ultrasonogram of a dog with hepatic adenocarcinoma shows two

hypoechoic focal nodules in the right lobe

65 An ultrasonogram of a dog with hepatic hemangiosarcoma shows liver with

normal echotexture with hypoechoic nodules left lobes on the medial aspect

66 An ultrasonogram of a dog with hepatocellular carcinoma shows large

irregular mass in the mid abdomen in proximity of middle and right hepatic

lobe and arising from liver.

67 An ultrasonogram of a dog with hepatocellular carcinoma shows the middle

two lobes with rounded margins but mixed echotexture.

68 An ultrasonogram of a dog with hepatocellular carcinoma shows a

hypoechoic nodule measuring 1 cm and below in different lobes

69 An ultrasonogram of a dog with hepatic lipidosis shows large focal hepatic

necrosis

70 FNAB of liver with hepatocellular carcinoma reveals the neoplastic cells

with massive fatty changes

71 FNAB of liver with hepatocellular carcinoma reveals the neoplastic cells

72 FNAB of liver FNAB of liver mass reveals adenocarcinoma

73 FNAB of liver with adenocarcinoma reveals the neoplastic cells

74 FNAB from cranial abdominal mass encroaching the liver revealing

liposarcoma with massive fatty change and necrosis

75 FNAB of liver from a 3 year-old female dachshand dog with suppurative

hepatitis/liver abscess reveals chronic hepatitis i.e., numerous neutrophils

and mononuclear cells

76 FNAB of liver shows bile duct hyperplasia, focal suppurative hepatitis with

hepatocellular regeneration of hepatocytes

77 FNAB of the liver reveals an old abscess with fibrinopurulent exudate

inside and many neutrophils

78 FNAB unremarkable changes in a dog with liver cirrhosis

79 FNAB of liver with hepatic lipidosis shows hepatocellular degeneration,

severe fatty changes and necrosis with loss of nuclei and in some places,

mild mononuclear cells infiltration, RBCs and neutrophils

Fig. No. Title

80 FNAB of liver reveals chronic hepatitis dominated by lymphocytes along

with few macrophages and neutrophils and hyperplasia of hepatocytes

81 FNAB of liver from a dog with cholecystitis reveals biliary epithelial

proliferation

82 Necropsy of a dog with cholangiohepatitis shows hepatic congestion with

distended and thickened gall bladder and congestion of liver, lungs, intestines and

spleen and splenic mass.

83 Histology of liver shows early hepatic cirrhosis, post necrotic collapse with loss of

hepatic architecture, hemorrhage, fibrosis and pseudolobulations, chronic

membrano-proliferative glomerulitis, necrosis of tubular epithelium and thickened

Bowman's capsule, massive stomach necrosis and chronic interstitial lung disease.

84 Histology of pancreas shows hemorrhage, leakage of pancreatic enzymes

with necrosis and fibrosis

85 Necropsy of a a dog with chronic hepatitis shows grossly enlarged and

congested liver with distended and wall thickened gall bladder,

splenomegaly with a few areas of focal infarcts, congested kidneys, mild

gastritis, mild enteritis, and red and gray hepatization of the lungs.

86 Histopathology showing marked congestion and multifocal areas of chronic

hepatitis. Spleen shows decrease in white pulp and relative increase in red

pulp with hemorrhage. Lungs reveal necrotizing suppurative

bronchopneumonia, edema, severe congestion; necrosis to the epithelium

with a lot of exudates in bronchioles, stomach revealed mild chronic

superficial gastritis (fibroblasts with a few inflammatory cells), stomach

wall edema and dilated ducts with debris.

87 Histopathology shows chronic superficial enteritis, mild focal interstitial

nephritis with degeneration, necrosis and sloughing of tubular epithelium

and pancreatic congestion.

88 Necropsy of a dog with chronic hepatitis shows gross enlargement of liver

and spleen, jaundice, anaemia and petechial hemorrhages on the gum and

pericardium.

89 Impression smears of liver reveal chronic hepatitis dominated by

lymphocytes along with few macrophages and neutrophils, hyperplasia of

hepatocytes and fatty change.

90 Histology of the liver reveals multifocal chronic hepatitis with

granuloma formation composed mainly of macrophages and lymphoid

cells.

91 Histology of the liver reveals chronic cholecystitis with hyperplasia of

overlying epithelia and intra-hepatobiliary obstructions.

92 Histology of lungs shows mild to moderate interstitial pneumonia with

emphysema, peribronchiolitis, activated macrophages, chronic

inflammation and mild edema.

Fig. No. Title

93 Necropsy of a dog with chronic active hepatitis shows grossly enlarged and

congested liver, pale spleen and jaundice.

94 Histology of liver reveals focal chronic hepatitis with severe chronic

congestion, anaemia, and accumulation of bile due to obstruction,

disruption of architecture, atrophy of cords, and degeneration and

necrosis of hepatocytes.

95 Histology of kidney reveals chronic renal failure characterized by chronic

interstitial nephritis, metastatic calcifications and tubular necrosis.

96 Histology of lungs shows Metastatic calcifications in the lungs, severe

congestion, edema and hemorrhages.

97 Necropsy of a dog with diabetes mellitus shows hepatic lipidosis,

hepatomegaly, friable liver, distended GB, jaundice, pancreatitis, and

swelling of mesenteric LNs and generalized congestion of the carcass.

Urinary bladder is also distended with urine and engorged.

98 An impression smear of the liver reveals massive fatty change with

disruption and necrosis of hepatocytes and sinusoidal congestion.

99 Histology of liver shows massive vacular degeneration/ fatty change

and necrosis in liver.

100 Histology of kidneys show hyaline casts in tubular lumen of the

kidney, tubular epithelial necrosis, membranoproliferative glomerulitis

and glomerular casts interlaced in bilirubin and hyaline casts in the

renal tubular lumen.

101 Histopathology of liver revealed chronic hepatic congestion, dilatation

of sinusoids and atrophy of hepatocytes.

LIST OF ABBREVIATIONS

@ : At the rate of

, : Comma

> : Greater than

< : Lesser than

≤ : Less than equal to

% : Per cent

; : Semicolon

± : Plus-minus

ACE : Angiotensin-converting-enzyme

A/G : Albumin/globulin

ALP : Alkaline phosphatase

ALT : Alanine transaminase

APTT : Activated partial thromboplastin time

AST : Aspartate transaminase

ARF : Acute renal failure

ATT : Ammonia tolerance test

BID : bis in die, (Latin for "twice daily")

B.wt : Body weight

BUN : Blood urea nitrogen

CAV : Canine adenovirus

CDV : Canine distemper virus

CHF : Congestive heart failure

CPV : Canine parvovirus

CRF : Chronic renal failure

CRTZ : Chemoreceptor trigger zone

DLC : Differential leucocyte count

E : Eosinophil

ECG : Electrocardiography

EDTA : Ethylenediamineteteraacetate

et al : and associates

FSBA : Fasting serum bile acids

Fig : Figure

FNAB : Fine needle aspiration biopsy

FNAC : Fine needle aspiration cytology oF : Fahrenheit

fl : Femtoliters

FDPs : Fibrin degradation products

g : Gram

g/dl : Gram per deciliter

GSD : German Shepherd dog

GADVASU : Guru Angad Dev Veterinary and Animal

Sciences University

GGT : Gamma-glutamyl transpeptidase

H2 : Histamine 2 receptors

Hb : Hemoglobin

HPF : High power field

HT3 : 5-hydroxytryptamine receptor antagonist

IBD : Inflammatory bowel disease

http://en.wikipedia.org/wiki/Bis_in_die

http://en.wikipedia.org/wiki/Gamma-glutamyl_transpeptidase

ICG : Indocyanine green

ICH : Infectious canine hepatitis

i.e. : That is

IgA : Immunoglobulin A

IgG : Immunoglobulin G

IgM : Immunoglobulin M

I/M : Intramuscular

IMHA : Immune mediated hemolytic anaemia

IU : International unit

K+ : Potassium

Kg : Kilogram

L : Lymphocyte

LDH : Lobular dissecting hepatitis

M : Monocyte

MCH : Mean corpuscular hemoglobin

MCHC : Mean corpuscular hemoglobin

concentration

MCV : Mean corpuscular volume

M/F : Male/Female

MPC : Mean platelet component

MPV : Mean platelet volume

mRNA : Messenger ribonucleic acid

mEq/L : Milli equivalents per liter

mg/dL : Milligrams per deciliter

m/kg : Milligram per kilogram

MHz : Mega hertz

mL : Milliliter

ml/kg : Milliliter per kilogram

N : Neutrophil

Na : Sodium

NAC : N-acetylcysteine

ºF : Degree Fahrenheit

PCV : Packed cell volume

PD : Polydipsia

pg : Pictogram

pH : Negative log of hydrogen ion

concentration

PO : Per os

PPSBA : Postprandial serum bile acids

PSS : Portosystemic shunts

PSVA : Portosystemic vascular anomalies

PT : Prothrombin

PU : Polyuria

RBC : Red blood cell

SAAG : Serum ascites albumin gradient

SAM-e : S-adenosylmethionine

SBA : Serum bile acids

SBP : Sulphobromopthalein

SDH : Sorbitol dehydrogenase

SE : Standard error

SID : Latin: semel in die "once a day".

SUN : Serum urea nitrogen

TB : Total bilirubin

TEC : Total erythrocyte count

TLC : Total leucocyte count

TP : Total protein

TSBAs : Total serum bile acids

U/L : Unit per liter

US : Ultrasound

USG : Ultrasonography

WBC : White blood cell

μmol/L : Micromole per liter

CHAPTER I

INTRODUCTION

Among all companion animals, dogs have perhaps taken the centre stage of

attraction among all people because of their cooperative and natural affinity for

human beings. Dogs perform many roles for humans such as hunting, herding, pulling

loads, protection, assisting police and military, companionship, and more recently

aiding handicapped individuals (Alan and Aaron 1983). This impact on human society

has given them the nickname "Man's Best Friend". Their loyalty, intelligence,

devotion and affection are incredibly rewarding.

In view of increasing urbanization, unscientific feeding, ever increasing

environmental pollution and abuse of common therapeutic agents and stress, like

human beings the pets are also becoming more susceptible to hepatobiliary diseases

and their importance in today‟s time cannot be ignored. The number of canine patients

is increasing day by day in veterinary clinics owing to increased concern and

attachment of the owners for alleviation of their beloved pets‟ sufferings. Canine liver

disease is one of the topmost common causes of non-accidental death in dogs as it

remains undetected during the early stages. The estimated frequency of canine

hepatitis varies with the investigated population and accounts for 1-2% (Poldervaart et

al 2009), and up to 12% in general population (Watson et al 2010).

Liver is the largest parenchymal organ in the body, carrying out diverse

number of biochemical processes essential for maintaining normal body homeostasis.

Some of these pivotal processes are synthesis of plasma proteins, fats and various

clotting factors; metabolism of carbohydrates, fats and amino acids, storage of

glycogen as a source of energy, secretion of bile for ideal digestion and detoxification

and/or excretion of drugs and toxins; it also helps the immune system fight diseases

2

(Remeth et al 2002). For its diverse metabolic activity, liver regulates the functioning

of most other vital organs of the body thereby playing a pivotal role in maintaining

the body‟s internal environment. The liver has a tremendous compensatory and

regenerative capacity to maintain its diverse functions even during a disease process.

It has been reported that liver can return to its normal size and maintain its function in

about two weeks after 70% partial hepatectomy due to replication of differentiated

hepatocytes and cholangiocytes (Hauptman et al 2001; Schotanus et al 2013). Though

it is an advantageous property of hepatic tissue, it poses a challenge to the clinician to

evaluate a hepatic insufficiency before a significant proportion of the liver is affected.

Owing to these facts, dysfunction of liver could lead to great morbidity and high

mortality rate if immediate consideration is not warranted.

Hepatic insufficiency implies the inability of the liver to carry out its

metabolic, excretory and detoxifying functions owing to a decrease in the number of

functional hepatocytes or because their normal activity is altered. Because the liver

works to rid the body of so many different substances, it is susceptible to damage

from many different sources.

Hepatic insufficiency as a clinical entity warrants a rapid evaluation strategy

to define the degree of insufficiency. The aim of clinicopathological evaluation of

hepatobiliary affections is to identify and characterize hepatic damage and

dysfunction, identify possible primary causes of secondary liver disease, differentiate

causes of icterus, evaluate potential anaesthetic risks, assess prognosis and response to

xenobiotics and monitor response to therapy. It is important to interpret all results in

the light of the other aspects of the diagnostic investigation, in particular the history

and physical examination. In most cases, a tentative diagnosis of primary hepatic

disease can be deduced by correlating the ultrasonographic abnormalities with the

3

history, physical examination findings, clinical hemato-biochemical results and

radiographic observations.

Though biochemical findings often prove to be useful aid in the diagnosis of

hepatobiliary affections, many of them are not specific indicators of liver disease. The

abdominal radiographs are useful to evaluate the morphologic abnormalities of the

liver and presence of abdominal effusion. However, for identification of specific

hepatopathies and thus establishment of definitive diagnosis of primary liver disease,

the histopathological examination of the liver biopsy specimens is usually required

(Kumar et al 2012).

Hepatic disease is often treatable and has a predictable prognosis when a

definitive diagnosis is made. In most cases, however, the cause of canine hepatitis is

idiopathic despite putting the efforts to define suspected aetiologies (Watson 2004;

van der Heijden et al 2012) and therefore the treatment is mostly non-specific,

empirical and symptomatic due to the lack of treatment options. The management of

hepatic insufficiency therefore achieves significance over the identification of the

causative factor as that may be time consuming or may even not become possible

under different clinical situations. The therapeutic protocol can be decided on the

basis of degree of hepatic insufficiency. The protocol involves management of hepatic

and other organ functions in addition to the control of the primary factor.

Liver diseases are common in dogs presented for treatment at the Teaching

Veterinary Hospital, Guru Angad Dev Veterinary and Animal Sciences University

(GADVASU), Ludhiana, Punjab, India, but the cause is usually unknown.

Unfortunately, little is known about the aetiology and progression of the canine

hepatic disease and very few therapies have been subjected to critical trials. The

profile of liver diseases in dogs presented to GADVASU small animal clinics needs to

4

be categorized. Further, studies clearly warranted to be undertaken to ascertain

whether primary or secondary hepatic diseases are more widespread in canines as well

as a cause of previously classified idiopathic liver disease.

In the light of these facts, the proposed study was undertaken with the following

objectives:

1. To study and categorize the hepatic insufficiency in dogs on the basis of clinical,

biochemical and pathological changes.

2. To correlate ultrasonographic findings with biochemical and pathological

changes.

3. To monitor the therapeutic response in diagnosed liver diseases.

CHAPTER II

REVIEW OF LITERATURE

Canine liver diseases are important cause of ailment and remain undetected

during the early stages. In the general population their incidence may go up to 12%.

The detailed review of literature pertaining to various aspects of hepatic insufficiency

in dogs and its management is discussed below.

2.1 SIGNALMENT

There are various Breeds of dogs which has increased susceptibility for

chronic hepatitis which include American and English cocker spaniel, West Highland

white terrier, Labrador retriever, Doberman pinscher and Scottish terrier (Andersson

and Sevelius 1991). Dog breeds at risk for developing chronic hepatitis may change

with time and geographic location due to genetic and environmental factors (Bexfield

et al 2012).

The disease was more common in middle aged to older animals and there was

a gender predisposition in male English and American cocker spaniels and female

Labrador retrievers (Andersson and Sevelius 1991).

Hereditary copper-associated hepatitis has been described in various breeds

(including Bedlington terrier, West Highland white terrier, Skye terrier, Doberman

pinscher, Dalmatian and Labrador) and suspected in others (Ettinger and Feldmann

2005; Hoffmann et al 2006). Ettinger and Feldmann (2005) also reported that Chinese

Shar Pei dogs are predisposed to hepatic amyloidosis. In a retrospective study,

Poldervaart et al (2009) reviewed that young American and English cocker spaniels

are at risk of chronic hepatitis with rapid progression to cirrhosis, males are

overrepresented and female dogs seem predisposed to chronic idiopathic and copper

associated hepatitis. George et al (1986) reported that Alsatians, Dachshunds and

Pomerians were frequently affected with jaundice.

6

Evidence from humans and rodents has indicated that aging leads to a marked

change in the liver structure and function (Schmucker 2005). In general, aged liver is

characterized by a decline in weight, blood flow, regeneration rate, and detoxification,

which have been related to an increased risk of liver abnormalities in the elderly

(Schmucker 2005). Mean age of dogs with hepatic diseases was reported as 51.9

months with sex ratio (M/F) below 1 in hepatitis and cirrhosis. Regarding breed

predisposition, Pomeranian and German Shepherded were over presented (Tiwari

2002). This is in agreement with Shih et al (2007) who reported that the median age

of Labrador retriever dogs suffering from hepatitis is 9.3 years (range, 3.9-14.0 years).

Alana (2004) examined a 12-year-old male castrated miniature schnauzer presented

with a history of abdominal distension. Serum biochemical analysis and abdominal

ultrasonography indicated hepatic disease. A wedge biopsy provided a diagnosis of

chronic active hepatitis.

Portosystemic shunts (PSS) are vascular communications taking blood directly

from the portal circulation to the systemic circulation, bypassing the liver in the

process. Portosystemic shunts may be acquired or congenital, intrahepatic or extra-

hepatic. Congenital PSS are diagnosed more commonly in purebred dogs than

crossbreeds, with a reportedly high incidence in Cairn terriers, Dachshunds, miniature

Schnauzers, Golden and Labrador retrievers, Irish wolfhounds, Maltese terriers and

Australian cattle dogs (Hunt et al 1998; Hunt et al 2004). A ductal plate malformation

was also described in five cases of dogs consistent with congenital hepatic fibrosis.

All five dogs were presented with clinical signs of portal hypertension (Brown et al

2010).

Neoplasms of the liver and biliary tracts are uncommon in domestic animals.

Frequency in the dog varies from 0.6% to 1.3% of all neoplasms (Patnaik et al 1980).

7

He also conducted a clinicopathological study on dogs with hepatic carcinomas and

reported that majority of dogs had hepatocellular carcinoma (80%), bile duct

carcinoma (65%), and sarcoma (61%). Seventy one per cent of dogs with carcinoid

were ten years or older.

2.2. AETIOLOGY

The aetiology of most cases of canine hepatitis remains unknown

(Poldervaart et al 2009). Involvement of the liver may be primary or it may be

secondary. Secondary involvement is termed “reactive hepatopathy” and is due to

liver‟s pivotal role as a primary organ of metabolism and detoxification. Known

causes identified in a small proportion of cases include the virus canine adenovirus

(Chouinard et al 1998), bacteria including leptospires (Bishop et al 1979) and

Helicobacter spp. (Fox et al 1996; Boomkens et al 2005), and several toxins and

drugs (Bunch 1993). Defects in copper metabolism have also been described in

several breeds, including the Bedlington terrier (Twedt et al 1979; van De Sluis et al

2002), Dalmatian (Webb et al 2002), Dobermann pinscher (Mandigers et al 2004),

Labrador retriever (Hoffmann et al 2006), Skye terrier (Haywood et al 1988) and

West Highland white terrier (Thornburg et al 1996). α-1 antitrypsin deficiency has

been linked to chronic hepatitis in the English cocker spaniel (Sevelius et al 1994).

Immune-mediated disease is suspected in some dogs, particularly Doberman

pinschers, but so far studies have failed to conclusively demonstrate a primary

immune-mediated aetiology (Andersson and Sevelius 1992; Weiss et al 1995; Dyggve

et al 2011). Hammer and Sikkema (1995) reported that primary hepatic neoplasms

were not common in dogs and cats and comprised only 0.8-2.3 % of all neoplasms of

these species. Metastasis to liver was much more common accounting for 7-36 % of

dogs having cancer. Many systemic diseases which primarily do not involve liver

8

itself such as gastrointestinal diseases of chronic inflammatory nature like

inflammatory bowel disease (IBD), acute pancreatitis, renal insufficiency, hypoxic

insult, sepsis etc. can precipitate reactive hepatopathy (Rothuizen and van den Ingh

1998). Congestive heart failure may induce hepatic congestion with structural and

functional derangements. The critical pathogenetic factor appears to be hepatic

hypoxia which causes centrolobular damage (Dunn et al 1973).

2.3. CLINICAL FINDINGS AND PHYSICAL EXAMINATION

Clinical signs of hepatopathies in dogs are extremely variable due to the

liver‟s extensive interaction with other organs and its unusual regenerative capacity

(Dial 1995; Fleming 2011). Some dogs show no clinical manifestations of liver

damage, especially in the very early stages of disease. Once symptoms do develop,

they usually are nonspecific. The general signs and physical examination findings

often associated with liver disease, irrespective of its cause, include one or more of

the following signs summarized in Table 1.

Table 1: Clinical sings associated with canine hepatic insufficiency

General clinical

signs

Gastro-

intestinal signs

Neurological

signs

Urological

signs

Cutaneous signs

Anorexia

Lethargy

Weakness

Weight loss

Abdominal

distension

Icterus

Abdominal pain

Hepatomegally

Pale mucus

membranes

Fever

Vomiting

Diarrhoea

Melena

Haematochezia

Haematemesis

Acholic faeces

Ataxia

Staggering

Behavioral

changes

Mental state

changes

(disorientation,

stupor, coma)

Irritability

Aggressiveness

Pacing

Head pressing

Blindness

Excessive

salivation

Generalized

seizures

Polyuria

Polydipsia

Pollakiuria

Stranguria

Dysuria

Hepatocutaneous

syndrome (rare)

http://www.ncbi.nlm.nih.gov/pubmed?term=Rothuizen%20J%5BAuthor%5D&cauthor=true&cauthor_uid=9584348

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Long-lasting anorexia, for example, caused by maldigestion (due to

chronic diarrhoea, foreign body presence, intestinal invagination, pancreatic

insufficiency) leads to changes in the metabolism of fats (Clarenburg 1992;

Center 1993; Hauptman et al 2001). These processes then often result in liver

steatosis (Yeager and Mohammed 1992). Serious enteritis cases enable bacteria

to penetrate through the altered intestinal mucosa and as a sequel to this damage

the liver tissue (Greene 1998). Systemic diseases such as the Tyzzer‟s disease,

salmonellosis, listeriosis and toxoplasmosis damage the liver tissue as well

(Hauptman et al 2001). It is therefore necessary, when examining patients with

clinical signs of affection of other organ systems, to find out whether it is not a

complicating factor to liver damage.

Dogs and cats with primary hepatic neoplasia are presented with vague

clinical signs of anorexia and lethargy. Vomiting and diarrhea are less common

whereas polydipsia (PD) and polyuria (PU) are seen in almost half of the dogs.

Jaundice and ascites are found in only eighteen to thirty per cent of dogs. The

most common findings on physical examination of the abdomen are

hepatomegaly and cranial abdominal mass (Patnaik et al 1980). Physical

examination is informative only in few dogs with liver disease. Icterus, hepatomegaly,

ascites as well as mucus membrane pallor are common findings, whereas petechiation

of skin and mucus membranes are extremely infrequent (Rothuizen and Meyer 2000).

Crawford (1985) diagnosed chronic active hepatitis with increased hepatic

copper concentration in 25 female and 1 male Doberman pinscher dogs.

Common clinical signs included PU/PD, weight loss, anorexia, icterus, and ascites.

Increased liver enzyme activities and abnormal liver function test results were the

most consistent clinicopathological changes.

http://europepmc.org/abstract/MED/4086350/?whatizit_url=http://europepmc.org/search/?page=1&query=%22Chronic%20active%20hepatitis%22

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Hepatic encephalopathy is a complex of neurological signs which results

from portosystemic shunting of blood in combination with reduction of

functional liver mass (Conn and Bircher 1988). Javier Lizardi-Cervera et al (2003)

reported, in a ten year study, that hepatic encephalopathy was a consequence of

cirrhosis in as many as 28 per cent of cases. It is a potentially reversible, or

progressive, neuropsychiatric syndrome characterized by changes in cognitive

function, behavior, and personality, as well as by transient neurological symptoms and

characteristic electroencephalographic patterns associated with acute and chronic liver

failure. Frequently, a precipitating factor can be identified. Once the precipitating

condition is resolved the encephalopathy also typically disappears.

Cholangiohepatitis in a dog is frequently presented with the signs of

abdominal discomfort, anorexia, fever, vomiting, icterus, dehydration, depression

and hepato-splenomegaly (Forrester et al 1992). Idiopathic hepatic fibrosis in dogs

is frequently associated with ascites, anorexia, weight loss and hepatic

encephalopathy (Rutgers et al 1993). The majority of dogs with hepatic disorder

express jaundice and ascites as hallmark along with other non-specific signs such as

anorexia, depression, weight loss and vomiting (Guilford 1993).

Vomiting associated with hepatopathies could be attributed to the direct

stimulation of the vomiting center via the chemoreceptor trigger zone (CRTZ) in

the fourth ventricle by endotoxins that were not cleared by liver (Batt and Twedt

1994).

The main causes of ascites in dogs include cirrhosis of the liver, chronic

circulatory insufficiency, peritoneal infections, metabolic disorders and tumors

(Glin´ska et al 2006). Leduc and De Troyer (2007) also stated that ascites is a

complicating feature of many diseases of the liver and peritoneum and commonly

causes dyspnea.

11

Kumar and Varshney (2006) conducted a study on 131 naturally occurring

ehrlichiosis in dogs manifesting anorexia, vomiting, pyrexia, melena, weight loss,

arrhythmia, ascites, peripheral nerve deficits, and lymphadenopathy. Pale mucosa,

ascites and hepatomegaly were marked in dogs‟ infection of Babesia gibsoni and

Ehrlichia canis. A retrospective analysis was conducted on 80 dogs suffering from

hepatic cirrhosis. Ascites was the most common clinical finding followed by icterus,

anorexia, neurological disturbances, dyspnea and subcutaneous edema respectively

(Silva et al 2007).

James el al (2008) conducted a study of 17 dogs presented with ascites due to

presinusoidal portal hypertension and identified idiopathic hepatic fibrosis or canine

chronic hepatitis as the underlying causes in the majority of cases. The prognosis was

generally poor and no histological, imaging or biochemical parameters were useful as

prognostic indicator. Dereszynski et al (2008) reported anorexia, lethargy,

vomiting, jaundice, diarrhea (melena, haematochezia), abdominal effusion,

peripheral edema, terminal encephalopathy and hemorrhagic diathesis in a dog

that consumed foodborne hepatotoxic aflatoxins. Common clinicopathologic

features included coagulopathic and electrolyte disturbances, hypoproteinemia,

increased serum liver enzyme activities, hyperbilirubinemia, and

hypocholesterolemia in dogs consumed foodborne hepatotoxic aflatoxins.

2.4. HAEMATOLOGICAL PROFILE

Haematological changes of dogs with hepatic insufficiency mostly include

mild regenerative anaemia (as a consequence of gastrointestinal bleeding or

rarely spontaneous bleeding due to coagulopathy) or more frequently normocytic

normochromic non regenerative anaemia suggestive of chronic disease (Ettinger

and Feldmann 2005). Non-regenerative microcytic hypochromic anaemia,

http://www.ncbi.nlm.nih.gov/pubmed?term=Dereszynski%20DM%5BAuthor%5D&cauthor=true&cauthor_uid=18447777

12

suggests chronic gastrointestinal blood loss. Leveille-Webster (2000) stated that

morphological changes of erythrocytes associated with liver diseases include

microcytosis, acanthocytosis, schistocytes and Heinz bodies. Microcytosis has

been described in hepatic vascular disease, chronic hepatitis and in dogs with

acquired shunting secondary to cirrhosis. Neutrophilic leukocytosis and

thrombocytopenia may be observed in chronic hepatitis (Bush 2002; Ettinger and

Feldmann 2005; Poldervaart et al 2009; Shaker and Khalifa 2012). Dogs suffering

from chronic hepatitis usually presented with nonspecific changes in

haematological parameters (Dill-Macky 1995) whereas, dogs with primary liver

cancer often associated with anaemia and neutrophilic leukocytosis (Kosovsky et

al 1989). However, most inflammatory diseases of the organism are associated

with leukocytosis; septic cases, on the other hand, are accompanied by

leukopenia (Center 1998). Bacterial infections cause neutrophilia with a left-shift

and a higher proportion of toxic neutrophils as well as monocytes. Anaemia

associated with hepatic neoplasia could be attributed to the chronicity or

excessive bleeding of tumor (Johnson 2000). Significant reduction in the mean

haemoglobin values was reported in dogs with different hepatic diseases in

comparison with healthy dogs. Similarly, slightly higher clotting time in dogs

with hepatic cirrhosis but within the normal range and without significant

difference from that of healthy control group was observed (Tiwari 2002).

2.5. BIOCHEMICAL CHANGES

2.5.1 Hepatic enzymology

Although elevated serum hepatobiliary enzyme activities are frequently

identified, they do not necessarily indicate clinically important hepatic disease. There

are several reasons for this discordance. First, increased serum hepatobiliary enzyme

13

activity can originate from non-hepatic tissues. Second, the liver's dual blood supply

and large blood flow make it uniquely sensitive to injury due to systemic disorders

and diseases in organ systems drained by the portal circulation, particularly the

gastrointestinal tract and the pancreas. Finally, drugs can induce excess hepatobiliary

enzyme production in the absence of liver damage.

Conventional tests for hepatic disease provide information about the integrity

of the hepatocytes (ALT, AST, and SDH) and the status of the biliary system (ALP

and GGT).

Alanine aminotransferase

Increases in serum ALT activity are considered liver-specific in dogs.

Alanine aminotransferase activity can increase with severe muscle necrosis, but

simultaneous evaluation of serum creatine kinase activity can rule out a muscle

source (Valentine et al 1990; Center 1996). Alanine aminotransferase is a

cytosolic enzyme, and leakage occurs with damage to hepatobiliary membranes.

The magnitude of serum ALT activity elevation is roughly proportional to the

number of injured hepatocytes (Center 1996) and is not considered significant

until it reaches double the normal value (Van Vleet and Alberts 1968). Valantine

et al (1990) reported that largest increase in serum ALT was seen with acute

hepatocellular injury and necrosis. Serum ALT activity may also increase

because of induction of enzyme synthesis by corticosteroid use and, possibly to a

lesser extent, by phenobarbital therapy (Center 1996; Muller et al 2000).

Increases in serum ALT activity have the highest sensitivity (80-100%) for

hepatic inflammation and necrosis, vacuolar hepatopathy, and primary neoplasia

(hepatocellular carcinoma, cholangiocarcinoma) but have less sensitivity (50-

14

60%) in cases of hepatic congestion, metastatic neoplasia, and portosystemic

vascular anomalies (Center 1996). Serum ALT has half-life of 2.5 days (Dossin

et al 2005).

Aspartate aminotransferase

The highest tissue concentrations of AST are present in the liver, skeletal

muscle, and cardiac muscle. Both cytosolic and mitochondrial liver isoenzymes

have been found in people, and presumably both isoenzymes occur in dogs as

well (Center 1996).

Serum AST activity increases from leakage secondary to hepatocyte

membrane injury, so it typically parallels serum ALT activity increases.

Increased serum AST activity, in the absence of increased ALT activity,

indicates an extrahepatic source, most likely muscle injury (Valentine

1990). Marked elevations in AST activity are suggestive of irreversible

hepatocyte injury with release of mitochondrial AST stores. Aspartate

aminotransferase half-life was reported to be 22 hours (Dossin et al 2005).

Measuring AST activity is somewhat more sensitive but less specific for

detecting hepatic disease than is measuring ALT activity (Center 1996; Leveille-

Webster 2000). Typically, there is little to no induction of serum AST with

corticosteroid or phenobarbital treatment (Badylak and Van Vleet 1981; Centeer

1996; Muller et al 2000).

Alkaline phosphatase

Serum alkaline phosphatase is a membrane-bound enzyme present in

many tissues. Three major isoenzymes contribute to total serum ALP: bone,

liver, and corticosteroid isoenzymes (Brunson et al 1980; Center et al 1992;

Center 1996).

15

Bone ALP accounts for about one-third of the total serum ALP and is

elevated with conditions associated with increased osteoblastic activity such as

bone growth, osteomyelitis, osteosarcoma, and secondary renal

hyperparathyroidism (Center 1996).

Liver ALP is a membrane-bound enzyme present on biliary epithelial

cells and hepatocytes. Liver ALP half-life is 70 hours (Center et al 1992; Center

1996). The largest increases in liver ALP activities are associated with focal or

diffuse cholestatic disorders and primary hepatic neoplasms (hepatocellular and

bile duct carcinoma). Less dramatic increases were found in cases of hepatic

necrosis, hepatitis, and nodular hyperplasia. Liver ALP can also be induced by

corticosteroid or phenobarbital administration (Badylak and Van Vleet 1981;

Center 1996; Muller et al 2000; Gieger et al 2000).

Corticosteroid ALP isoenzymes is produced in the liver and is located on the

hepatocyte plasma membranes lining the bile canaliculi and sinusoids (Center

1996). It has a similar half-life to liver ALP and contributes to total serum ALP in

dogs exposed to exogenous corticosteroids or in cases of spontaneous

hyperadrenocorticism (Center 1996). However, increased corticosteroid ALP activity

has also been associated with chronic illness, possibly secondary to stress and

concomitant increases in endogenous glucocorticoid secretion (Brunson et al 1980;

Center et al 1992; Center 1996).

Increased ALP activity is one of the most common abnormalities detected on

serum chemistry profiles in ill dogs and its measurement has a high sensitivity (80%)

for hepatobiliary disease, but its specificity is low (51%). Elevated ALP activity with

a concurrent increase in serum GGT activity increases the specificity for liver disease

to 94% (Center et al 1992).

16

Gamma-glutamyltransferase

Highest tissue levels of GGT in dogs are present in the kidney, pancreas, with

lesser amounts in the liver, gallbladder, intestines, spleen, heart, lungs, skeletal

muscles, and erythrocytes (Jerry et al 2008).

Serum GGT activity is largely derived from the hepatobiliary system. In dogs,

hepatic GGT is located on the hepatocyte canalicular membrane. Gamma

glutamyltransferase activity appears to have a lower sensitivity but higher specificity

(87%) for detecting hepatobiliary disease than ALP activity does (Center et al 1992).

The most marked elevations of GGT activity result from diseases of the

biliary epithelium such as bile duct obstruction and cholecystitis (Center

1996). Moderate elevations can also be found with primary hepatic neoplasia

(hepatocellular and biliary carcinoma) and corticosteroid induction (Brunson 1980;

Badylak and Van Vleet 1981; Center et al 1992; Center 1996). Mild elevations are

found in cases of hepatic necrosis and anticonvulsant administration (Center 1996;

Muller et al 2000; Gieger et al 2000).

The diagnostic value of GGT has been assessed in clinical patients with and

without liver disease (Center 1986). Experimental studies in dogs and cats

undergoing acute, severe diffuse necrosis have shown either no change is serum GGT

or only mild increases in activity (1-to 3-fold normal) that resolve over the ensuing 10

days.

Sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) has been identified in several human and

animal tissues. It is located primarily in the cytoplasm and mitochondria of the liver,

17

kidney, and seminal vesicles. The use of the SDH assay is based on the finding that

SDH activity in the serum is normally low but increases during acute episodes of liver

damage. Measurement of SDH activity is therefore useful as a specific indicator of

liver-cell damage (Joseph et al 1979).

Arginase

This is yet another marker of hepatocellular injury that is less prone than ALT

and AST to elevations in „secondary‟ hepatopathies. Levels of arginase decline to

normal during recovery after injury. Persistent elevations, therefore, may have greater

negative prognostic significance than do persistent elevations of ALT or AST (Center

2007).

2.5.2 Hepatic functions

Hepatic function can be assessed by estimating the excretory capacity

(bilirubin, bile acids, NH3) and synthetic functions (NH3/urea, albumin, fibrinogen,

and prothrombin) of the liver.

Bilirubin

Bilirubin concentration can also be used to assess liver function. Serum

bilirubin is dependent upon the rate of heme pigment formation, albumin

binding, hepatobiliary circulation and uptake, hepatic storage, conjugation, and

elimination. Therefore, hyperbilirubinemia can result from increased

production (prehepatic); decreased uptake, conjugation, and storage

(hepatic); or decreased elimination (posthepatic). Animals suspected of having

liver disease, total bilirubin concentration was shown to have high specificity but

very low sensitivity for liver disease with predictive value of a negative test

(Center et al 1991).

Conjugated and unconjugated bilirubin can be measured by using van den

Bergh reagents. Elevated concentration of the unconjugated form may indicate

18

increased heme pigment liberation or delayed hepatic uptake and processing

(Center 1989). Acute hemolytic disorders are most commonly responsible for

increased unconjugated bilirubin; however, this occurs early in the disease

process (Center 1989). Usually, by the time a dog is examined by a veterinarian,

equilibrium has been established between conjugated and unconjugated forms of

bilirubin.

Consequently, information from physical examination, history, and liver

enzymes generally make differentiating forms of bilirubin unnecessary (Center

1989, Bunch 1992). Dickson et al (1989) stated that elevated bilirubin levels along

with increased levels of ALP, cholesterol and urinary bilirubin suggests cholestasis,

while increased ALT/AST ratio suggests necrosis. He also stated that the degree of

increase in serum bilirubin values has prognostic significance in chronic liver injuries,

but not in acute injuries. Urinary bilirubin and urobilinogen can be detected reliably

by using commercially available dipsticks. A positive test indicates hepatic or biliary

tract dysfunction. Urinary bilirubin is a more sensitive indicator of liver injury than

serum bilirubin. An increase in urinary bilirubin is nearly always indicative of a

corresponding increase in the serum direct fraction attributable to intrahepatic or

extrahepatic cholestasis (Remeth et al 2002).

Serum ammonia and urea concentration

Serum ammonia concentration can also be used to assess hepatic function,

but a single baseline sample can be normal even in a patient with signs of hepatic

encephalopathy. Products other than ammonia may be responsible for the clinical

signs (Tyler 1990). The ammonia tolerance test (ATT) is a provocative test of

hepatic function. It is performed by measuring the fasting serum ammonia

followed by measuring the serum ammonia after the oral administration of

19

ammonium chloride. It is a very sensitive indicator of hepatic function and portal

circulation; significant elevations were detected following 60% hepatectomy in

dogs, but not after 40% (Prasse et al 1983). Unfortunately, because ammonia

samples are not stable, they must be analyzed immediately. Furthermore, there is

a potential risk of inducing neurological signs with ammonium chloride

administration in a patient with impaired liver function. The variability of results

because of improper sample handling restricts the use of ATT to facilities that

have the capability of performing the analysis.

Urea formation is related to hepatic metabolism of ammonia and low

serum urea nitrogen concentration (<10 mg/dl) may result when ammonia is not

metabolized (Schall 1976). Hall (1985) reported a significant decrease in serum

urea nitrogen (SUN) in anorectic dogs with normal liver function due to

decreased protein intake. He also noted that dogs with decreased hepatic mass

might have normal levels of SUN if they were dehydrated or had concurrent

renal dysfunction. These observations were in agreement with Johnson and

Sherding (1994) who stated that blood urea nitrogen (BUN) concentration may

be decreased secondarily to liver disease. It has been reported that 40% of the dogs

with hepatic encephalopathy have ammonium biurate crystals in urine, which can be

reliably used for its diagnosis (Rothuizen 2004).

Sulphobromopthalein and Indocyanine green

Sulphobromopthalein (SBP) is an exogenous indicator of hepatic

function. After SBP has been injected, its serum concentrations are measured

against time. The rate at which the drug is eliminated assesses a complex series

of mechanisms, including albumin binding, portal circulation, hepatocyte uptake,

cytosolic protein binding, conjugation, and biliary excretion (Center 1989).

20

Eikmeier (1960) carried out fifteen different tests on large numbers of dogs to

determine their suitability to diagnose liver disease. He reported that for the

detection of liver damage only the determination of serum bilirubin and the SBP

test proved satisfactory. Unfortunately SBP now has limited availability because

it has been reported to cause anaphylactic and local reactions in man (Center

1989).

Indocyanine green (ICG) has fewer side effects than SBP, but it is

technically a more difficult assay to perform in the laboratory (Center 1986).

Both tests are affected by obesity, edema, ascites, albumin concentration, and

sampling techniques (Center 1986; Sutherland 1989). False positive results will

occur in an icteric animal because SBP and ICG compete with bilirubin for

uptake, metabolism, and excretion (Center 1986).

Serum bile acids

Measurement of SBA is a relatively easy, safe, non-invasive and rapid

means of assessing hepatic function. Bile acids are stable in serum for long

periods of time and can be frozen. They are, consequently, ideal for private

practitioners who send samples to regional laboratories. Bile acids are equivalent

to the ATT in detecting deficiencies in hepatic mass or circulation (Center et al

1985; Center 1989; Mayer 1986) and are less variable than BSP or ICG (Center

1989). The test does not involve the administration of any dye, chemicals, or

other exogenous substances and, therefore, has no risk to the patient.

Bile acids are produced by hepatocytes and represent an end product of

hepatocellular cholesterol metabolism (Wilson 1981). Use of the SBA test is

indicated whenever hepatobiliary disease is suspected. The test has high

sensitivity and specificity. The results of the SBA test are usually unequivocal

21

when performed properly with a fasting and two-hour postprandial sample.

Occasionally, confusion arises when the test is conducted on a patient with a

healthy liver. For instance, one sometimes finds a higher concentration of bile

acids in the fasting sample than in the postprandial sample. This may occur with

a spontaneous contraction of the gall bladder during a prolonged fast (Center et

al 1991). However, the fasting serum bile acids (FSBA) concentration should not

exceed the laboratory reference range for the postprandial serum bile acids

(PPSBA) if the liver is healthy. Another source of variability can be attributed to

individual differences in the response time of the gall bladder to feeding. Clinical

studies are lacking to assess whether this is significant.

It is also possible for the SBA concentration of an animal with impaired

liver function to be within the reference range (false negative). Transient

decreases in bile flow may lower the concentration. The serum concentration of

bile acids can also be lowered by impaired ileal function (Center et al 1991).

These two conditions are generally rare.

Elevations in the SBA concentration occur when there is defective

hepatoportal circulation, loss of functional hepatic mass, hepatobiliary

obstruction, or laboratory error. If hepatic disease is suspected and the SBA

concentration is elevated, hepatic biopsy is indicated. It has been speculated that

minor elevations can occur in patients treated with glucocorticoids (Johnson et al 1985).

Glucocorticoids may disrupt bile acid metabolism by altering canalicular permeability

and causing cholestasis. In dogs, this should be supported by an elevation in the

concentration of serum ALP. A recent study found that topical glucocorticoids elevated

ALP, but did not affect SBA (Meyer et al 1990).

Specificity of SBA as an indicator of hepatobiliary disease in dogs and cats has

22

been reported (Center 1990). Rutgers et al (1988) also described SBA concentration as

more sensitive indicator of hepatobiliary function than the enzyme profile. Bile acid

concentrations >25-30 μmol/L in dogs is suggestive of hepatobiliary disease, i.e.

decreased functional mass, alterations in portal circulation, or cholestasis. It was also

observed in the same study that the dogs with bile acid concentrations < 15 μmol/L do

not have evidence of hepatic pathology on biopsy, whereas dogs with values > 25

μmol/L usually have hepatic pathology. Dogs with bile acid values between 15-25

μmol/L are in an equivocal zone (i.e., may or may not have hepatic pathology).

Determination of total serum bile acids (TSBAs) in the presence of jaundice is

warranted only when haemolysis could not be ruled out, because bilirubin and bile

acids do not share hepatic transport systems and in haemolytic disease TSBAs should

remain normal (Leveille-Webster 2000).

Serum cholesterol

Serum cholesterol levels are variable in diseases of the liver. Cholesterol is

excreted from the organism primarily through the biliary system and its rise is usually

associated with diseases of this system. Hypocholesterolemia is associated with a long-

lasting liver disease. The reason for this is the drop in the production or absorption from

the intestines or higher conversion to bile acids. The most frequent liver disorder

associated with hypocholesterolemia is the PSS, in which increased conversion to bile

acids is the primary mechanism (Leveille-Webster 2000). Hypercholesterolemia is

commonly associated with cholestatic disease (Hall 1985). Moreover, increased

production of cholesterol might occur with retention of lecithin in bile.

Blood glucose

Liver plays a critical role in maintenance of the blood glucose concentration,

and marked hypoglycaemia is sometimes associated with liver failure.

23

Hypoglycaemia has been reported in dogs with hepatic insufficiency associated with

vascular shunts (Jerry et al 2008) and more than 75% of hepatic mass must be lost

before occurrence of hypoglycaemia is noted which might be attributed to decreased

gluconeogenesis and decreased insulin clearance (Dial 1995). Hypoglycaemia has

also been reported as a paraneoplastic syndrome in dogs with hepatic neoplasia

(Center 1996) and as an early indicator of hepatic failure in severe acute hepatobiliary

injury (Leveille-Webster 2000).

The most common cause of hyperglycaemia and glycosuria associated with

hepatic insufficiency in dogs is diabetes mellitus. Mild hyperglycaemia can occur in

some dogs up to 2 hours after consumption of diets containing increased quantities of

monosaccharides and disaccharides, corn syrup, or propylene glycol; during

intravenous (IV) administration of total parenteral nutrition fluids; in stressed,

agitated, or excitable dogs; in animals in the early stages of diabetes mellitus; and in

animals with disorders and drugs causing insulin resistance (glucocorticoids,

progestins, megesterol acetate). Hyperglycaemia is also associated with

hyperadrenocorticism, diestrus in bitch, pheochromocytoma, pancreatitis, exocrine

pancreatic neoplasia, renal insufficiency and head trauma (Nelson and Couto 2008).

2.5.3 Serum protein gradient

The plasma albumin concentration is determined by the hepatic synthetic

rate that normally is in equilibrium with degeneration. Hypoalbuminemia may be

caused by defective albumin synthesis associated with severe hepatocellular

disease or may be caused by increased albumin loss resulting from

glomerulopathy (protein-losing nephropathy), severe intestinal inflammation, or

intestinal lymphangiectasia (protein-losing enteropathy). In severe chronic

hepatopathy, there is a tendency for elevations in IgM, IgG, and IgA. Both

24

decreased albumin and increased globulin results in a decrease in the

albumin/globulin (A/G) ratio (Jerry et al 2008). Hypoproteinemia associated

with liver disease usually accompanied by hypoalbuminemia and that is helpful

in differentiating acute from chronic diseases (Hall 1985). Hypo- or

hyperproteinemia will be found in dogs suffering from neoplasia, whereas

hyperglobulinemia is more consistent finding in those dogs (Kosovsky et al

1989). Dill-Macky (1995) reported hypoproteinemia, hypoalbuminemia and

hypergammaglobulinemia in advanced stage of chronic hepatitis in dogs.

Chronic hepatic disorders such as cirrhosis and portosystemic vascular anomalies

(PSVA) are most commonly accompanied with serum hypoalbuminemia

(Tennant 1997). Hypergammaglobulinemia associated with chronic liver disease

was attributed to enhanced systemic immunoreactivity due to Kupffer‟s cell

processing of portal antigens or secondary to autoantibody production (Leveille-

Webster 2000).

Ascites is now being classified as "high gradient" and "low gradient" based on

the serum ascites albumin gradient (SAAG). If the difference between serum albumin

and ascitic fluid albumin is > l.1g /dl it is called high gradient ascites, whereas if the

difference is < 1.1g/dl it is termed as low gradient ascites (Burgess 2004). SAAG is

considered as a marker of portal hypertension and used as an index to replace the

exudates- transudate concept in ascitic fluid (Tan and Lapworth 2010). Moreover,

SAAG >1.1 g/dl is suggestive of portal hypertension which can be a consequence of

chronic liver disease (Saravanan et al 2012).

2.6 PERITONEAL FLUID ANALYSIS

Characterizing the peritoneal fluid is an important step in the determination of

primary causes of liver function affection. Analysis of cell numbers and their kinds

25

(inflammatory or neoplastic ones, etc.), protein concentration, specific gravity

measurement and biochemical values (amylase, lactic dehydrogenase and glucose

concentration) have been proved to be of high diagnostic value (Perman 1989; Glińska

et al 2006). Cytologic examination of peritoneal fluid is believed to be the most sensitive

and specific method in establishing the neoplastic aetiology of ascites (Glińska et al

2006). Similarly, he reported that changes in triglycerides and cholesterol concentration

were caused by ascites due to liver disease. When the effusion in the abdominal cavity

is formed by transudation, it may suppose low albumin production by the liver

parenchyma leading to low protein concentration in the blood serum and subsequent

fluid passage from blood to body cavities (Rebar 1989). Contrary to this, bacterial

peritonitis results in exudation. Peritoneal fluids associated with most of hepatic

disorders in dogs were noted to have hypoproteinemia, with a protein content < 2.5 g/dl

i.e., either transudate or modified transudate (Dill-Macky 1995; Johnson 2000).

Rothuizen and Meyer (2000) observed that hepatic congestion was associated with

sanguineous peritoneal fluids.

2.7 COAGULATION ABNORMALITIES

The liver is the source of most proteins taking part in the blood coagulation

(fibrinogen, prothrombin, factors V, VII, IX, X, XI, and XII together with factors II,

VII, IX and X, which are K-vitamin dependent) and blood coagulation inhibitors

(antithrombin III, plasminogen, α2-macroglobulin, α2-antiplasmin) (Feldman 1980).

Synthesis of coagulation proteins tends to be diminished in liver disease, and

decreased plasma prothrombin synthesis is associated with a corresponding increase

in the prothrombin time. Fibrinogen is an acute phase reactant, and its concentration

in plasma may be greatly increased in chronic inflammatory diseases or in neoplasia.

Plasma fibrinogen is generally normal in mild or moderate liver disease. Because of

26

rapid turnover of fibrinogen and prothrombin, the concentration of these proteins in

plasma may decrease rapidly in fulminant hepatic injury. The turnover of albumin is

longer and the concentration of albumin is diminished primarily in chronic liver

disease in which there is significant loss of hepatocellular mass (Jerry et al 2008).

Measurement of protein C has been validated for use as a biomarker for the

assessment of experimental liver injury (Saha et al 2007), and when combined with

other conventional laboratory tests, it was shown to be of value in the recognition of

PSS and other severe clinical forms of hepatobiliary disease in dogs (Toulza et al

2006). Badylak et al (1983) conducted a study on dogs with naturally occurring

hepatopathy and reported that 50 and 75 per cent had abnormal prothrombin (PT) and

activated partial thromboplastin time (APTT), respectively. Hepatobiliary disorders in

dogs may associate with defects in platelet aggregation (Willis et al 1989) and

increase in fibrin degradation products (FDPs) (Center 1996)

2.8 URINALYSIS

Many patients with hepatobiliary disease have PU and PD and therefore a low

urine specific gravity. Some dogs with PSVA have detectable ammonium biurate

crystalluria on urine sediment examination due to concurrent hyperuricaemia and

hyperammonemia hence several examinations of fresh urine samples may be

necessary in order to detect crystalluria. It is normal for some dogs (particularly male

dogs) to have some conjugated bilirubin in their urine, but presence of

hyperbilirubinuria is an indicative finding of excessive extravascular haemolysis or

hepatobiliary disease (Santilli and Gerboni 2003).

2.9 ELECTROLYTE AND ACID BASE DISORDERS

Hepatic diseases rarely cause electrolyte disturbances. Acid base abnormalities

associated with liver disease are most often respiratory alkalosis, metabolic alkalosis

and metabolic lactic acidosis (Narrins and Gardner 1981).

27

2.10 SEROLOGY AND MOLECULAR DIAGNOSIS

Serological and molecular approach of the blood collected is used in a routine

way for the purpose of health state screening. It is very precise and the center of its use

lies in the diagnostics of viral infections including canine distemper virus (CDV)

and canine adenovirus types-1 (CAV-1); Toxoplasma gondii (Headley et al 2013);

vector borne diseases such as leishmaniasis (Hamarsheh et al 2012), leptospirosis

(Richard et al 2006), babesiosis and ehrlichiosis (Vargas et al 2012; Hamel et al

2012), Hepatozoon canis, Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema

reconditum and Mycoplasma hemocanis (Hamel et al 2012).

2.11 MEDICAL IMAGING

2.11.1 Radiography

A combination of laterolateral and ventrodorsal projections is suitable for liver

imaging. The size, the position and the density characteristics of the liver are

evaluated. As a general rule, the liver image considerably beyond the rib arch may be

considered to be liver enlargement (Popesko et al 1990).

Evaluating some hepatomegaly in a more exact way, it is necessary to

examine the axis of the stomach in relation to the axis of the body, which changes in

such cases. Radiographs reveal also masses that are associated or only adjacent to the

liver (tumours, abscesses), as well as position changes due to a hernia and a torsion of

liver lobes (Miles 1997). Radiography may confirm the presence of ascitic fluid in the

abdominal cavity manifested as a loss of clarity and detail of the abdominal cavity

(Meredith and Rayment 2000). However, lack of abdominal contrast and insensitivity

to detect subtle changes limits the precision of abdominal radiography. It is difficult to

evaluate the entire liver as much of the liver is silhouetted by the diaphragm, stomach

and right kidney (Konde and Pugh 1996).

http://www.ncbi.nlm.nih.gov/pubmed?term=Hamarsheh%20O%5BAuthor%5D&cauthor=true&cauthor_uid=22937916

28

Contrast angiography may be used to visualize radiologically the vascular

system and demonstrate vascular shunts (Center 1993; Leveille-Webster 2000).

Capnoperitoneography, the special contrast radiographic procedure, enhances

the visceral visualization of abdominal organs in general and is very useful in the

evaluation of liver lobes and its borders, especially the diaphragmatic border (Kumar

et al 2012). In suspected cases of hepatic neoplasia, thoracic films to evaluate the

pulmonary metastasis are also desired.

2.11.2 Ultrasonography

Ultrasonography is an excellent non-invasive technique that makes it possible to

characterize the liver parenchyma structure, liver size, and also masses or focal changes

such as abscesses, tumours and cysts (Miles 1997). Ultrasonography helps to localize

lesions larger than 0.5 cm in size (Center 1998) and differentiating focal from diffuse

disease and obstructive from non-obstructive icterus (Kumar et al 2012). This

technique may be used to obtain biopsy specimens from the liver tissue and masses

adjacent to the liver. Nyland and Gillet (1982) stated that ultrasonography is useful in

diagnosing posthepatic biliary obstruction. Distension of the gall bladder with loss of

tapering of the neck into the cystic duct was the first indication.

2.11.3 Doppler ultrasonography

Doppler ultrasonography is a non-invasive technique for the evaluation of

tissue perfusion. In comparison with other organs there are two kinds of blood

circulation in the liver. The portal venous system has low blood pressure in vessels

without strong pulsation. In the arterial system on the other hand, there is a marked

stronger pulsatile blood flow because of higher blood pressure in vessels. In patients

suffering from liver cirrhosis the intrahepatic resistance of vessels increases up to

five times and, proportionately to it, the portal system blood pressure rise leads to

29

portocaval shunt formation. Examination of hepatic arterial blood flow is also

possible using either transcutaneous or intravascular Doppler ultrasonography

techniques (Hubner et al 2000).

2.12 Liver biopsy and histopathological examination

Biopsy is a method aiding in the determination of a precise diagnosis and

disease prognosis (Rockey et al 2009). Liver biopsy is indicated in cases of abnormal

enzymatic activities associated with liver functions and their persistence for as long as

30 days and more, hepatomegaly of undetermined origin, liver complications of

systemic diseases, suspected neoplasia, therapy response determination and disease

progression (Hoefer 1992; Kerwin 1995). Liver biopsy can be further used to

differentiate acute from chronic disorders, to stage neoplastic disease and to assess

response to therapy. Selection of the best procedure for obtaining a liver biopsy

depends on numerous factors including liver size, presence of coagulopathy, any focal

or diffuse lesion, presence of biliary tract obstruction, or any other intra-abdominal

abnormalities. The selection of the biopsy method also depends on likelihood of

surgical resection of a mass, tolerance of general anaesthesia, available equipment and

expertise of the clinician (Nelson and Couto 1998). The various biopsy methods

include fine-needle aspiration, blind percutaneous needle biopsy using Tru-Cut biopsy

needle, ultrasound-guided needle biopsy, keyhole needle biopsy and laparoscopic-

guided biopsy (Johnson and Sherding 1994). The biopsy specimens so procured are

subjected to standardized processing and histopathological examination for yielding

definitive diagnosis of hepatic affections.

2.14 MANAGEMENT OF HEPATOBILIARY DISEASES

A variety of drugs are used in both acute and chronic liver disease, each of

which has specific indications as well as contraindications.

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2.14.1 Anti-inflammatory drugs

The most widely used anti-inflammatory drugs for treating chronic liver

disease are:

Corticosteroids

Corticosteroids have immune-modulating and antifibrotic properties. They

have a potent indirect antifibrotic action by reducing prostaglandin and leukotriene

production from inflammatory cells and a weak direct antifibrotic action by inhibiting

mRNA and enzymes. Corticosteroids are very rarely indicated for acute liver disease

since they are often associated with portal hypertension. The only study documenting

the use of corticosteroids is in cases of canine chronic hepatitis, given at 2.2 mg/kg for

seven to 14 days. They resulted in a significant increase in survival time (Strombeck

et al 1998). However, the ideal dose and duration of treatment remain unknown.

Immune-mediated hepatitis has not yet convincingly been shown to exist in dogs. It is

often difficult to assess „remission‟ in veterinary cases, particularly as corticosteroids

induce hepatic enzymes and so confuse attempts to follow the disease

clinicopathologically. Adverse effects of steroids in liver disease include increased

protein catabolism, fluid retention, gastrointestinal ulceration, risk of infections and

steroid hepatopathy.

Other drugs with anti-inflammatory activity in addition to their other actions

that may be used in animals with liver disease include ursodeoxycholic acid (Meyer et

al 1997), antioxidants such as S-adenosylmethionine (SAM-e) (Center et al 2005) and

zinc, and colchicine (Bexfield and Watson 2009).

Azathioprine

Azathioprine has been used in dogs with chronic hepatitis but, until an

autoimmune aetiology has been definitively described in this species, it is difficult to

31

justify the use of this or other immunosuppressive medications such as cyclosporine

(Bexfield and Watson 2009).

2.14.2 Antibiotics

Antibiotics are indicated when a bacterial infection is a primary cause or

secondary complication of canine liver disease. They are also commonly used in the

treatment of hepatic encephalopathy. Where possible, antibiotics should be chosen

based on the results of culture and sensitivity testing. However, they are often chosen

based on knowledge of the likely sensitivity profile of implicated organisms. The

bacteria involved are usually of enteric origin and it is particularly important to try to

culture bile in cases of ascending cholangitis both before and during treatment to

avoid eruption of antimicrobial resistance in these patients (O‟Neill et al 2006).

Ampicillin, amoxicillin, cephalexin, fluoroquinolones and metronidazole are used in

dogs with liver disease because of their efficacy against enteric organisms and

concentration in bile. Antibiotics that rely on hepatic clearance or those which are

potentially hepatotoxic should be avoided. These include tetracyclines,

sulphonamides, chloramphenicol and erythromycin (Bexfield and Watson 2009).

2.14.3 Antioxidants

Antioxidants include vitamin E, zinc, silymarin (milk thistle) and SAM-e.

Oxidant stress is increased in cases of liver disease due to the effects of inflammation,

reduced blood flow and mitochondrial damage by refluxed bile acids (Bexfield and

Watson 2009). The use of antioxidants therefore seems logical, although, in general,

there is no clear evidence that they improve the quality of life or survival of an

animal.

SAM-e increases hepatic and red blood cell glutathione levels, and is widely

available as a neutraceutical for dogs. It is particularly helpful for toxic hepatopathies

32

in humans, such as phenobarbital-induced hepatopathy, and recent work suggests it

might be helpful for steroid hepatopathy in dogs (Center et al 2005). Unfortunately,

there is scarcity of data about the use of both SAM-e and silymarin in dogs with acute

toxic hepatopathies.

Vitamin E has an effective antioxidant activity in dogs with liver disease

(Twedt et al 2003). It should also be used in dogs suffering from copper storage

disease, as levels of vitamin E are reduced in hepatocytes in such patients. Vitamin E

is given at a dose rate of 400 - 600 U/day in medium-sized dogs.

Zinc has an antioxidant activity as well. However, not all antioxidants are

necessarily innocuous in animals with liver disease for instance; ascorbic acid may

increase liver damage by accumulating iron, so it is best to avoid vitamin C

supplementation (Bexfield and Watson 2009)..

2.14.4 Lathyrogenic (Antifibrotics) drugs

Beside the antifibrotic action of corticosteroids and azathioprine, more specific

antifibrotics are available. Colchicine is an alkaloid which binds tubulin and has the

potential to reverse fibrosis. It is useful in some dogs and should probably be reserved

for those found to have moderate to marked fibrosis following biopsy results.

Although it improves survival in human cirrhotic patients, there are limited reports on

its use in dogs. It should be used carefully as adverse effects, including

myelosuppresion, anorexia and diarrhoea, can occur (Bexfield and Watson 2009).

Colchicine is used at a dose rate of 0.025-0.03 mg/kg/day and has a reputation for

causing nausea, vomiting and diarrhoea in dogs. Some authorities suggest that

colchicine should not be used until evidence to support its use is published.

2.14.5 Choleretics and bile acid modifiers

Ursodeoxycholic acid (ursodiol) is a bile acid modifier. It has been used safely

in many canine and feline cases of liver disease, but is not licensed for use in small

33

animals. It is a hydrophilic bile acid that displaces toxic hydrophobic bile acids and

stimulates bile flow thus acts as a choleretic agent. These two actions reduce cell

damage and oxidative stress resulting from the retention of bile acids in the liver. It

also has an immunomodulatory action by reducing immunoglobulin and interleukin

production and the expression of major histocompatibility complex type-1 on

hepatocytes. Recent studies show additional antioxidant activity with a synergistic

action of SAM-e and vitamin E. Although ursodeoxycholic acid has been widely and

safely administered in dogs, only a single case report documents its use in this species

(Meyer et al 1997). Larger studies are required to clarify its indications and efficacy.

Currently, ursodeoxycholic acid is potentially indicated in most cases of liver disease,

particularly those associated with biliary stasis. However, it should be avoided in dogs

with complete biliary obstruction as it has a potential to cause gall bladder rupture,

although complete cholestasis is rare in animals (Bexfield and Watson 2009).

Ursodeoxycholic acid is administered orally at a dose rate of 15 mg/kg PO SID or

divided BID.

2.14.6 Copper chelators

Copper chelators include 2,2,2-tetramine tetrahydrochloride (2,2,2-T;

Trientene), D-penicillamine and zinc. Copper chelators are not licensed for use in

dogs as they can cause significant side effects if they are not used carefully.

Penicillamine is the one with the most pharmacokinetic information in dogs, and is

the most readily available copper chelator. Unfortunately, it is not helpful in acute

crisis as chelation can take weeks to months to occur (Mandigers et al 2005),

therefore, 2,2,2-T may be more useful in these circumstances. Zinc is generally used

as a prophylactic in dogs with copper storage disease, and commercially produced

hepatic support diets often contain increased zinc for this reason. D-penicillamine and

34

2,2,2-T are given 30 minutes before feeding at a dose rate of 10-15mg/kg PO BID

whereas zinc gluconate and zinc acetate are given at 10mg/ kg BID, one hour before

feeding.

2.14.7 Control of gastrointestinal haemorrhage

Sources of gastrointestinal haemorrhage (e.g., parasites, ulcers) should be

treated or removed whenever possible. Lactulose (5-20 ml PO every 8 to 12 hours

until the stools are slightly soft), a synthetic disaccharide, should be given. This

osmotic laxative will hasten gastrointestinal emptying, allowing less time for

absorption of ammonia produced in the colon by intestinal bacteria. More

importantly, fermentation of lactulose lowers intestinal pH, trapping ammonia (as

ammonium ions) in the colonic contents, and changing the colonic bacterial flora

favourably. An antibiotic that is not absorbed from the intestinal tract and is active

against urea-splitting bacteria such as neomycin (25 mg/kg PO BID) is also useful in

some patients (Bexfield and Watson 2009).

2.14.8 Dietary management

Appropriate dietary management is as important as drug therapy in dogs with

liver disease. Each case is an individual and the diet should be adjusted accordingly.

In particular, diets with inappropriate and excessive protein restriction may limit

hepatic regeneration and result in protein–calorie malnutrition in animals with liver

disease. Commercially produced hepatic support diets may be too low in protein and,

again, protein supplementation may be necessary. However, these liver diets have

other beneficial features such as increased amounts of zinc and B vitamins, therefore,

in practice, dogs with liver disease may be fed a commercial liver diet with extra

high-quality protein supplementation; or a high-quality, digestible diet (e.g., a diet

marketed for intestinal disease). Suitable high-quality proteins for liver disease (i.e.,

35

those that have all the essential amino acids and are also digestible) include cottage

cheese, chicken and soya.

The effectiveness of dietary therapy should be monitored by controlling

clinical signs and checking that the patient maintains its bodyweight and blood protein

levels. Many drugs and dietary manipulations are indicated for the management of

liver disease in dogs, each of which has specific indications and actions. Care must be

taken when using drug therapy as many products have the potential to cause serious

complications.

2.14.9 Miscellaneous

Stravitz (2008) suggested that the administration of N-acetylcysteine should be

considered in patients with early-stage hepatic encephalopathy regardless of

aetiology. Similarly, Bexfield and Watson (2009) reviewed that N-acetylcysteine is an

antidote for paracetamol toxicity and may also be helpful in cases of potentiated

sulphonamide toxicity. They also reviewed that ascites should be treated with

spironolactone with or without furosemide or thiazide diuretics and abdominocentesis

should be performed when ascites is so severe that it interferes with respiration

process.

Because the liver is physiologically and anatomically diverse, there is no

single test that adequately identifies hepatic disease or its primary cause. Therefore, a

battery of tests is used to diagnose the hepatobiliary affections.

36

CHAPTER III

MATERIALS AND METHODS

3.1 PLACE OF WORK

The study was carried out on the dogs reported in year 2013-14 at the

Department of Veterinary Medicine at Teaching Veterinary Hospital, Guru Angad

Dev Veterinary and Animal Sciences University, Ludhiana.

3.2 SECTION OF ANIMALS FOR STUDY

On an average more than fifty dogs were presented every day for the treatment

of various ailments at the Small Animal Clinic. A total of 140 dogs suspected with

diseases of liver, biliary tract and hepatic vasculature were selected for the study and

investigated according to the following protocol:

3.3 CLINICAL EVALUATION

The client information and comprehensive clinical examination of each animal

was performed as follows:

3.3.1 Client information

The detailed client information i.e., case number, date of presentation, name,

address and cell phone number of the dog‟s owner were recorded in “Hepatic

Insufficiency Case Record” to follow up the case during the period of treatment till

recovery or death or at least till the settlement of animal‟s condition.

3.3.2 History taking

Complete history of the pet was obtained from the owner which included age,

breed, sex, weight, vaccination and deworming status, history of previous illness (if

any) and history of current illness, duration of illness and the treatment given (if any).

Clinical signs were recorded in chronological order of occurrence especially pyrexia,

jaundice, appetite status, vomiting/regurgitation or hematemesis and frequency (if

37

any), epistaxis, defecation status (colour, consistency and frequency), weight loss/

cachexia, water intake, urination status (colour, quantity, frequency and difficulty in

urination if any), history of coughing and/or exercise intolerance, abdominal

distension, dietary protocol, presence of external parasites, change in attitude,

episodes of weakness and seizures or collapse or any other nervous manifestations.

3.3.3 Physical examination

A thorough physical examination of selected patients was conducted as

proposed by Nelson and Couto (2009). Aspects of the examination included: Rectal

temperature, mucous membrane colour and capillary refill time, heart/arterial pulse

rate and quality and hydration status were recorded. Auscultation of the heart (rate,

rhythm, adventitious sounds) and lungs was performed and findings were recorded.

Patients were also examined for jugular venous distension or pulsation, superficial

lymph nodes enlargement and the presence of skin bruises, petechiation and/or

ecchymoses and hepatocutaneous syndrome. Abdominal palpation and ballottement

were carried out to check for the presence of any pain (hepatodynia), fluid

accumulation, organomegaly or any abnormal mass. Oral cavity was also examined

for ulcerations and abnormal odours. Signs of hepatic encephalopathy (including

dementia, seizures, changes in personality and motor disturbances, etc.) as well as any

other indication of reactive hepatopathy were thoroughly investigated.

The detailed information of clinical evaluation of the case was recorded as per

the enclosed proforma in Annexure 1.

3.3.4 Sample collections

Blood sampling: For collection of blood, the patient was properly restrained either in

lateral or sternal recumbency without chemical restraint. A blood sample (5mL) was

38

collected under aseptic condition from either cephalic or recurrent tarsal vein. The

freshly collected blood was divided into four portions:

(i) One part poured into Potassium Ethylene-Diamine Tetra-Acetate (K3EDTA)

containing vials (AcCuvet-PLUS, Peerless Biotech) for haematology and

examination of haemoprotozoan parasites.

(ii) Second portion poured into sodium fluoride-containing vials (Bioplus) for

glucose estimation.

(iii) Third portion poured into 3.2% sodium citrate-containing vials (AcCuvet,

Quantum Biological, Chennai, India) for estimation of coagulation parameters

{prothrombin time (PT), activated partial thromboplastin time (APTT) and

fibrinogen}.

(iv) Fourth portion of blood samples was collected without anticoagulant in dry

disposable sterile syringes (Romo-Jet™, Romsons Juniors India) and was

allowed to clot for 2 hours and centrifuged at 2500 rpm for 3 minutes for

harvesting serum. Serum samples were used within three hours for estimation of

biochemical parameters and the rest was stored at -20°C as stock samples and

for estimation of total serum bile acids (TSBAs).

Urine sample: The urine samples from suspected dogs was collected aseptically in 5

ml sterile syringes (Dispovan, Ballabgarh, Faridabad, India) attached to 23G sterile

hypodermic needles using ultrasound guided cystocentesis. Samples were

immediately taken to biochemistry lab for evaluation.

Peritoneal fluids: The peritoneal fluid of ascitic dogs was collected aseptically in

sterile K3EDTA coated vials (AcCuvet-PLUS, Peerless Biotech) with the animal in

lateral recumbency using USG guided needle aspiration or on some occasions blindly

with the animal in lateral recumbency, or in standing position in those patients with

39

severe abdominal distention. Twenty three gage needles attached to 5 ml syringes

were used for puncturing the abdomen caudal to umbilicus on ventral midline.

3.3.5 Laboratory analysis

Comprehensive hepatobiliary testing includes a complete blood count, serum

biochemical profile, urinalysis; coagulation profile tests; abdominal imaging; ascitic

fluid examination and culture and liver fine needle aspiration biopsy for clinical

pathology.

3.3.5.1 Complete blood count (CBC)

Haematologic markers were estimated using fully automated haematology

analyzer (ADVIA®2120 haematology system, Siemens Healthcare Diagnostics Inc.,

with Veterinary Package Software, Abbott Laboratories, IL, USA). Haematologic

markers include: haemoglobin (Hb), total leucocytic count (TLC), differential

leucocytic count (DLC), total erythrocytic count (TEC), packed cell volume (PCV),

mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean

corpuscular haemoglobin concentration (MCHC) and platelet count. Platelets were

also qualitatively evaluated in many cases.

Differential leucocytic count (mature and immature neutrophils) and

morphologic abnormalities of peripheral blood cells (toxic changes, inclusions and

neoplasia) were discovered by microscopic examination with the oil immersion lens

of well-prepared films of peripheral blood stained with Leishman‟s stain as per the

method described by Jain (1986).

3.3.5.2 Biochemistry panel

Serum samples were analyzed to determine the activities of alanine

aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase

(ALP), gamma-glutamyltransferase (GGT), total bilirubin (TB) concentration, total

40

plasma proteins (TP) and albumin (ALB) concentration, albumin/globulin (A/G) ratio,

blood urea nitrogen (BUN), creatinine, glucose, cholesterol and sodium and

potassium concentration. These variables were measured from nearly all of the

patients in this study using fully automated chemistry analyzer (Johnson & Johnson

VITRᵠS 750Xrc) and fully automated chemistry system with the help of reagent kits

(Johnson & Johnson diagnostic kits, Mumbai, India).

Total serum bile acids (TSBAs) concentrations were analyzed from 25

(17.86%) fasting dogs by an enzymatic spectrophotometric method to identify those

with hepatobiliary dysfunction.

Out of 140 serum samples, twenty-five samples from different breeds of dogs

confirmed of having hepatobiliary disease (based on combined data from serum

biochemistry profile, haematology, ultrasonography and urinalysis) were picked up at

random and were sent to the SRL Diagnostics Reference Laboratories for analysis

(SRL limited, Maharashtra, India).

3.3.5.3 Examination of peritoneal fluid

Samples of peritoneal fluid were examined for colour, turbidity, odor, total

protein content, cytology, differential cell count and aetiological agents particularly

bacteria. Five ml of peritoneal fluid was centrifuged at 1500 rpm for 3 minutes (using

R-8C BL Laboratory centrifuge, Remi) and a thin smear was prepared from the

sediment, stained with Leishman‟s stain (BTL Research lab Vadorda, Gujrat, India)

and dried on slide warming plate equipped with thermostat. The prepared slides were

examined microscopically under oil immersion lens to help determine the potential

underlying cause of the fluid accumulation. Peritoneal fluid samples were also

submitted for bacterial culture in those cases suspected to be of inflammatory or

infected nature. Suitable bacteriological culture media (including heart and brain

41

infusion agar, nutrient agar and blood agar) were used for culturing and incubated for

48-72 hours to check for any bacterial growth.

3.3.5.4 Urinalysis

The urine so collected was subjected to routine physical, chemical and

microscopic examination. In the gross physical examination, urine was evaluated for

colour, odor, and the presence of turbidity or any deposits. In the chemical analysis,

urobilinogen, bilirubin, glucose, protein, ketones, specific gravity, erythrocytes,

leucocytes, urine pH, bacteria and nitrite were detected using dipstick method

(Johnson & Johnson diagnostic kits, Mumbai, India; Multistix® 10 SG Reagent Strips

for urinalysis, Siemens Healthcare Diagnostics Inc., USA; CLINITEK STATUS

analyzer, Bayer Healthcare LLC).

For microscopic examination, urine samples were centrifuged at 2000 rpm for

5 minutes. The supernatant was discarded and the sediments were thoroughly re-

suspended in the urine. A drop of this reconstituted sediment was transferred onto a

microscopic glass-slide and covered with glass slip. Initially, the quantity and the type

of casts were assessed under low power (10x) of light microscope. High power (40x)

was used to detect the presence of any abnormal structure. Slides were also examined

for presence of crystals, microorganisms, urothelial cells, pus cells and erythrocytes.

3.3.5.5 Examination of haemoprotozoa

Blood samples from icteric and pyretic dogs were examined for the presence of

haemoprotozoan parasites infections (Babesia, Hepatozoon canis and Ehrlichia canis)

as prescribed by Soulsby (1982). Haemoprotozoa were routinely examined through

preparation of thick and light blood smears by placing a drop of blood approximately

4 mm in diameter on a clean microscopic slide near the end. The drop is then spread

by using another slide “spreader” at a 45° angle and backing it into the drop of blood

42

so the spreader catches the drop and it spreads by capillary action along its edge.

Smears were air dried, labeled clearly and stained with a high-quality Leishman‟s

stain. Slides were dried by putting them on slide warming plate, and examined

microscopically directly under oil immersion lens (1000x magnification).

3.3.5.6 Coprological examination

Examination of fecal samples for the presence of parasite eggs, larvae, cysts

and oocysts was routinely performed through fecal floatation test as prescribed by

Soulsby (1982). Approximately two grams of fecal sample were collected directly

from the rectum of dogs into clean plastic containers using gloved finger and brought

to the clinical laboratory. For demonstration of various parasitic eggs, larvae, cysts

and oocysts in fecal smears, collected fecal matter was placed in porcelain mortar and

saturated salt solution was then added (Specific gravity 1.20). The combination was

stirred thoroughly and poured into a clean plastic tube. A clean glass coverslip was

put on the top surface of tube touching the surface of fluid. The suspension was then

allowed to sit for about 20 minutes so that any parasitic eggs present in the feces float

to the top of the fluid. Lastly, the top layer of fluid was placed on a clean microscope

slide and examined under light microscope.

3.3.5.7 Estimation of clotting profile

Prothrombin time (PT), activated partial thromboplastin time (APTT) and

fibrinogen concentration were estimated by the following protocol:

a) Prothrombin time

Prothrombin time was estimated by manual method using UNIPLASTIN®

reagent. Reagent vials were brought to room temperature (20-30°C). Vials contents

were mixed to obtain homogeneous suspension and sufficient amount of the reagent

was aspirated from the reagent vial and put in a clean and dry test tube. Reagent was

43

prewarmed in a water bath and brought to 37°C before use in the test procedure and

reagent vial immediately recapped and kept at 2-8°C. 100µl of platelet-poor plasma

(PPP) was put in a 12 x 75 mm tube and placed in a water bath (37°C) for 3 to 5

minutes. 200µl of UNIPLASTIN®

reagent (prewarmed for 3 minutes at 37°C) was

added forcibly to the tube and stopwatch was simultaneously switched on. Contents of

the tube were mixed by gentle shaking of the tube. Tube was then gently tilted back

and forth and the stopwatch was instantaneously stopped as the first fibrin strand was

visible and gel clot formation starts. Time elapsed was recorded in “seconds”. Test

was repeated in the same manner on duplicate samples and average was calculated to

report the value.

b) Estimation of activated partial thromboplastin time

Activated partial thromboplastin time was estimated by manual method using

reagents LIQUICELIN-E® and TULIP

® calcium chloride solution. Prior to use, the

reagent was mixed by gentle swirling and sufficient amount of the reagent was

aspirated from the vial into a clean and dry test tube. Reagent was brought to room

temperature prior to prewarming at 37°C for testing purposes. Separate test tubes

containing LIQUICELIN-E® and TULIP

® calcium chloride solution were brought to

37°C in a water bath. 100µl of test plasma and 100µl LIQUICELIN-E®

was put in a

12 x 75 mm tube and briefly shaken to mix the reagent and plasma; and placed in the

water bath at 37°C for 3 to 5 minutes. After incubation, 100µl of prewarmed calcium

chloride was added forcibly into the plasma and LIQUICELIN-E®

mixture and

stopwatch was simultaneously turned on. Tube was shaken briefly and gently to mix

the contents and kept at 37°C for 15 seconds. After 15 seconds of incubation, tube

was gently removed and tilted back and forth until a gel clot forms. Time elapsed for

clot formation was recorded in seconds and the test was repeated in the same manner

on duplicate samples. Average was calculated to report the value.

44

c) Estimation of fibrinogen

Fibrinogen was determined by Schalm method. Blood collected into sodium

citrate tubes was filled in two microhematocrit tubes and centrifuged at 2000 rpm for

10 minutes to clear the plasma. For determination of total plasma protein, a drop of

plasma was placed on the prism of a refractrometer. Another capillary tube was

suspended in a water bath adjusted at 58°C for 3 minutes. Capillary tube was

immersed in such a way that an entire column was surrounded by the warm water.

Capillary tube was then centrifuged to spin down the precipitated fibrinogen. Protein

concentration was measured in fibrinogen-free plasma prepared in second tube using

refractrometer. Fibrinogen concentration was estimated by calculating the difference

between the two plasma protein readings.

3.4 RADIOGRAPHIC, ULTRASONOGRAPHIC AND ELECTROCARDIO-

GRAPHIC STUDIES

Radiographic, ultrasonographic and electrocardiographic studies were

undertaken in most of the dogs suffering from hepatic dysfunction.

3.4.1 Radiology

Lateral and/or ventrodorsal (VD) radiographic views of abdomen and

sometimes of chest were taken to evaluate the size and shape of liver and heart and to

rule out the presence of any abnormal growth and metastasis. The various

radiographic findings were recorded and documented in the hepatic insufficiency case

records. Radiographic examination was conducted on selected cases using 160 mAs

X-ray machine (Siemens, Bharat electronics Ltd, India). Radiographic factors were

kept as per the requirement of case ranging from 10-16 mAs and 60-80 KVp at a

constant focal film distance of 32 inches for abdominal exposure. Potter bucky grid

and high speed intensifying screen were employed. For lateral and VD chest

radiograph, 18-24 mAs and 68-75 KVp were used.

45

3.4.2 Abdominal ultrasonography

The majority of suspected subjects were scanned using the ultrasound

technique after shaving of abdominal area from the costal arch to the pelvic inlet to

confirm with certainty the diagnosis of hepatic insufficiency. Patients were restrained

in ventrodorsal and lateral position with the help of the pets‟ owners and an attendant.

A coupling medium (gel, Ultrasound Transmission Aqueous Gel-Ethicon Division of

Johnson and Johnson Ltd., Aurangabad) was put on the areas to be scanned to ensure

an intimate contact of transducer with the skin. Ultrasonography was performed with

ultrasound machine with 3.5 or 7.5 MHz microconvex linear array transducer

depending on the patient. The liver was examined by positioning the transducer on the

ventral midline immediately caudal to the xiphoid and scanned craniodorsally.

Complete sweeps (sliding) and fanning of ultrasound beam through the liver was

made routinely in both sagittal and transverse planes. Hepatic size, surface regularity,

structure regularity, hepatic veins and echodensity; presence of portosystemic

collateral shunts and ascites, gall bladder changes, and portal lymph node size in

addition to the scanning of other abdominal organs including spleen, kidneys, adrenal

glands, gastrointestinal tract, pancreas, urinary bladder, uterus in females suspected of

having pyometra and prostate gland in males, mesenteric lymph nodes or any

abdominal mass using a concept/MCV Veterinary Ultrasound Scanner (Dynamic

imaging Co., Scotland, UK) gray scale, M real-time B-mode scanner).

Ultrasonographic images were recorded on thermographic printing papers of UPP-110

S series (Sony Corp. Tokyo, Japan) with a UP-895 CE (Sony Corp, 6-7-35,

Kitashinagawa-Ku, Tokyo, Japan) video graphic printer. The amplitude of returning

echoes was classified as normal (normoechoic), increased (hyperechoic), and

decreased (hypoechoic) or absent (anechoic) when compared to the normal echo

amplitudes for those organs.

46

3.4.3 Liver fine needle aspiration cytology/biopsy (FNAC/FNAB)

In many cases suspected for liver disease (altered echogenicity, irregular liver

margins, abnormal mass or high activities of hepatic enzymes) percutaneous

ultrasonographic-guided fine-needle aspirates (using 23 G hypodermic needle or

spinal needle connected to a 5 CC syringe in a few cases with severe abdominal

distension or giant dogs, Figure 1 A) were taken whenever it was possible to assist

confirming the diagnosis. In all patients, pet owners provided verbal informed consent

for the procedure. Before FNAB, bleeding tendencies were routinely evaluated by

careful review of the history, physical examination, blood smear (to confirm platelets

≥100,000/μl) and a routine coagulation profile (PT, APTT and fibrinogen). Animals

suspected to have acquired bleeding tendencies (platelets count < 100,000/μl or

having prolonged bleeding time) were exempted from tissue sampling. Only one case

with platelet count of less than 100,000/μl was injected with vitamin K1 followed by

sampling after 72 hrs. In general, FNA specimens were taken first and subjected to

immediate assessment. After localization, the needle was gently passed through the

lesion four to six times (Fig. 1 B). The needle was withdrawn and direct smears were

prepared from the samples and passed to the laboratory. Prepared smears were stained

with high quality Leishman‟s stain and examined by an expert cytopathologist.

3.4.4 ELECTROCARDIOGRAPHY (ECG)

Electrocardiography was performed for the majority of ascitic dogs and those

suspected of having cardiac diseases on the bases of case history, physical

examination and auscultation findings (like arrhythmia, tachycardia and adventitious

sounds). Dogs were restrained in right lateral recumbency on wooden table without

chemical restraint and with the help of the pet‟s owner and trained attendant.

Electrocardiography was recorded by using Bailey‟s hexaxial lead system. Limb leads

47

were placed on elbow joints (olecranon process) of both forelimbs and slightly above

the stifle joints of both hindlimbs after applying gel. The upper limbs were held

perpendicular to the long axis of the patient and parallel to the floor to avoid changing

of mean electrical axis with each pair of limbs parallel and not contacting each other.

No electromagnetic disturbances were allowed and proper earthing of the instrument

was done. Recordings were performed at paper speed of 50 mm/sec and amplitude of

10 mV and all the abnormalities were detected from lead II tracing.

Based on the combination of above findings, a confirmatory diagnosis was drawn

and the animals were subjected to the conventional medical therapy.

3.5 BLOOD CROSSMATCHING (BCM)

Blood cross matching was tested in the laboratory to avoid any complications

of incompatibility. Blood cross matching was divided into two parts: the major

crossmatch consists of mixing the patient‟s plasma with the donor‟s red blood cells;

the minor crossmatch consists of mixing the donor‟s plasma with the patient‟s red

blood cells. Of the two tests, the major blood crossmatch is much more important in

determining survival of the transfused red blood cells. Blood was collected into

EDTA tubes from recipient and potential donor. Tubes were centrifuged at 1500 rpm

for 5 min to separate plasma from RBCs. Blood plasma was then removed with a

pipette and transferred to a clean, labeled glass and observed for any. Consequently,

RBCs was washed 3 times with phosphate buffer saline (PBS) by adding 5 ml of PBS,

mixed thoroughly and centrifuged for 2 minutes. Saline is then removed, leaving

pellet of RBCs at bottom of tube which is then resuspended with PBS to make a 3–5%

RBC suspension. For each donor 3 tubes were labeled; major, minor, and recipient

control and to each tube 2 drops (50 µl) of plasma and 1 drop (25 µl) of RBC

suspension were added as follows:

48

a. Major crossmatch: recipient plasma + donor RBCs

b. Minor crossmatch: donor plasma + recipient RBCs

c. Control: recipient plasma + recipient RBCs

Gentle mixing and incubation for 15–20 minutes at 37°C in a warm water bath were

carried out, followed by centrifugation for 15 seconds at 1500 rpm. Supernatant was

examined for and the bottom RBCs were gently resuspended by tapping tube and

examined macroscopically for agglutination.

An autocontrol sample of recipient RBCs and plasma was included because

some recipients may have autoagglutination interfering with the BCM. When the

recipient control was positive (i.e., agglutination was present), the donor was replaced

because conclusions about blood compatibility between patient and donors could not

be established. Any and/or agglutination in the major or minor BCM (but not the

control) indicated an incompatibility and the need to choose a new donor.

3.6 TREATMENT AND MANAGEMENT

Medical therapy; type and duration of treatment of dogs diagnosed with

hepatopathy were almost similar with some modifications as per case depending on

the type and stage of the disease process, the severity and the other organ systems

involved. All dogs were treated symptomatically and palliatively. Treatment

adjustments were made if histology of FNAB resulted in a different diagnosis.

Response of therapy was evaluated on the basis of clinical improvement, laboratory

findings and periodical feedback from the pet owner.

The animals were randomly divided into two groups:

Animals in group I were given only conventional therapy

Animals in group II were subjected to conventional treatment along with N-

acetylcysteine tablets (Mucinac®) and in those cases with cholestasis,

ursodeoxycholic acid (Udiliv®

) was also added to the treatment protocol.

49

The clinical cases diagnosed with liver cirrhosis, neoplasia or cholelithiasis

and those which were complicated by urolithiasis and/or neoplasms were treated for

stabilization and were referred to the department of Veterinary Surgery and Radiology

for surgical management.

Therapeutic regimens

i) Antibiotics

Ampicillin (Roscillin®) was given twice daily (BID) at the dose rate of 20

mg/kg body weight intramuscularly during the course of treatment (IM).

In some cases amoxicillin (Amoxyrumforte®) was given at 20 mg/kg, BID, IM

as a substitute to ampicillin.

If the case was complicated with gastrointestinal disorder, enrofloxacin

(Enrosol®, Quentas

®, or Floxidin

®) at dose rate of 5 mg/ kg BW, BID along

with rabeprazole and domperidone combination (Rablet-D®

) was given.

Metronidazole (Metrogyl®

) was given at the dose rate of 10 mg/kg BW, BID

intravenously (IV) for 3-4 days in combination with Roscillin in those cases

with high TLC count.

Amikacin @ dose rate of 5mg/kg BW, BID was added to the above mentioned

antimicrobials in some cases when endotoxemia was suspected and was given

intramuscularly.

ii) Antioxidants

Silymarin (Silybon®

) syrup @ the dose rate of 10 mg/kg or Vitamin E

(Evion®) tablet at dose rate of 400 U once daily (SID) for 15 days was given.

iii) Liver tonics

Liverolin® or Liv-52

® syrup, two tea spoons, BID, for 15 days

Liver extract (Belamyl®) was given by IM injection, SID @ dose rate of 1.0

ml and 2.0 ml for small and large sized dogs respectively.

50

Vitamin B complex (Polybion®

) injection was given to anorectic dogs by IM

route, SID for 3-4 days.

iv) Fluid therapy and blood transfusion therapy

a. Fluid therapy was given as per condition based on skin tent test, and serum

biochemical profile results:

Dogs with history of anorexia for many days were treated with 5 per cent

dextrose normal saline (DNS) intravenously (IV) @ dose rate of 15-20 ml/kg

body weight to rehydrate and provide energy source.

Dextrose solution (50 %) was used by IV route at the dose rate of 1-2 ml/kg

over 1-2 hour infusion as a source of energy in patients with complete

anorexia.

Normal saline solution (NSS) and lactated Ringer‟s was administered

according to the acid base status.

b. Whole blood transfusion was carried out whenever possible in patients with

severe anaemia when haemoglobin content was less than 7 g/dl or PCV less than

20%. Blood was transfused at dose rate of 20 ml /kg BW. Before blood

transfusion, a single dose of dexamethasone sodium phosphate (Doxona®) was

administered intravenously at rate dose of 0.05 mg/kg to avoid any adverse

reaction.

v) Antipyretics and anti-inflammatory

Dipyron (Novalgin®/Vetalgin

®) was given in pyretic cases.

Prednisolone @ 2mg/kg was incorporated in treatment plane in cases with

immune mediated hemolytic anaemia (IMHA) and during blood transfusion.

Meloxicam (Melonex®) injections were a part of treatment in those patients

suspected with disseminated intravascular coagulation (DIC).

51

vi) Deworming (Plozyl®

) tablets; 1 tablet per 10 kg/BW was administered for dogs

with hookworms.

vii) Canine babesiosis was treated with diminazine aceturate (Berenil®) @ dose rate

of 1.2 mg/kg BW as a single dose (STAT) in one group and in other group of

dogs along with N-acetylcysteine.

viii) Canine monocytic ehrlichiosis treated with Doxycycline (Doxycip®

) tablets at

the dose rate of 10 mg/kg, SID for two weeks.

ix) Diuretics

Furosemide (Lasix®) was administered as a single dose @ the rate of 1-

2mg/kg BW in severe ascitic dogs.

Combination of furosemide and spironolactone (Lasilactone®) tablets @ the

dose rate of 1-2 mg/kg was given to severe ascitic cases with a tendency to

hypokalaemia.

Diuretics were always given after rehydration of patients.

x) Antemetics like metoclopramide (Perinorum®) at the dose rate of 0.2-0.4mg/kg,

and histamine receptor-2 blockers (ranitidine, aciloc®) at dose rate of 0.2-0.4

mg/kg BW, IV, BID were given in cases of gastroenteritis.

xi) Abdominocentesis was performed to remove large amounts of fluid in some

cases with severe ascites as it was interfering with the patient‟s ability to breath.

xii) A source of protein powder (Proteinex®), 1 tea spoon was given to patients with

hypoproteinemia and owners were advised to feed the animal egg white.

xiii) Diabetic dogs were treated with NPH insulin, subcutaneously (SC) at regular

intervals.

xiv) Congestive heart failure was controlled by angiotensin converting enzyme

(ACE) inhibitors (enalapril®) tablet, @ the dose rate of 0.25-0.5 mg/kg BW,

BID, digoxin tablets @ the dose rate of 0.05 mg/kg BW and L-carnitine

(carnisure®

) in addition to the diuretics depending on the situation.

52

xv) Cathartics.

Lactulose syrup was given to constipated patients @ the dose rate of 1-2 ml/kg

BW, BID.

xvi) Anticholestatics

Ursodeoxycholic acid (Udiliv®) tablets were given @ the dose rate of 15

mg/Kg, SID for 15 days in jaundice cases and dogs with chronic hepatopathy.

xvii) N-acetylcysteine tablets (Mucinac®) @ the rate dose of 10 to 15 mg, PO, SID

for minimum of 15 days was added to the treatment regimens in one group of

dogs.

3.7 FOLLOW-UP

Periodic blood and serum evaluation, together with ongoing monitoring of the

dog‟s appetite, body weight, behaviour, attitude, stability during the session,

complications and side effects (if any) and overall condition, were all part of the

ongoing “treatment” of chronic canine hepatopathies. However, in some cases in

which the owners were living in remote areas, a telephone call follow-up at weekly

interval was regularly done and clinical improvement was considered based on the

owner‟s witness and local veterinarian report. An attempt was also made to identify

potential prognostic indicators. Outcome after treatment (complete remission or

recurrence of clinical signs, or residual disease), survival time after diagnosis, date of

death, and presumable cause of death (clinical signs before death related to hepatitis

or not) were all recorded in hepatic insufficiency case record.

3.8 POST MORTEM EXAMINATION

With the consent of the owner, post mortem was performed in six dogs that

died during the course of treatment. Detailed post mortem gross lesions were

recorded and representative tissue samples were collected and placed in plastic

containers containing 10 per cent neutral buffered formalin solution. Impression

53

smears of the liver parenchyma (cut surface liver) were also taken, air dried and

stained later with Leishman‟s stain. Tissue samples were taken to the histopathology

lab for further processing and investigation with an expert pathologist for evaluating

tissue alteration. Fixed tissues from necropsy samples were trimmed, embedded in

paraffin wax blocks, sectioned at 5 μm, and stained with hematoxylin and eosin (H &

E) stain to characterize the inflammatory infiltrate and to evaluate the presence and

distribution of fibrosis, necrosis, bile duct hyperplasia, and pigment. Histologic

findings were described as morphologic diagnoses using light microscopy.

3.9 STATISTICAL ANALYSIS

The data was collected in a worksheet in MS Excel and further analyzed there

and in SPSS® Statistics 16 software package. The data obtained were expressed as

mean+SE. The significance of results was evaluated by applying Student‟s t-test and

/or ANOVA (Duncan‟s multiple range test; DMRT) to determine significant

difference among means (P 0.05) (Singh et al 1991).

CHAPTER IV

RESULTS AND DISCUSSION

Large numbers of dogs suffer from hepatic disorders due to varied reasons. On

an average more than 50 dogs are presented everyday at Small Animal Clinics,

Teaching Veterinary Hospital, Guru Angad Dev Veterinary and Animal Sciences

University, Ludhiana, Punjab, for the treatment of various ailments.

The present study entitled “Clinico-Pathological and Therapeutic Studies on

Hepatic Insufficiency in Dogs” was carried out on 140 dogs who fulfilled the selection

criteria for canine hepatic dysfunction. The dogs exhibiting systemic signs of illness

specially jaundice and/ or ascites and other clinical manifestations of pale mucous

membranes, fever, anorexia, lethargy, and weight loss, PU and PD, vomiting, melena,

coagulopathies or other manifestation suggestive of hepatic disease were examined

clinically and subjected to a complete laboratory analysis.

4.1 SIGNALMENT

4.1.1 Age

Hepatic disorders can occur at any age. In the present study, the age of dogs

suffering from liver diseases varied from 6 months (0.5 year) to 14 years. Kearns (2009)

reviewed that ICH mostly affects dogs aged less than one year of age and unvaccinated

leading to severe hepatic necrosis and can also cause ocular and renal changes.

Poldervaarrt et al (2009) reported in a retrospective study that mean age of onset of

chronic hepatitis in dogs was 7.7 years (range, 0.4-14.2) and 2.3 years (range, 0.5-7.2)

for lobular dissecting hepatitis (LDH). Chronic hepatitis occurs over a period of months

to years and is a leading cause of morbidity and mortality in dogs (Bexfield et al 2012).

The rational interpretation for increased incidence of liver diseases in older animals

could be attributed to the frequent exposure of liver to different and multiple insults,

55

both primary and reactive, over time. The age wise distribution of liver dysfunction

cases presented in this study is depicted in Figure 2. Most of the cases presented (62,

44.29%) fall in the young age group (0-4 years), followed by middle age (4-8 years)

group (49, 35%) and geriatric age (> 8 years) dogs (20.71%). The average age at which

cases were presented was 4.73 years. Tiwari (2002) reported mean age of dogs with

hepatic diseases as 51.9 months (4.3 years) which is close to the mean age reported in

this study. In another study, Strombeck and Gribble (1978) reported 5.3 years as a mean

age which is not significantly different from our study.

In a retrospective analysis on ascites due to presinusoidal portal hypertension in

dogs, James et al (2008) found that 70.5 % of dogs were 4 years of age or younger at

the time of presentation and idiopathic hepatic fibrosis of canine chronic hepatitis was

the underlying cause in the majority of cases. In the present study, the average age of

animals suffering from primary hepatopathies were calculated as 4.7 years with the

majority falling in the young age group. Nineteen out of 42 dogs with reactive

hepatopathies (i.e., 45.2%) were in the middle age group of 4-8 years with the mean

age of 6.49 years. Tiwari (2002) reported that the average age of dogs suffering from

hepatic diseases was more (5 years) than those dogs with extrahepatic disease i.e.,

reactive hepatopathies (2 years). In our study, the mean age of dogs with chronic liver

diseases irrespective of the cause was 4.39 years (i.e., middle age group). Anderson and

Sevelius (1991) reported that majority of cases suffering from chronic hepatitis were

presented between 5-7 years (middle age group) which is in agreement with the results

reported in this study.

However, evidence from humans and rodents has indicated that aging leads to a

marked change in the liver structure and function. In general, aged liver is

characterized by a decline in weight, blood flow, regeneration rate, and detoxification,

which have been related to an increased risk of liver abnormalities in the elderly

56

(Schmucker 2005). Shih et al (2007) reported that the median age of Labrador retriever

dogs suffering from hepatitis as 9.3 years (range, 3.9-14.0 years). In the present study,

the mean age of dogs diagnosed with hepatic neoplasia (primary/secondary) was 6.6

years (range, 3.5-9 years) with 7 dogs (46.7%) falling in geriatric age (>8 years),

followed by middle age 5 (33.33%) and young age 3 (20%). Patnaik et al (1980)

reported that average age of majority of dogs with hepatic neoplasia was more 10 years

or older.

Fig. 2 Age wise distribution of cases with hepatic insufficiency

4.1.2 Breed susceptibility

The present study population comprised eleven pure breeds and mixed breed

dogs (Table 2, Figure 3). Among the various breeds affected, Labrador retriever

constituted the maximum with 71 (50.71%) cases, followed by German shepherd 22

(15.71%), Mongrel 12 (8.57%) and Pug 10 (7.14%). Other breeds observed with

hepatic insufficiency in the present study were Saint Bernard 5 (3.57%), Spitz 4 (2.86),

Pomeranian, Dachshund and Rottweiler each contributing 3 (2.14%), Cocker spaniel 2

(1.43%). Dalmatian, French mastiff, Apsorussian, Pit bull terrier and Lhasa apso

contributed one (0.71%) case each. This finding is also in accordance with studies

57

conducted by different workers (Andersson and Sevelius 1991; Tiwari 2002; Hoffman

et al 2006 and Bexfield et al 2012) who showed that Labrador retriever was at an

increased risk for developing chronic hepatitis along with other breeds like Doberman

Pinscher, Bedlington Terrier, Dalmatian, Cocker spaniel, Sky Terrier, Standard Poodle,

German Shepherd dogs, English Springer Spaniel and Beagles. However, higher ratio

of Labrador dogs in the present study is attributed to the higher population of Labrador

breed in and around Punjab. The reason for chronic hepatitis to be widely found in

these breeds can be familial tendencies (Andersson and Sevelius 1991). To our

knowledge some breeds reported in this study like French mastiff and Asporussian

were not susceptible for developing chronic hepatitis; however, dog breeds at risk for

developing chronic hepatitis may change with time and geographic location due to

genetic and environmental factors.

Table 2: Breed wise distribution of cases with hepatic insufficiency

Breed Total (n=140)

Labrador retriever 71 (50.71%)

German Shepherd dogs 22 (15.71%)

Mongrel 12 (8.57%)

Pug 10 (7.14%)

Saint Bernard 5 (3.57%)

Spitz 4 (2.86%)

Pomeranian 3 (2.14%)

Dachshund 3 (2.14%)

Rottweiler 3 (2.14%)

Cocker spaniel 2 (1.43)

Dalmatian 1 (0.71%)

French mastiff 1 (0.71%)

Asporussian 1 (0.71%)

Pit bull terrier 1 (0.71%)

Lhasa Apso 1 (0.71%)

Total number of patient 140

Figures in parenthesis indicate percentage. (n) refers to number of dogs.

58

Fig. 3: Breed wise distribution of cases with hepatic insufficiency

4.1.3 Sex predisposition

Amongst the total of 140 patients included in the present study, males

constituted 87 (62%), whereas females were 53 (38%) (Figure 4). Sex wise distribution

of various hepatic diseases is presented in (Table 3, Figure 5). Vaden et al (1997) in a

study on renal failure in dogs revealed that higher incidence of males suffering from

renal failure could be because of the reason that the intact males are more likely to

wander, a behaviour that offers greater exposure to infections or toxic environmental

hazards and puts them at a greater risk. Similarly, higher incidence of hepatic

dysfunction in male dogs could be attributed to same reason; particularly the liver can

be a victim of many primary as well as secondary (reactive) causes. Higher population

of male dogs in and around Punjab appeared to be the reason for higher prevalence in

male dogs.

59

Table 3: Sex wise distribution of various hepatic diseases (n=140)

Category of the disease Total Male Female

Primary hepatopathies 98 (70%) 61 (62.24%) 37 (37.76%)

Reactive hepatopathies 42 (30%) 26 (61.90%) 16 (38.10%)

Chronic hepatitis/

hepatosis 42 (30%) 23 (54.76%) 19 (45.24%)

Acute hepatitis/hepatosis 37 (26.43%) 24 (64.86%) 13 (35.14%)

Cholecystitis 16 (11.43%) 11 (68.75%) 5 (31.25%)

Neoplasia 15 (10.71%) 9 (60%) 6 (40.00%)

Cholangiohepatitis 9 (6.43%) 7 (77.78%) 2 (22.22%)

Liver abscess 9 (6.43%) 6 (66.67%) 3 (33.33%)

Liver cirrhosis 6 (4.29%) 4 (66.67%) 2 (33.33%)

Obscured hepatopathy 4 (2.86%) 2 (50%) 2 (50%)

Cholelithiasis 2 (1.43%) 1 (50%) 1 (50%)


Fig. 4: Sex wise distribution of cases included in the study

4.2 EPIDEMIOLOGY OF HEPATIC INSUFFICIENCY IN DOGS

In the present study, out of 140 dogs, 98 (70%) constituted the primary liver

disorders out of which 61 (62.24%) were males and 37 (37.76%) were females,

60

whereas 42 (30%) represented the reactive hepatopathies out of which 26 (61.90%)

were males and 16 (38.10%) females (Table 3, Fig. 5). Similar observation was

reported by Chohan and Bansal (2005) who conducted an epidemiological study on 35

cases of dogs suffering from liver dysfunction and found that the majority of dogs

(74%) had primary liver disorders and the rest (26%) represented the reactive

hepatopathies. According to Hess and Bunch (2000), many diseases compromising the

liver dysfunction originate outside the hepatobiliary system and require further

investigations to characterize them as primary or reactive ones. Mayer and Twedt

(2000) conducted a study on liver biopsy in 150 dogs and observed that 25% of

presented cases were suffering from reactive hepatopathies. These observations in

general are not different from the observations reported in our study.

In the present study, among the hepatobiliary diseases in dogs chronic

hepatitis/hepatosis formed the largest group (42, 30%). Out of 42 cases with chronic

hepatitis/hepatosis, 23 (54.76%) were males and 19 (45.24%) were females (Table 3,

Fig. 5). Five cases were further diagnosed as chronic active hepatitis (based on

cytological/histological examination). In addition, 61.90% (26) of these cases

constituted primary hepatopathies and the remaining 16 (38.10%) formed reactive

hepatopathies (Figure 6). Poldervaart et al (2009) conducted a retrospective study

(2002-2006) on primary hepatitis in dogs and reported that chronic hepatitis was over

presented compared to acute hepatitis in referred population. However, the proportion

and distribution of these components vary widely and is necessary to include in the

diagnosis the activity and stage of the disease as well as the possible aetiology.

Out of 37 dogs diagnosed with acute hepatitis/hepatosis, 24 (64.86%) were

males and 13 (35.14%) were females (Table 3, Fig. 5). Primary hepatopathies

61

constituted 67.57% (25/37) of these cases followed by reactive hepatopathies which

accounted for 32.43% (12) (Figure 6).

Cholecystitis formed 16 cases, out of which 11 (68.75%) were males and 5

(31.25%) females (Table 3, Fig. 5).

Hepatic neoplasia was observed in 15 (10.71%) cases out of which 9 (60%)

were males and 6 (40%) females (Table 3, Figure 7). Metastatic neoplasias were

observed in 9 (60%) cases whereas primary hepatic neoplasia in 6 (40%) cases (Fig. 7).

O'Brien and Matheson (2004) also reported that metastatic neoplasias comprise the

most common category of liver malignancy in the dog. The determination of liver as

primary site was based on the clinical signs and biochemical findings relating to the

liver, ultrasonographic and histologic findings, and elimination of the possibility of

other sites as primary. Spleen was the main organ of metastasis. However, in cases of

hemangiosarcoma and adenocarcinoma, in which both the spleen and liver usually were

involved, if the liver had the largest single lesion, it was considered the primary site;

smaller, multiple well-defined nodules in the liver were considered metastases. In

addition to these factors establishing the liver as the primary site, one or more of the

following features were taken into consideration in making a diagnosis of

hepatocellular carcinoma: the histologic patterns of the neoplasm; intrahepatic and

extrahepatic metastases; cellular pleomorphism and anaplasia; mitotic activity; absence

of histologic boundaries; and absence of usual hepatic lobular pattern. Out of 15 dogs

suffering from heptic neoplasia, 6 dogs were diagnosed with hepatocellular carcinoma

followed by hemangiosarcoma (5) and adenocarcinoma (4). Patnaik et al (1980) also

reported that hepatocellular carcinoma constituted the highest percentage compared to

the others investigated. Hepatic neoplasias were confirmed by USG guided FNAB of

the liver. In the present study, the incidence of hepatic neoplasia was quite high as

62

compared to other studies. Patnaik et al (1980) reported and reviewed that the

neoplasms of the liver and biliary tract are uncommon in domestic animals and in dogs

it constituted 0.6% to 1.3% of all neoplasms. However, the higher incidence reported in

the present study could be due to the mere coincidence or it might possibly be

attributed to the increased environmental pollution by herbicides and other hazards.

Dogs with cholangiohepatitis contributed 9 (6.43%) cases out of which 7

(77.78%) were males and 2 (22.22%) females (Table 3, Fig. 5).

Liver abscess/suppurative hepatitis contributed 9 (6.43%) cases out of which 6

(66.67%) were males and 3 (33.33%) were females (Table 3, Fig. 5). Farrar et al (1996)

also reported that hepatic abscesses are rare in dogs. In pups the most common cause is

thought to be extension of bacterial infection from omphalophlebitis. In adults,

suggested routes of infection include: haematogenous spread from other sites of

infection, ascending infection from the biliary tract and infection secondary to necrosis

from trauma, torsion or neoplasia affecting the liver. Immunosuppression due to

diabetes mellitus was thought to be an underlying factor contributing to spread of

infection (Grooters et al 1994).

Liver cirrhosis constituted 6 (4.29%) cases out of which 4 (66.67%) were males

and 2 (33.33%) were females (Table 3, Fig. 5). Liver cirrhosis is the late stage of

chronic hepatitis and it‟s a long process with low incidence.

Obscured hepatopathy and cholelithiasis contributed by 4 (2.86%) and 2

(1.42%) cases respectively with equal sex contribution (Table 3, Fig. 5). Obscured

hepatopathies are rare as most of hepatic diseases are diagnosed. Other workers

(Strombeck and Guilford 1990; Neer 1992; Kirpensteijn et al 1993; Voros et al 2001)

also reported low frequency of cholelithiasis in dogs.

64

4.3 HISTORY AND CLINICAL PRESENTATION

The history plays a pivotal role in diagnosing hepatic insufficiency in dogs.

Most clinical signs observed in dogs with liver failure are nonspecific but can include

decreased appetite, vomiting, diarrhoea, weight loss, dehydration, and yellow

discoloration of the eyes, skin and gums.

4.3.1 Past history

Previous history of dogs with hepatic insufficiency presented in the present

study revealed that 43 (30.7%) dogs had never been ill, 22 (15.7%) were presented

with unknown history, 15 (10.7%) came with a history of progressive abdominal

distention and 9 (6.4%) had history of fever (Table 4, Fig. 8).

Inappetence (partial anorexia) was seen in 8 (5.7%) dogs whereas history of

vomiting as well as fits were reported in 6 (4.29%) dogs each. History of weakness

and enteritis were a complaint in 4 (2.67%) cases each. Complete loss of appetite,

gastroenteritis, canine parvovirosis, haematochezia, ivermectin overdosage, exercise

intolerence, tick infestation, whelping followed by endometritis/pyometra, ataxia,

maggot wounds was reported in 3 (2.14%) cases each. Subcutaneous abscess,

hematemesis and respiratory distress each were reported in two dogs 2 (1.43%).

Other historical findings like corneal opacity, obesity, babesiosis, epistaxis,

gastrointestinal parasitism, erythema, alopecia, glucocorticoid (prednisolone)

overdosage, blood transfusion, marked weight loss, pica and abdominal pain each was

reported in a single case (0.71%). Concerning history of vaccination and deworming

status, out of 140 dogs; 102 (72.9%) had regular and proper vaccination, 20 (14.3%)

had irregular vaccination, 15 (10.7%) were not vaccinated and 3 (2.1%) were

presented with unknown history of vaccination (Figure 9). Similarly, 101 (72.1%)

dogs had regular and proper deworming, 23 (16.4%) were irregularly dewormed, 14

(10%) were not dewormed and 2 (1.4%) with unknown history of deworming (Figure

10).

65

The history of feeding regimen revealed that out of 140 dogs, 74 (52.86%)

dogs were fed vegetarian diets and the remaining 66 (47.14%) were fed on mixed

food diets. Detailed feed history was investigated for 103 dogs and the policy of

feeding regimen is depicted in Figures 11 and 12.

Table 4: Previous history of dogs with hepatic insufficiency (n=140)

Previous history Number

Never being ill 43 (30.71%)

Unknown 22 (15.71%)

Abdominal distension 15 (10.71%)

Fever 9 (6.43%)

Inappetence 8 (5.71%)

Vomiting 6 (4.29%)

Fits 6 (4.29%)

Weakness 4 (2.86%)

Enteritis 4 (2.86%)

Anorexia 3 (2.14%)

Gastroenteritis 3 (2.14%)

CPV 3 (2.14%)

Haematochezia 3 (2.14%)

Ivermectin overdosage 3 (2.14%)

Exercise intolerance 3 (2.14%)

Tick infestation 3 (2.14%)

Whelping followed by endometritis 3 (2.14%)

Ataxia 3 (2.14%)

Maggot wounds 3 (2.14%)

Subcutaneous abscess 2 (1.43%)

Hematemesis 2 (1.43%)

Respiratory distress 2 (1.43%)

Corneal opacity 1 (0.71%)

Obesity 1 (0.71%)

Babesiosis 1 (0.71%)

Epistaxis 1 (0.71%)

Gastrointestinal parasitism 1 (0.71%)

Erythema and alopecia 1 (0.71%)

Prednisolone overdosage 1 (0.71%)

Blood transfusion 1 (0.71%)

Weight loss 1 (0.71%)

Pica 1 (0.71%)

Abdominal pain 1 (0.71%)


66

Fig. 8: Previous history of dogs with hepatic insufficiency (n=140)

68

4.3.2 CLINICAL SIGNS AND PHYSICAL EXAMINATION

The clinical signs of dogs with primary hepatic diseases were highly variable

and did not differ from that of reactive hepatopathies (Table 5 a,b,c, Figure 13). Holt

et al (1995) and Varshney and Hoque (2002) also observed that clinical signs

associated with liver dysfunctions were very variable, non-specific and vague related

to neurological and gastrointestinal signs.

Out of 140 dogs with hepatic insufficiency in the present study, complete

anorexia was reported in 64 (45.7%) dogs, whereas 61 dogs (43.6%) showed varying

degree of inappetence and the rest 15 dogs (10.7%) had normal appetite status.

Vomiting was observed in 64 (45.7%) dogs and out of these, 7 dogs (5 %) showed

hematemesis. According to Batt and Twedt (1994), the vomiting associated with liver

dysfunction could be attributed to the direct stimulation of the vomiting center by

chemoreceptor trigger zone (CRTZ) in the fourth ventricle of the brain by endotoxins

that were not cleared by the diseased liver. The present study showed that dogs

suffering from hepatitis and other hepatic disorders were having more consistency in

signs of inappetence/anorexia, vomiting, icterus, depression, abdominal distension,

pyrexia, hepatomegaly and hepatodynia. Similar observations were also reported by

other workers (Crawford et al 1985; Forrester et al 1992; Clarenburg 1992;

Rothuiezen and Meyer 2000, Kumar and Varshney 2006).

In the present study, slightly icteric mucus membranes were evident at a

minimum plasma bilirubin concentration of 1.9 mg/dL. However, Hardy (1983)

reported that icterus occurred when bilirubin accumulates in plasma or tissues, but

was rarely clinically perceptive until serum concentration was 3 mg/dL or more.

Hepatodynia (pain in the liver) is due to stretching of hepatic capsule and is the most

striking presenting complaint of patients with hepatic congestion (Dunn et al 1973).

69

Regarding the faecal consistency, normal soft faeces was observed in 81 dogs

(57.9%) and firm faeces in 38 (27.1) dogs. Diarrhoea was observed in 16 (11.4%) and

constipation in 3 (2.1%). Absence of defecation due to prolonged anorexia and normal

defecation with alternate diarrhoea was observed in one case (0.7%) each. This

indicated that vomiting occured more frequently as compared to diarrhoea in liver

compromised dogs. Sixty-one dogs (43.57%) had black tarry stools (melena) and 44

(31.4%) showed normal brown stools. Yellow faeces was observed in 14 (10%) dogs,

haematochezia in 7 (5.0%), acholic faeces (Fig. 14) and green faeces in 4 (2.9%),

brown yellowish stool in 4 (2.8%) and combination of melena and haematochezia in 2

(1.4%) cases. Vomiting and black tarry stools were among the predominant signs

observed in the dogs with azotaemia. Melena and haematochezia observed in this

study could be ascribed to the gastrointestinal ulceration or coagulopathies (Selvelius

1995).

In the present study, 12 dogs (8.5%) had mild renal impairment, 10 (7.1%) had

chronic renal failure (CRF), 8 (5.7%) showed acute renal failure (ARF) and 7 (5%)

revealed severe renal impairment secondary to liver disease. In these cases, liver was

primarily or reactively insulted. Common gastrointestinal complications of liver

and/or kidney disease in dogs and cats include reduced appetite with reduced food

intake, nausea, vomiting, uremic stomatitis and halitosis, gastrointestinal

haemorrhage, diarrhoea, and haemorrhagic colitis (Polzin and Osborne 1995; Kumar

and Varshney 2006; Dereszynski et al 2008; Plozin 2011).

In the present study, acholic faeces were observed in 4 cases, one case with

hepatic adenocarcinoma and another case with hepatic lipidosis secondary to diabetic

ketoacidosis. The remaining two cases had cholestasis but the exact cause was

unknown. According to Van Den Ingh et al (1986) the condition of acholic faeces was

70

most often caused by partial or complete occlusion of the choledochal duct due to

neoplastic diseases, inflammatory processes, or eventration and incarceration of

the liver.

Regarding frequency of urination, normal urination was seen in 51 dogs

(36.4%), followed by oliguria 47 (33.1%) and polyuria 33 (23.6). Pollakiuria was

reported in 7 (5%) dogs, followed by urinary incontinence and urine retention with

one (0.7%) case each. Polyuria and polydipsia both were also a part of the past history

in many cases particularly those with chronic liver disease. The causes of pollakiuria

are numerous depending on the sex and age and occasionally seen as a consequence

of PSS. Polyuria/polydipsia could be ascribed to impaired adrenal steroid metabolism,

altered portal vein osmoreceptor, loss of renal medullary concentration gradient,

encephalopathy (Hess and Bunch 2000) and potassium depletion (Bunch 2003).

Colour of urine varied among the cases depending on the stage. Normal

coloured urine was observed in 72 (51.4%) dogs whereas dark yellowish urine was

seen in 54 (38.8) dogs. Light yellowish urine was observed in 5 (3.6%) dogs.

Transparent urine and haematuria was detected in 3 (2.1%) dogs each. Brownish

coloured urine as well as greenish coloured urine was seen in 1 (0.7%) dog each.

Colour of urine can be affected with many factors (including dehydration, jaundice

and coagulopathies). Thirty seven dogs were presented with normal water intake

frequency. Frequency of water intake was reduced in 52 (37.1%) dogs and polydipsia

was reported in 51 (36.4) dogs. Severe abdominal pain (hepatodynia) was observed

only in 5 (3.6%) cases out of which one dog expressed position of relief (Fig. 15).

Skin bruises were observed only in 9 (6.4%) cases (Fig. 16 A & B) and were

ascribed to the rupturing of underlying blood vessels, decreased synthesis of blood

coagulation proteins and blood coagulation inhibitors (Feldman 1980).

71

Petechiation and ecchymoses were seen in 13 (9.28%) dogs (Fig. 18 A & B)

and epistaxis in a single (0.7%) dog. These findings are in line with Kavanagh et al

(2011) who reviewed that hepatobiliary diseases can lead to hypocoagulable states.

Unkempt hair coat was seen in 54 (38.6%) which could be due to prolonged

inappetence/anorexia, poor body condition and gastrointestinal disorders.

Palpation of superficial lymph nodes revealed generalized lymphadenopathy

in 1 (0.7%) case which, on FNAB, was found to be canine lymphosarcoma. Five

(3.57%) dogs showed enlargement of popliteal lymph nodes and in one case both

prescapular and popliteal lymph nodes were enlarged. Lymph node enlargement can

also be seen with systemic infection.

Dyspnoea was observed in 10 (7.1%) dogs with severe abdominal distension

due to peritoneal effusion. Coughing and peripheral oedema was detected in 7 (5%)

dogs each.

In a retrospective study on 80 dogs with hepatic cirrhosis, ascites was found to

be the most common clinical finding, followed by icterus, anorexia, neurological

disturbances, dyspnoea and subcutaneous oedema (Silva et al 2007). Dyspnoea was

attributed to the overpressure of ascitic fluids on the diaphragm and respiratory

muscles and in some cases to respiratory tract infection, whereas subcutaneous

oedema was a consequence of hypoproteinemia associated with liver disease (Hall

1985).

Abdominal distension was observed in 50 (35.8%) cases of ascites and 7 (5%)

cases of haemoperitoneum (Fig. 19 A & B). Witte et al (1971) reported that in most

cases of cirrhosis, both hepatic (high protein) and mesenteric lymph (low protein)

were produced at an increased rate and that ascites developed when the return of

lymph to systemic venous circulation failed to keep pace. Sevelius (1995) had also

72

observed that ascites was one of the predominant clinical findings in chronic hepatitis

dogs.

Corneal opacity (blue eye syndrome, Fig. 17 A) was observed in 2 (1.4%)

young unvaccinated dogs suspected for acute infectious canine hepatitis (ICH) and

was completely cured after 4 months of treatment (Fig. 17 B) with systemic

antibiotics and 5% normal saline eye drops. Infectious canine hepatitis is the only

virus with primary tropism for the liver and can cause ocular changes (Kearns 2009).

Out of the seven cases with haemoperitoneum, hemangiosarcoma and

adenocarcinoma was diagnosed in 2 (28.57%) cases each, hepatocellular carcinoma,

suppurative peritonitis and Ehrlichia canis infection was diagnosed in a single (0.7%)

case each. Aronsohn et al (2009) in a retrospective analysis observed that

hemangiosarcoma, hepatocellular carcinoma, splenic haematoma, carcinomatosis and

splenic torsion were among the most common causes of haemoabdomen which are in

accordance with the observations reported in the present study. Rupture of the hepatic

capsule and consequent haemorrhage can produce haemoperitoneum. Mylonakis et al

(2007) in a retrospective study of 61 cases observed that canine monocytic

ehrlichiosis causes thrombocytopenia which can cause generalized bleeding.

Exercise intolerance was observed in 5 (3.57%) dogs as a result of anaemia,

heart disease (ascites/pleural effusion) and/or other organ systems complications.

Anaemic animals have decreased ability of blood to supply tissues with adequate

oxygen for proper metabolic functions (Hoffbrand and Pettit 1993). As a consequence

there will be lethargy, weakness, exercise intolerance, anorexia, heart murmur,

dyspnoea and pale mucous membranes (Keskar et al 1985, Raskin 1994). Prolonged

capillary refill time is attributed to the dehydration and haemoconcentration.

73

Table 5: Clinical manifestations of dogs with hepatic insufficiency (n=140)

Parameter Clinical findings Total

Appetite status Normal appetite 15 (10.7%)

Anorexia 64 (45.7%)

Inappetence 61 (43.6%)

Stools

Colour &

Consistency

Black tarry stools 61 (43.57%)

Brown stools 44 (31.4%)

Yellowish 14 (10%)

Haematochezia 7 (5%)

Acholic faeces 4 (2.9%)

Green faeces 4 (2.9%)

Brown yellowish 4 (2.8%)

Both melena and haematochezia 2 (1.4%)

Vomiting No vomiting 76 (54.3%)

Yellow coloured 56 (40%)

Red coloured 7 (5%)

Frothy 1 (0.7%)

Frequency of urination

Normal urination 51 (36.4%)

Oliguria 47 (33.1%)

Polyuria 33 (23.6%)

Pollakiuria 7 (5%)

Incontinence 1 (07%)

Retention 1 (07%)

Urine colour Transparent 3 (2.1%)

Yellowish 72 (51.4%)

Dark yellowish 54 (38.6%)

Light yellowish 5 (3.6%)

Haematuria 3 (2.1%)

Brownish 2 (1.4%)

Greenish 1 (0.7%)

Water intake Normal 37 (26.4%)

Reduced 52 (37.1%)

Polydipsia 51 (36.4%)

Hepatodynia Severe abdominal pain (position of relief) 5 (3.6%)

Skin bruises Skin erosion and ulceration 9 (6.4%)

Hair coat Unkempt hair coat 54 (38.6%)

Lymph nodes enlargement Normal size 133 (95%)

Generalized lymphadenopathy 1 (0.7%)

Popliteal LNs enlargement 5 (3.57%)

Prescapular and popliteal LNs enlargement 1 (0.7%)

Dyspnoea Difficult breathing 10 (7.1%)

Coughing Coughing 7 (5%)

Peripheral oedema Swelling of hind limbs and subcutaneous tissues 7 (5%)

Corneal opacity Blue eye syndrome 2 (1.43%)

Diathesis Petechiation 6 (4.3%)

Petechiation and ecchymosis 7 (5%)

Epistaxis 1 (0.7%)

Abdominal distension Ascites (with or without organomegaly) 50 (35.8%)

Hemoperitonium (with or without organomegaly) 7 (5%)

Severe hepatomegaly 1 (0.7%)

Exercise intolerance Fatigue while exercising 5 (3.57%)

CRT <2 Sec 127 (90.7%)

2 Sec 8 (5.7%)

>3 Sec 5 (3.57%)


74

Fig. 13: Clinical manifestations of dogs with hepatic insufficiency

75

4.4 PHYSICAL EXAMINATION

4.4.1 General attitude, posture and body condition

In the present study when the demeanour of the dogs with hepatic

insufficiency was observed, it was found that 67 (47.86%) were alert and 73 (52.1%)

exhibited varying degree of depression, dullness and lethargy. Apathy was seen in 108

(77.1%) dogs, followed by restlessness in 53 (37.8) dogs. Tremors as well as

expression of agitated behaviour was observed in 8 (5.7%) dogs each. Aggression and

head pressing were reported in 4 (2.9%) dogs each. Other neurological signs seen

were ataxia observed in 38 (27.1%) and staggering in 21 (15%) dogs. Dementia

(change in mentation) constituted 4.4% (6 cases) and blindness was noted in 4 (2.9%)

cases. Stupor was detected in 19 (13.6%) dogs and collapse in 11 (7.9%), whereas

coma was observed in 9 (6.4) and seizures in 8 (5.8%) dogs. Circling and convulsions

were observed in 3 (2.1%) and 2 (1.4%) cases respectively. However, hepatic

encephalopathy was observed in 6 (4.3) dogs. Conn and Bircher (1988) stated that

hepatic encephalopathy is a complex of neurological signs, which could results from

reduction in functional mass of the liver. The signs suggestive of hepatic

encephalopathy like depression, motor disturbances, seizures, behavioural changes,

dementia, hypersalivation were not very consistent even in the same case i.e., wax and

wane. These observations are in agreement with other workers (Barrett et al 1976;

Taboada 1991; Rothuizen and Van Den Ingh 1998; Javier Lizardi-Cerveraet et al

2003) who reported similar findings.

On examination of the body condition of affected dogs, it was observed that

93 (66.4%) showed weight loss, 68 (48.6%) exhibited weak condition, 40 (28.6) were

very weak, 24 (17.1%) found to be active, 7 (5%) showed cachexia, 2 (1.4%) were in

lateral recumbency and 1 (0.7%) in sternal recumbancy. These observations could be

76

ascribed to the inadquate nutrient intake or assimilation and enhanced tissue

catabolism (Hess and Bunch 2000).

4.4.2 Hydration status

On examination of the hydration status by assessing the skin turgor of the dogs

suffering from hepatic dysfunction, 46 (32.9%) had less than five per cent

dehydration, 36 (25.7%) dogs were mildly dehydrated, 50 (35.7%) dogs were

moderately dehydrated, and 8 (5.7%) were severely dehydrated. According to Giebler

(1995) and Rothuizen and Meyer (2000), disruption of liver and kidney functions may

be manifested by signs of diarrhoea, polyuria, polydipsia and dehydration. This can be

attributed to poor skin coat in most of the dogs with liver dysfunction. Anorexia,

fever, vomiting, icterus, ascites and diarrhoea, which are frequent manifestations of

liver disease, can precipitate dehydration (Forrrester et al 1992). According to

Fleming et al (1989), on examination of a dog with renal failure; one may find

dehydration, hypothermia and mucosal injection. In the present study, many dogs

were found to suffer from primary or secondary renal impairment.

4.4.3 Examination of mucous membrane and oral cavity

In the present study, most (52, 37.1%) of the dogs had pale conjunctival and

oral mucus membranes. The mucus membranes of oral cavity, sclera and vagina were

icteric in 45 (32.2%) dogs (Fig. 20 a & b) and congested in 24 (17.1%) dogs, whereas

19 (13.6) dogs showed normal (pink) mucus membranes. The pale mucous membrane

is usually due to anaemia associated with chronic liver disease due to gastrointestinal

haemorrhage or excessive haemorrhage from neoplasm (Johnson 2000). Cotter (2000)

stated that anaemia of hepatic diseases occurs as a result of decrease in ATP leading

to shortened red cell life span. In addition, various reactive hepatopathies encountered

in this study, such as immune mediated haemolytic anaemia (IMHA), sepsis,

77

neoplasia, gastrointestinal parasitism, haemoprotozoa (Babesia and Ehrlichia), viral

infection, and pyometra etc, can all precipitate anaemia. Center (1994) mentioned that

many systemic infections, such as rickettsial diseases, viral diseases, pyometra, etc,

were accompanied with liver response eliciting an increase in liver enzyme profile.

Jaundice was the outcome of bilirubin accumulation in the blood and extravascular

space due to increased production, decreased clearance, or impaired conjugation by

the liver/or any problem in the bile flow (Rothuizen and Meyer 2000; Yadav et al

2011). In the present study, jaundice was observed in 26.19% of acute hepatic

dysfunction cases and in 20.59% of chronic cases. These observations concur with the

findings of Sevelius (1995) who reported that jaundice is less common in chronic

hepatitis cases. Congestion of mucus membranes usually accompanied with febrile

diseases.

On performing oral examination, halitosis was encountered in 46 (32.9%)

dogs, whereas 91 (65%) dogs had normal mouth odour. Three (2.1%) dogs revealed

sweet fruit odour as a consequence of diabetic ketoacidosis. Oral ulceration was

observed only in 5 (3.57%) dogs. Oral ulceration can be a complication of uraemia.

Uraemia was seen in many dogs specially in those with renal failure. These changes

were in line with the findings of Polzin (2011). Petechial haemorrhages and

ecchymosis of oral mucus membranes were also reported in one case 1 (0.7%) (Fig.

21 A & B). Because the liver is the source of most proteins taking part in the blood

coagulation, hepatic failure may lead to bleeding disorders (Feldman 1980).

4.4.4 Abdominal palpation and ballottement

Abdominal palpation and ballottement of the liver was unfruitful in 76

(54.28%) cases. Thirty four (24.28%) cases showed hepatomegaly (Fig. 22) which

was later confirmed with plain radiography and/or ultrasonography. Nineteen cases

78

(13.57%) revealed peritoneal effusion (ascites or haemoperitoneum) which was

confirmed by USG guided abdominocentesis. Hepatodynia was detected in 7 (5%)

dogs that did not reveal clinical signs of abdominal pain. Abdominal mass at the

region of liver was detected only in 3 (2.1%) cases. Severe abdominal effusion

frequently interferes with the palpation of liver and liver masses. These observations

are in agreement with Rothuizen and Mayer (2000) who noted that physical

examination was informative only in few dogs suffers from liver diseases. Apart from

liver changes and peritoneal effusions, splenomegaly was detected in two cases

(1.4%), bilateral renomegaly with calcification in one and intestinal intussusception in

1 (0.7%) dog each.

4.4.5 Vital body parameters

4.4.5.1 Rectal temperature

The mean ± SE values of rectal temperature (ºF) of dogs suffering from

hepatic dysfunction associated with acute cases were 102.66+0.15 ºF and in chronic

cases 102.42+0.13 ºF (Table 6, Fig. 23). However, 84 (60%) cases were within the

normal range, 40 (28.6%) dogs had pyrexia and the rest 16 (11.4%) had subnormal

body temperature. According to Twedt (1981), pyrexia could be a consequence of

hepatocellular damage, infection, sepsis or absorption of intestinal bacterial toxins.

Subnormal body temperature was often observed at last stages of diseases.

4.4.5.2 Heart rate

The mean ± SE values of heart rate (per minute) of dogs suffering from

hepatic dysfunction associated with acute cases were 125.49+3.09 per minute and in

chronic cases 140.45+2.28 per minute (Table 6, Fig. 23). However, mean values of

heart rate were within the normal limits and these also varied with size, breed and

general health condition of animal. In the present study, 75 (53%) dogs had

79

tachycardia. Tachycardia could be ascribed to the anaemia which requires pumping of

more blood by the heart and also to the diseased heart itself in those cases with heart

involvement. Three (2.14%) dogs had bradycardia and the rest 62 (44.29%) revealed

normal heart rate.

4.4.5.3 Pulse rate

The mean ± SE values of pulse rate (per minute) of dogs suffering from

hepatic dysfunction associated with acute cases were 124.32+1.63 per minute and

with chronic cases 139.49+62 per minute (Table 6). Since pulse rate is a consequence

of the heart rate, an increase in heart rate will cause an increase in pulse rate.

4.4.5.4 Respiration rate

The mean ± SE values of respiration rate (per minute) in of dogs suffering

from hepatic dysfunction associated with acute cases were 52.95+1.40 per minute and

in chronic cases 50.33+1.54 per minute (Table 6, Fig. 23). However, 100 (71.4%)

dogs had tachypnoea and 2 (1.4%) showed hypopnea. This increase in respiration rate

is a compensatory process due to the fact that majority of the dogs selected for the

present study were anaemic and/or icteric and to compensate for the oxygen demand

the respiration rate might had been elevated.

Table 6: Vital body parameters of dogs in various hepatic disease (n=140)

Stage Rectal

temperature (ºF)

Heart rate

(per minute)

Respiration

rate

(per minute)

Pulse rate

(per minute)

Normal

range

101-103 70-120 18-34 70-120

Acute

stage

102.66+0.15 125.49+3.09 52.95+1.40 124.32+1.63

Chronic

stage

102.42+0.13 140.45+2.28 50.33+1.54 139.49+62

80

Fig. 23: Vital body parameters of dogs in various hepatic diseases (n=140)

81

4.4.6 Thoracic auscultation

Cardiac auscultation on dogs suffering from primary liver disorders revealed

no abnormalities in majority of cases. However, cardiac arrhythmia was detected in 5

(7%) cases and muffled heart sound in 2 (1.4%) cases. The cause of arrhythmia and

muffling of heart sound in these cases was due to CHF. Physiological sinus

arrhythmia was reported in a single case. Auscultation of chest revealed normal lung

sound in 126 (90%) dogs, harsh lung sound in 9 (6.4%), lung crackles in 4 (2.8%) and

muffled lung sound in 1 (0.7%) case. These abnormal lung sounds were ascribed to

respiratory tract diseases which coexisted as other complications. Chest x-ray of these

cases revealed interstitial and sometimes bronchial lung patterns in many cases and

pleural effusions in 2 (1.4%) cases.

4.5 LABORATORY EXAMINATION

4.5.1 Examination of haemoprotozoa

Thin blood smear was prepared from all pyretic dogs with or without tick

infestation and was subjected to microscopic examination to rule out haemoprotozoan

parasites infection. Ten (7.14%) dogs were diagnosed with B. gibsoni infection and 5

(3.75%) revealed E. canis infection. However, negative result did not confirm the

negative status of the animals, since 28 (41.17%) dogs were found to be infested with

Rhipicephalus ticks and many other dogs had past history of ticks‟ infestation with or

without treatment exposure.

4.5.2 Haemato-biochemichal changes in dogs with hepatic insufficiency

4.5.2.1 Haematological changes in dogs with hepatic insufficiency

Morphologic abnormalities of WBCs and RBCs detected by evaluation of the

blood film can be helpful in determining the cause of anaemia and indicate specific

disease processes in some cases. Haematological changes of dogs with hepatic

82

insufficiency, irrespective of the cause and category, revealed anaemia and

neutrophilic leukocytosis with mild or moderate to severe left shift and varied degree

of toxic changes in neutrophils.

Anaemia is one of the most common clinical signs encountered in many

infectious and non-infectious diseases in dogs characterized by pallor mucous

membrane, weakness, lethargy, tachycardia and tachypnoea. Out of 140 dogs with

hepatic insufficiency, 12 (8.57%) dogs with acute hepatitis/hepatosis did not show

anaemia. Rest of dogs (128, 91.43%) with acute and chronic hepatopathies showed

anaemia as there was pale mucus membranes and significant (p<0.05 or p<0.01)

decrease in Hb values (Table 9, 10, 11). The morphological classifications of anaemia

for 128 dogs with hepatic insufficiency based on morphology of RBCs and

regeneration of bone marrow are depicted in “Table 7 and Figure 24”. Out of these

128 anaemic dogs, 71 (55.47%) had nonregenerative anaemia and remaining 57

(44.53%) showed anaemia with regeneration.

Anaemic animals were grouped into different groups based on morphology of

RBCs and regeneration of bone marrow (Table. 7, Figure 24). Out of the 128 dogs,

the most common type of regenerative anaemia was normocytic normochromic in 34

(17.86%), followed by macrocytic hypochromic in 18 (14.06%), normocytic

hypochromic in 4 (3.13%) and microcytic normochromic in 1 (0.78%). The common

type of nonregenerative anaemia was normocytic normochromic and accounted for 41

(32.03%), followed by macrocytic hypochromic in 15 (11.72%), normocytic

hypochromic in 9 (7.03%) and microcytic normochromic in 6 (4.69%). In the present

study, the most common type of anaemia associated with acute hepatitis was

normocytic normochromic regenerative anaemia, followed by macrocytic

hypochromic regenerative anaemia, whereas most of chronic cases (chronic hepatitis,

liver abscess, liver cirrhosis and hepatic neoplasia) revealed normocytic

83

normochromic nonregenerative anaemia. These observations are in line with Ettinger

and Feldmann (2005) who stated that haematological changes of dogs with hepatic

insufficiency mostly include mild regenerative anaemia (as a consequence of

gastrointestinal bleeding or rarely spontaneous bleeding due to coagulopathy) or

more frequently normocytic normochromic nonregenerative anaemia suggestive

of chronic disease. They also stated that non regenerative microcytic

hypochromic anaemia suggests chronic gastrointestinal blood loss. Dill-Macky

(1995) reported nonspecific haematological changes in chronic hepatitis.

Regeneration of bone marrow was determined by observation of nucleated RBCs,

reticulocytosis and high MCHC values. However, anaemia present in acute hepatic

diseases was mostly associated with excessive haemorrhage (Johnson 2000).

Table 7: Morphological classification of anaemia in dogs with hepatic insufficiency

(n=128)

Sl. No Type of

anaemia RBC morphology Total

1. Regenerative

anaemia

Normocytic, normochromic 34 (26.56%)

Macrocytic, hypochromic 18 (14.06%)

Normocytic, hypochromic 4 (3.13%)

Microcytic, normochromic 1 (0.78%)

2.

Non

Regenerative

anaemia

Normocytic, normochromic 41 (32.03%)

Macrocytic, hypochromic 15 (11.72%)

Normocytic, hypochromic 9 (7.03%)

Microcytic, normochromic 6 (4.69%)

Total 128 (100%)

Figures in parenthesis indicate percentage. (n) refers to number of dogs

84

Fig. 24: Morphological classification of anaemia in dogs with hepatic insufficiency

85

The significant (p<0.05 or p<0.01) decline in the mean Hb values in all

categories of hepatic insufficiency on the day of presentation as compared to healthy

control group beside the changes in other haematological indices (i.e., Hb, PCV,

MCV, MCH and MCHC; Table 8, 9, 10) indicated that animals were usually

anaemic. Tiwari (2002) reported significant reduction in the mean haemoglobin levels

of different hepatic diseases as compared to healthy dogs. The low levels of

haemoglobin and hence development of anaemia in hepatobiliary diseases has been

attributed to increased degradation of red blood cells, the possible causes of which

may be the increased transit time of erythrocytes through the spleen due to reduced

portal blood flow and/or increased fragility of red blood cells due to high levels of

bile acids (Rothuizen and Mayer 2000).

In addition, decreased erythrocyte survival (Felsher et al 1968, Hume et al

1970), impaired bone marrow response (Kimber et al 1965), decreased nutrient

uptake due to inappetence, reduced availability of micronutrients from liver (Hess

and Bunsh 2000) or inadequate erythropoietin production due to decreased

production of α2 globulins (precursor of erythropoietin) has also been proposed as the

possible causes for anaemia associated with hepatobiliary diseases. Higher values of

MCHC could be attributed to the regeneration.

Immune mediated haemolytic anaemia (Fig. 25) which is a secondary cause of

hepatopathy was seen in 2 cases due to blood transfusion and heavy infestation with

Babesia gibsoni infection. Stained blood smear examination revealed clumping of

RBCs, spherocytosis, nucleated RBCs, reticulocytosis and leukomoid response with

degenerative left shift.

Poikilocytosis (acanthocytes, schistocytes, Heinz bodies) which is a common

sign of hepatic dysfunction was frequently observed during microscopic examination

of the blood films which is in agreement with Leveille-Webster (2000) who stated

86

that morphological changes of erythrocytes associated with liver diseases include

microcytosis, acanthocytosis, schistocytes and Heinz bodies.

Thrombocytopenia was observed in the majority of cases with hepatic

insufficiency, but among the various categories of hepatic disease, significant

(p<0.01) decrease in platelet counts was observed only in dogs with liver cirrhosis

(Table 9, 10, 11). However, thrombocytosis with activated platelets was also

encountered in some cases. These observations are in agreement with Kavanagh et al

(2011) who reviewed that hepatobiliary disease can have profound effects on

coagulation function leading to hypercoagulable or hypocoagulable states. He also

stated that overall coagulation status with hepatobiliary disease depends on both the

type and severity of disease and the presence of associated complications. Multiple

alterations in platelet number and function have been found in human patients with

liver disease (Lisman and Leebeek 2007; Witters et al 2008), although in a series of

22 cats with hepatic disease, only one cat had a low platelet number (Lisciandro et al

1998). Similarly, in 28 dogs with naturally occurring hepatic disease and two dogs

with congenital liver shunts, there were no abnormalities in platelet numbers reported

(Badylak et al 1983; Niles et al 2001; Kummeling et al 2006). Several mechanisms

have been suggested for thrombocytopenia in patients with liver disease, including

(1) increased platelet sequestration in the spleen as a result of congestive

splenomegaly; (2) reduced production of thrombopoietin by the liver; (3) increased

platelet breakdown due to auto-antibodies, and (4) increased consumption resulting

from low-grade DIC (Lisman and Leebeek, 2007; Witters et al 2008). In addition to

platelet count, modern hematology analyzers can provide information on activation

status of the platelets (Zelmanovic and Hetherington 1998; Moritz et al 2005; Yilmaz

et al 2008). Activated platelets are characterized by an increase in mean platelet

volume (MPV) and a decrease in mean platelet component (MPC). Changes in MPV

and low platelet concentration were consistent with an intravascular activation in

patients with moderate affected liver function (Jørgensen et al 1984).

87

In the present study, qualitative evaluation of thrombocytes for 47 (33.57%)

dogs with hepatic dysfunction revealed that 29 (61.70%) dogs had thrombocytopenia,

13 (27.66%) had adequate platelet counts and 5 (10.64%) dogs showed

thrombocytosis with activated thrombocytes. These observations are in agreement

with Willis et al (1989) who demonstrated the presence of qualitative defects in

platelet aggregation in dogs with hepatobiliary disorder. Thrombocytopenia and

anisocytosis were also seen in majority of cases with haemoprotozoa (Babesia

gipsoni and Ehrlichia canis) infections which is in agreement with Zygner et al

(2007) who reported similar findings.

Other coagulation profile parameters (i.e., PT, APTT and fibrinogen) were

checked in 49 dogs (28 male and 21 female). Results of coagulation analysis are

presented in Table 8. Twenty-nine (59.18%) dogs with hepatic dysfunction had at

least one abnormal coagulation parameter. Coagulopathy is indicated by decreased

fibrinogen and/or prolonged PT and APTT times. Out of the 49 dog subjected to

coagulation profile estimation, 17 (34.69%) had acute hepatitis/hepatosis, 17

(43.69%) chronic hepatitis/hepatosis, 7 (14.29%) cholangiohepatitis, 5 (10.20%) liver

cirrhosis and 3 (6.12%) non-specific reactive hepatopathies (B. gibsoni infection).

Hyperfibrinogenemia was observed only in 2/3 dogs among the 49 tested

dogs. These two dogs were positive for B. gibsoni infection. Mean fibrinogen value

(0.96±0.02 g/L) was significantly (p<0.05) lower in dogs with liver cirrhosis as

compared to healthy control dogs. The observations recorded in the present study are

in line with the observations reported by other workers (Prins et al 2010; Ruiz de

Gopegui et al 2007).

Mean values of both PT and APTT concentrations were above upper control

values in all categories of hepatic disease, only APTT was significantly (p<0.05)

prolonged in liver cirrhosis compared to healthy control dogs. Disseminated

intravascular coagulation (DIC) was suspected in dogs with low fibrinogen and low

88

platelet counts, combined with prolonged clotting time. The liver plays an important

role in maintaining haemostasis. Hepatocytes not only produce fibrinogen,

prothrombin and the factors V, VII, IX, X, XI and XIII, but are also responsible for

the activation of the vitamin K-dependent factors II, VII, IX and X and protein C

(Prater 2000). In liver disease, factor and inhibitor synthesis and clearance of

activated factors in both the coagulation factors and fibrinolytic system is impaired,

both quantitatively and qualitatively (Mammen 1994, Badylak et al 1983, Kemkes-

Matthes et al 1991, Lisciandro et al 1998, Niles et al 2001, Kummeling et al 2006).

The extent of coagulation abnormalities depends upon the degree of disturbed liver

function. Patients with hepatic failure may present with the entire spectrum of factor

deficiencies and may even develop DIC. In humans, the most severe abnormalities

are found in patients with liver cirrhosis (Kemkes-Matthes et al 1991, Mammen,

1994). In addition, long-standing biliary tract obstruction can decrease absorption of

fat-soluble vitamins including vitamin K, which is required for activation of certain

coagulation factors (Neer 1992, Ward 2006).

Differential leucocytic count revealed neutrophilic leucocytosis in almost all

the cases (Table 8, 9, 10). The neutrophilic leukocytosis which is characteristic of

acute inflammatory conditions (Center 1998; Johnson 2000; Bush 2002; Verma 2005

and Poldervaart et al 2009) was observed in 137 (97.85%) cases of dogs with hepatic

dysfunction which is in agreement with many workers (Al-sarramf et al 1974; Ihde et

al 1974; Okudak et al 1977; Patniak et al 1980; Kosovsky et al 1989; Farrar et al

1996; Ettinger and Fledmann 2005; Silva et al 2007; Shaker and Kkalifa 2012).

Neutrophilia, lymphopenia and esinopenia which were observed in almost all the

cases could be attributed to the stress response or concurrent viral infections.

Monocytosis which is usually observed in ehrlichiosis was rarely seen in the present

study. Eosinophilia was observed in a few cases with parasitic infestation and allergic

conditions. Neutropenia was observed in few cases with severe septicemic conditions.

89

Table 8: Fibrinogen and clotting times in dogs with different hepatic diseases (n=49)

Parameter

(Mean values)

Control values/

pooled plasma (n=6)

Acute hepatitis/

hepatosis (n=17)

Cholangiohepatitis

(n=7)

Chronic hepatitis/

hepatosis (n=17)

Cirrhosis

(n=5)

Non-specific reactive

hepatopathies (n=3)

PT (s) 6.9±1.3

6.8-9.7

7.1±3.3

6.8-10

7.4±2.3

6.9-10.2

9.7±2.9

7.1-11

10.9±1.1

9.2-13

8.1±3.2

6.7-28.9

APTT (s) 12.4±2.3

10.2-16.9

13.8±1.9

13.3-15.3

13.9±4.1

7.9-26.1

17.2±3.9

17.1-21.4

18.3±1.6*

17.4-21.6

13.2±3.2

7.8-26.3

FP (g/L) 2.1±2.3

1.2-2.9

2.2±1.1

1.6-3.2

2.6±3.4

1.6-2.2

2.6±1.4

1.0-3.4

0.96±0.02*

1.6-6.3

4.5±2.2 *

1.7-9.5

*Significant at 5% (P<0.05); ** Significant at 1% (P<0.01)

90

4.5.2.2 Biochemical changes in dogs with hepatic insufficiency

The biochemical profile (including liver enzymes, TB, TP and ALB, A/G

ratio, BUN, creatinine, GLU and cholesterol) of control healthy dogs and dogs

suffering from various liver dysfunctions for the various categories of liver disease on

the day of presentation is depicted in Tables 9, 10, 11 and 12. The importance of

enzymatic and biochemical analysis in classification of liver disease has been studied

extensively (Sherding 1985; Sevelius 1995; Kramer and Hoffman 1997; Varshney et

al 2001; Tiwari 2002).

Mean values of TB (mg/dL) were significantly (p<0.01) higher in dogs with

acute hepatitis/hepatosis (4.99±1.41 g/dL), chronic hepatitis/hepatosis (4.06±1.07),

cholecystitis (3.13±0.93), primary (3.77±0.61) and reactive hepatopathies (4.20±1.14)

as compared to control healthy dogs (0.37±0.09). The mean values of TB were also

significantly (p<0.05) higher in dogs with liver cirrhosis (1.90±0.87) and hepatic

neoplasia (1.99±0.57) as compared to control healthy dogs (0.37±0.09), but no

significant rise in the mean value of TB was seen in dogs with cholangiohepatitis

(4.61±1.89), liver cirrhosis (1.90±0.87) and obscured hepatopathies (1.77±1.28) as

compared to control healthy dogs (0.37±0.09).

Hyperbilirubinemia observed in cases of hepatitis could be attributed to

hepatocytes damage or biliary obstruction associated with hepatic inflammation (Bush

2002). The levels of rise differed significantly for different liver dysfunctions which

could be used to classify primary hepatopathies into various categories. Generally, it‟s

hard to differentiate liver disease on the bases of total bilirubin level (Center 1994);

however, some workers (Patniak et al 1980, Johnson et al 1982; Crawfod et al 1985;

Hardy 1983) have described considerably high levels of bilirubin in some chronic

diseases such as chronic active hepatitis, copper storage diseases and hepatic

neoplasms. However, studies have shown that total bilirubin was found to be normal

in three fourth of dogs with hepatic neoplasia (Whiteley et al 1989).

91

In the present study, mean values of liver enzymes (U/L) varied significantly

among the dogs with hepatic dysfunctions based on the severity and stage of liver

disease and the involvement of other organ systems.

Dogs suffering from acute hepatitis/hepatosis showed significant (p<0.01)

increase in the mean values of ALT (333.97±44.28 U/L), AST (266.80±52.45 U/L),

ALP (768.56±77.53 U/L) and GGT (93.64±29.51 U/L) as compared to control healthy

dogs (Table 9). Dogs with chronic hepatitis/hepatosis also revealed significant

increase (p<0.01) in the mean values of ALT (185.05±30.08 U/L), AST

(201.17±46.36 U/L), ALP (450.55±58.18 U/L) and GGT (26.27±5.63 U/L) as

compared to control healthy dogs (Table 9).

Cholangiohepatitis cases showed significant (p<0.05) increase in the mean

values of ALT (249.67±95.50 U/L), AST (145.88±33.90 U/L) and ALP

(546.33±181.42 U/L) as compared to healthy control group, but a non-significant rise

was observed in the mean value of GGT (177.25±94.11 U/L) as compared to healthy

control dogs (Table 9).

Dogs with liver cirrhosis revealed significant (p<0.05) increase only in the

mean value of AST (85.67±19.12 U/L), whereas the mean values of ALT, ALP and

GGT were non-significantly increased as compared to healthy control group (Table

9). Similar findings were reported by Twedt (1985). This unpronounced increase in

liver enzymes indicated the absence of significant on-going inflammation or

intrahepatic cholestasis or decreased viable parenchymal mass.

Dogs with liver abscess showed significant (p<0.05) increase in the mean

value of ALT (109.22±26.96 U/L) and significant (p<0.01) increase in AST

(109.62±22.27 U/L) and ALP (726.78±189.30 U/L) values as compared to control

healthy dogs. A non-significant increase was observed in the mean value of GGT

(100.00±63.80 U/L) as compared to control group (Table 10).

92

Table 9: The mean±SE values of hemato-biochemical parameters in healthy and hepatic insufficiency dogs on the day of presentation

Parameter Control

(n=6)

Acute Hepatitis

(n=37)

Chronic hepatitis

(n=42)

Cholangiohepatitis

(n=9)

Liver cirrhosis

(n=6)

Hb (g/dL) 12.22±0.29 10.02±0.76* 8.49±0.53** 8.11±1.15** 9.68±1.86*

TEC (106/µL) 5.59±0.10 4.92±0.37 4.18±0.26** 4.01±0.53 4.05±0.95

TLC (103/µL) 13.9±0.67 22.7±2.63** 29.7±4.90** 24.3±5.80 3.30±6.5*

N (%) 58.67±3.04 88.27±1.49** 89.79±1.32** 90.78±1.96** 89.67±2.75**

L (%) 38.67±2.23 11.14±1.42** 10.93±1.71** 16.00±8.65* 10.00±2.58**

M (%) 0.00±0.00 0.00±0.00 0.10±0.07 2.00±1.76 0.00±0.00

E (%) 2.67±0.84 0.59±0.31 0.05±0.05 0.44±0.44 0.67±0.42

PCV (%) 39.93±0.82 32.73±2.00** 25.06±1.28** 26.54±3.19** 28.54±4.87*

MCV (fL) 71.20±0.49 51.55±1.70** 53.82±1.59** 53.90±1.97** 49.15±2.89**

MCH (pg) 21.70±0.32 20.36±0.42 20.02±0.38** 17.69±1.78 19.32±0.08**

MCHC (g/dL) 30.47±0.24 38.64±0.89** 37.46±1.20** 36.37±1.48** 39.94±2.16**

Platelets (105/ µL) 2.94 + 0.39 2.12+327 1.71±0.26 1.32±0.15 1.94±0.27**

GLU (mg/dL) 100.50±3.12 128.08±23.83 131.97±18.04 116.00±20.96 83.67±5.18*

BUN (mg/dL) 13.67±0.96 60.46±11.99** 49.54±9.67** 32.33±6.93* 28.67±6.31

Creatinine (mg/dL) 0.92±0.04 2.15±0.46* 1.58±0.18** 1.70±0.39 1.60±0.16**

TP (g/dL) 6.37±0.29 5.64±0.22 4.96±1.06* 4.79±0.30** 4.78±0.37**

ALB (g/dL) 3.35±0.24 3.62±0.16 1.78±0.11** 1.93±0.20** 1.62±0.15**

Globulin (g/dL) 2.92±0.34 2.01±0.13 4.18±1.05 2.86±0.20 3.17±0.26

A/G ratio 1.26±0.21 0.88±0.07 0.56±0.03* 0.70±0.09 0.52±0.04*

TB (mg/dL) 0.37±0.09 4.99±1.41** 4.06±1.07** 4.61±1.89 1.90±0.87

ALT (U/L) 18.00±3.34 333.97±44.28** 185.05±30.08** 249.67±95.50* 108.00±72.63

AST (U/L) 36.67±3.91 266.80±52.45** 201.17±46.36** 145.88±33.90* 85.67±19.12*

ALP (U/L) 52.50±9.67 768.56±77.53** 450.55±58.18** 546.33±181.42* 376.50±185.64

GGT (U/L) 2.50±0.50 93.64±29.51** 26.27±5.63** 177.25±94.11 34.67±29.50

Cholesterol (mg/dL) 176.67±24.99 189.94±21.46 107.88±8.85* 144.00±18.05 124.83±28.53


93

Table 10: Hemato-biochemical parameters in healthy and hepatic insufficiency dogs on the day of presentation (Mean±SE)

Parameter

Control

(n=6)

Liver abscess

(n=9)

Cholecystitis

(n=16)

Neoplasia

(n=15)

Hb (g/dL) 12.22±0.29 8.94±1.18* 8.79±1.02** 9.92±1.11*

TEC (106/µL) 5.59±0.10 4.32±0.52 4.36±0.54 4.54±0.54

TLC (103/µL) 13.9±0.67 30.10±5.74* 23.20±4.07** 37.70±6.36**

N (%) 58.67±3.04 94.78±1.00** 85.50±2.57** 93.60±1.64**

L (%) 38.67±2.23 4.56±0.87** 13.75±2.21** 6.27±1.66**

M (%) 0.00±0.00 0.11±0.11 0.00±0.00 0.00±0.00

E (%) 2.67±0.84 0.22±0.22 0.75±0.51 0.13±0.13*

PCV (%) 39.93±0.82 28.68±3.97* 27.57±2.59** 29.74±3.99*

MCV (fL) 71.20±0.49 69.05±5.85 52.85±2.21** 52.81±1.60**

MCH (pg) 21.70±0.32 20.80±0.91 20.19±0.37** 19.90±0.39**

MCHC (g/dL) 30.47±0.24 31.36±2.21 38.93±1.36** 36.45±1.30**

Platelets (105/ µL) 2.94 + 0.39 2.26±0.47 1.36±0.41 3.30±0.72

GLU (mg/dL) 100.50±3.12 99.1±9.23 111.56±9.04 115.93±22.25

BUN (mg/dL) 13.67±0.96 87.89±40.69 36.13±12.79 55.83±15.67*

Creatinine (mg/dL) 0.92±0.04 4.43±2.05 1.28±0.32 2.59±0.61*

TP (g/dL) 6.37±0.29 6.04±1.33 4.45±0.22** 5.41±0.30*

ALB (g/dL) 3.35±0.24 3.19±0.60 1.51±0.12** 2.24±0.20**

Globulin (g/dL) 2.92±0.34 2.02±0.22 2.94±0.20 3.17±0.17

A/G ratio 1.26±0.21 0.76±0.23 0.57±0.09* 0.71±0.06*

Total bilirubin (mg/dL) 0.37±0.09 5.13±2.32* 3.13±0.93** 1.99±0.57*

ALT (U/L) 18.00±3.34 109.22±26.96* 78.25±17.68** 138.53±54.18*

AST (U/L) 36.67±3.91 109.62±22.27* 132.40±36.45* 277.17±133.13**

ALP (U/L) 52.50±9.67 726.78±189.30** 458.56±100.69** 581.20±147.03

GGT (U/L) 2.50±0.50 100.00±63.80 39.07±14.79** 103.13±50.44

Cholesterol (mg/dL) 176.67±24.99 155.89±48.66 126.57±14.47 150.08±19.06


94

Cholecystitis dogs showed significant (p<0.01) increase in the mean values of

ALT (78.25±17.68 U/L), ALP (458.56±100.69 U/L) and GGT (39.07±14.79 U/L) as

compared to control healthy dogs (Table 9). Significant (p<0.05) increase in the mean

value of AST (132.40±36.45) was also observed (Table 10).

Dogs with hepatic neoplasia revealed significant (p<0.05) increase in the

mean values of ALT (138.53±54.18 U/L) and significant (p<0.01) increase in the

mean values of AST (277.17±133.13 U/L) and ALP (581.20±17.03 U/L) as compared

to control healthy dogs (Table 9). Mean value of GGT (103.13±50.44 U/L) was non-

significantly increased (Table 10).

In primary hepatopathy cases, significant (p<0.05) increase in the mean values

of ALT (185.87±21.86 U/L), AST (193.79±30.25 U/L), ALP (544.32±44.89 U/L) and

GGT (81.51±16.64 U/L) was observed as compared to healthy control dogs (Table

11). Dogs with reactive hepatopathies also showed significant (p<0.01) increase in

the mean values of ALT (228.05±36.94 U/L), AST (111.43±23.10 U/L) and ALP

(669.17±83.58 U/L) when compared to healthy control dogs (Table 10). Significant

(p<0.05) increase in the mean value of GGT (53.80±20.04U/L) was also seen as

compared to healthy control dogs (Table 11). Dogs with obscured hepatopathies

showed fluctuated values in liver enzymes ranging from normal to slightly elevated

(Table 11).

The findings of liver enzymes reported in this study are in agreement with

Center (1996) who documented increased activity of ALT and AST in dogs with

hepatic inflammation and necrosis. The ALT is a liver specific cytosolic enzyme in

dogs (Kramer and Hoffman 1997). Acute hepatitis could be differentiated from

chronic hepatitis by the fact that the former resulted in a greater rise in total bilirubin,

ALT and AST as compared to the changes in chronic hepatitis. Valentine et al

95

(1990) reported that largest increase in serum ALT was seen in acute

hepatocellular injury and necrosis caused by various stimuli or factors and the

magnitude of elevation was roughly proportional to the number of injured

hepatocytes. Litchfield and Gartland (1974) also concluded that ALT assay was

useful in detecting acute hepatic damage in dogs after administration of a single

dose of carbon tetrachloride at dose rate of 0.2 ml/kg body weight. Measuring

AST activity is somewhat more sensitive but less specific for detecting hepatic

disease than is measuring ALT activity (Center 1996; Leveille-Webster 2000). In

the present study, it was observed that the levels of AST were in trend with levels of

ALT. Patnaik et al (1980) also reported increased activity of both ALT and AST in

dogs with hepatocellular carcinoma and bile duct carcinoma but ALT values were

greater than AST values. Similar observations were reported by Dial (1995) who

reported that increases in serum AST in dogs and cats paralleled increase in the serum

ALT and like ALT;it was associated with leakage following altered membrane

permeability.

Elevation of ALP in cases of hepatitis is associated with inflammation and

damage of hepatocytes (Strombeck and Gibble 1978). In dogs, ALP is a membrane

bound enzyme found on hepatocytes and luminal surface of biliary epithelial cells

(Sanecki et al 1987). Elevation of ALP in patients more than one year old was usually

of hepatic origin unless the patient had bone disease (Price and Sammons 1976).

Biochemical changes were indicative of hepatic inflammation, with increases in ALT,

AST, ALP and bilirubin reflecting hepatocellular damage and cholestasis. Similar

observations were reported by Farrar et al (1996) in 14 dogs with hepatic abscesses.

GGT is located on the hepatocytes canalicular membrane and serum elevation

of both ALP and GGT have been associated with increased de novo synthesis as well

96

as elution from membranes (Leveille-Webster 2000). High values of serum ALP and

GGT have been documented in similar conditions causing cholestasis such as

cholangiohepatitis, biliary cirrhosis, biliary obstruction, cholecystitis and

cholelithiasis (Shull and Hornbuckle 1979; Guelfi et al 1982; Braun 1983; Johnson

1992; Center 1996; Tennant 1997; Ward 2006). Similar findings were observed in the

present study.

Mean value of TP (g/dL) was significantly (p<0.01) lower in dogs with

cholangiohepatitis (4.79±0.30 g/dL) and liver cirrhosis (4.78±0.37 g/dL, Table 9),

cholecystitis (4.45±0.22 g/dL, Table 10) and primary hepatopathies (5.09±0.17 g/dL,

Table 11) as compared to healthy control dogs (6.37±0.29 g/dL). Similarly, mean

value of TP was significantly (p<0.05) lower in dogs with hepatic neoplasia

(5.41±0.30 g/dL, Table 10) and obscured hepatopathies (3.78±0.74 g/dL, Table 11) as

compared to healthy control dogs (6.37±0.29 g/dL). A non-significant decline in the

mean value of TP was observed in dogs with acute hepatitis/hepatosis (5.64±0.22

g/dL, Table 9), chronic hepatitis/hepatosis (5.96±1.06 g/dL, Table 9), liver abscess

(6.04±1.33 g/dL, Table 10) and reactive hepatopathies (6.33±1.06 g/dL, Table 11) as

compared to healthy control dogs (6.37±0.29 g/dL).

Mean value of ALB (g/dL) was significantly (p<0.01) decreased in dogs with

chronic hepatitis/hepatosis (1.78±0.11 g/dL, Table 9), cholangiohepatitis (1.93±0.20

g/dL, Table 9), liver cirrhosis (1.62±0.15 g/dL, Table 9), cholecystitis (1.51±0.12

g/dL, Table 10), hepatic neoplasia (2.24±0.20 g/dL, Table 10), primary hepatopathies

(2.00±0.10 g/dL, Table 11) and reactive hepatopathies (2.17±0.13 g/dL, Table 11) as

compared to healthy control dogs. Significant (p<0.05) decline in the mean value of

ALB was also seen in dogs with obscured hepatopathies (1.75±0.46 g/dL, Table 11)

as compared to healthy control dogs.

97

Table 11: Hemato-biochemical parameters in healthy and hepatic insufficiency dogs on the day of presentation (Mean±SE)

Parameter

Control (n=6) Primary

hepatopathies (n=98)

Reactive

hepatopathies (n=42)

Obscured

hepatopathies (n=4)

Cholelithiasis

(n=2)

Hb (g/dL) 12.22±0.29 9.06±0.38** 9.63±0.68** 9.35±1.85 12.4 14.4

TEC (106/µL) 5.59±0.10 4.34±0.19** 4.21±0.34 4.42±1.00 5.93 6.82

TLC (103/µL) 13.9±0.67 28.50±2.24** 25.00±3.87** 25.20±7.64 11.01 25.5

N (%) 58.67±3.04 90.08±0.81** 88.21±1.49** 81.50±4.27** 80 96

L (%) 38.67±2.23 9.23±0.74** 14.21±2.49** 16.50±3.30** 18 2

M (%) 0.00±0.00 0.07±0.04 0.38±0.38 0.00±0.00 0 0

E (%) 2.67±0.84 0.55±0.16 0.00±0.00 1.00±1.00 2 2

PCV (%) 39.93±0.82 28.28±1.16** 29.30±1.86** 28.61±10.55 36.9 43.54

MCV (fL) 71.20±0.49 54.52±1.19** 54.61±2.14** 55.88±15.82 52.60 62.08

MCH (pg) 21.70±0.32 20.09±0.21** 19.84±0.51** 20.00±0.63 21.6 19.4

MCHC (g/dL) 30.47±0.24 38.86±0.89** 41.83±2.03** 33.30±5.14 41.00 31.25

Platelets (105/ µL) 2.94 + 0.39 2.0±0.19 1.71±0.29 1.44±0.56 - -

GLU (mg/dL) 100.50±3.12 109.33±5.77 160.31±27.65* 108.75±21.47 310 60

BUN (mg/dL) 13.67±0.96 48.44±6.59** 56.22±10.40** 25.50±7.09 4 9

Creatinine (mg/dL) 0.92±0.04 2.01±0.28** 2.21±0.38** 1.45±0.45 0.6 0.7

TP (g/dL) 6.37±0.29 5.09±0.17** 6.33±1.06 3.78±0.74* 6.9 5.3

ALB (g/dL) 3.35±0.24 2.00±0.10** 2.17±0.13** 1.75±0.46* 2.3 2.1

Globulin (g/dL) 2.92±0.34 3.01±0.07 4.16±1.05 2.03±0.29 4.6 3.2

A/G ratio 1.26±0.21 0.68±0.04* 0.71±0.05* 0.83±0.12 0.50 0.66

Total bilirubin (mg/dL) 0.37±0.09 3.77±0.61** 4.20±1.14** 1.77±1.28 1.5 0.2

ALT (U/L) 18.00±3.34 185.87±21.86** 228.05±36.94** 71.00±26.00 54 118

AST (U/L) 36.67±3.91 193.79±30.25** 111.43±23.10** 37.00±8.62 97 24

ALP (U/L) 52.50±9.67 544.32±44.89** 669.17±83.58** 204.25±68.19 1148 490

GGT (U/L) 2.50±0.50 81.51±16.64** 53.80±20.04* 15.75±7.65 82 27

Cholesterol (mg/dL) 176.67±24.99 131.16±6.95 179.92±20.02 139.75±9.71 140 175


98

Non-significant difference was observed in the values of ALB in dogs with

acute hepatitis/hepatosis (3.62±0.16 g/dL, Table 9) and liver abscess (2.19±0.60 g/dL,

Table 10) as compared to healthy control dogs.

Mean value of globulins was increased in all chronic cases (chronic

hepatitis/hepatosis, liver abscess, liver cirrhosis and hepatic neoplasia) as compared to

healthy control dogs (Table 8, 9). Hall (1985) also was of the opinion that

hyperglobulinemia was frequently encountered in chronic liver disease. Similarly,

mean value of A/G ratio was decreased in chronic cases (chronic hepatitis/hepatosis,

liver abscess, liver cirrhosis) as compared to control healthy group.

Owing to the fact that liver is the main site for synthesis and degradation of

the proteins, it can influence levels of total proteins in many ways (Tennant 1997).

Center (1994) stated that determination of total proteins gives an idea about the

overall protein balance and alone it doesn‟t provide much information in comparison

to albumin and globulin levels. Hypoalbuminemia in hepatic or extrahepatic diseases,

reflects acute phase response or liver dysfunction, owing to switch of liver to

synthesis of acute positive phase proteins rather than negative phase protein i.e.,

albumin (Eckersall and Conner 1988, Sevelius and Anderson 1995). Decreased

plasma albumin levels in hepatopathies have also been observed by other workers

(Nalinikumari et al 1998; Sevelius 1995, Tenant 1997). Besides, decreased nutrient

uptake associated with hepatopathies may be a possible factor for hypoalbuminemia

(Kindmark and Laurell 1972, Skrede et al 1975, Chio and Oon 1979).

Hypoproteinemia occurs mainly due to hypoalbuminemia (Strombeck et al (1976). A

low serum albumin concentration due to liver disease indicates diffuse and chronic

hepatopathies (Prasse et al 1983). Dial (1995) documented that hypoalbuminemia can

be used to differentiate acute from long-term liver disease. Hypoalbuminemia in liver

dysfunctions can also reflect either increased volume of distribution than impaired

hepatic synthesis as observed in ascites or dilutional hypoalbuminemia owing to

99

retention of sodium and water or leaking of albumin directly from hepatic lymph into

ascites (Center 1994). Dill-Macky (1995) observed hypoproteinemia,

hypoalbuminemia and hypergammaglobulinemia in advanced stage of chronic

hepatitis in dogs. Similarly, Kosovsky et al (1989) reported that both

hyperproteinemia and hypoproteinemia were reported in dogs with liver tumour,

whereas hyperglobulinemia was found to be more consistent findings in these dogs.

The hyperglobulinemia in chronic liver disease was always attributed to

increased levels of gamma globulin fractions (Anderson and Sevelius 1992; Ward

2006) which may be associated with enhanced systemic immunoreactivity due to

abnormal Kupffer‟s cell processing of portal antigens or secondary to auto antibody

production (Dial 1995, Leveille-Webster 2000).

Mean value of BUN (mg/dL) was significantly (p<0.01) higher in acute

hepatitis/hepatosis (60.46±11.99 mg/dL, Table 9), chronic hepatitis/hepatosis

(49.54±9.67 mg/dL, Table 9), primary hepatopathies (48.44±6.59 mg/dL, Table 11)

and reactive hepatopathies (56.22±10.40, Table 11) as compared to healthy control

dogs. The mean value of BUN was also significantly (p<0.05) higher in

cholangiohepatitis (32.33±6.93 mg/dL, Table 9) and hepatic neoplasia (55.83±15.67

mg/dL) as compared to healthy control dogs. Non-significant decrease in the mean

value of BUN was observed in dogs with liver cirrhosis (28.67±6.31 mg/dL Table

10), liver abscess (87.89±40.69 mg/dL, Table 10), cholecystitis (36.13±12.79 mg/dL,

Table 10) and obscured hepatopathies (25.50±7.09 mg/dL, Table 10) as compared to

healthy control dogs was seen.

Mean value of creatinine was significantly (p<0.01) higher in chronic

hepatitis/ hepatosis (1.58±0.18 mg/dL, Table 9), liver cirrhosis (1.60±0.16, Table 9),

primary hepatopathies (2.01±0.28 mg/dL, Table 11) and reactive hepatopathies

(2.21±0.38, Table 11) as compared to healthy control dogs. Mean value of creatinine

was also significantly (p<0.05) higher in hepatic neoplasia (55.83±15.67, Table 10) as

100

compared to healthy control dogs. A non-significant rise in the mean value of

creatinine was observed in cholangiohepatitis (1.70±0.39 mg/dL, Table 9), liver

abscess (4.43±2.05 mg/dL, Table 10), cholecystitis (1.28±0.32 mg/dL, Table 10) and

obscured hepatopathies (1.45±0.45 mg/dL, Table 11) as compared to healthy control

dogs.

Low BUN can occur in canine liver disease, reflecting a reduced ability to

synthesize urea from ammonia in hepatic urea cycle (Bexfield and Watson 2006).

Willard (2010) also attributed low BUN in dogs with chronic hepatitis to the

decreased intake of proteins and or excessive loss due to decreased synthesis.

However, severe liver damage can precipitate multiple organ system failure and the

high concentration of BUN and creatinine observed in the present study indicated

effect of hepatic insufficiency on renal function. Hall (1985) observed that anorectic

dogs with normal liver function could have a significant decrease in BUN because of

decreased protein intake, and dogs with decreased hepatic mass might have normal

levels of SUN if they were dehydrated or had concurrent renal dysfunction.

Mean plasma glucose value was significantly (p<0.05) decreased in liver

cirrhosis (83.67±5.18 mg/dL) cases as compared to healthy controls dogs (Table 9).

Other categories of hepatic disease i.e., acute hepatitis/hepatosis, chronic

hepatitis/hepatosis, cholangiohepatitis, liver abscess, neoplasia, cholecystitis, primary

hepatopathies, reactive hepatopathies and obscured hepatopathy (Table 9, 10 and 11)

did not show significant differences from normal values. However, in the present

study, both hypoglycaemia and hyperglycaemia were detected. Dial (1995) reported

that more than 75% of hepatic mass must be lost before occurrence of hypoglycaemia

which might be attributed to decreased gluconeogenesis and decreased insulin

clearance, whereas Leveille-Webster (2000) reported hypoglycaemia as an early

indicator of hepatic failure in severe acute hepatobiliary injury. However, the most

common cause of hyperglycaemia and glycosuria associated with hepatic

101

insufficiency in dogs is diabetes mellitus. Diabetes mellitus was diagnosed in the

present study as an underline cause of reactive hepatopathy. Mild hyperglycaemia can

occur in some dogs up to two hours after consumption of diets containing increased

quantities of monosaccharides and disaccharides, corn syrup, or propylene glycol;

during intravenous (IV) administration of total parenteral nutrition fluids; in stressed,

agitated, or excitable dogs; in animals in the early stages of diabetes mellitus; and in

animals with disorders and drugs causing insulin resistance (glucocorticoids,

progestins, megesterol acetate). All of these conditions were reported during history

taking. Hyperglycaemia also associated with hyperadrenocorticism, diestrus in bitch,

pheochromocytoma, pancreatitis, exocrine pancreatic neoplasia, renal insufficiency

and head trauma (Nelson and Couto 2008). Among these, renal insufficiency and head

trauma were also seen.

Mean value of cholesterol (mg/dL) was significantly (p<0.05) decreased in

chronic hepatitis (107.88±8.85 mg/dL) as compared to healthy control dogs (Table 9).

Other categories of hepatic insufficiency revealed a non-significant decrease or

increase in the mean value of cholesterol as compared to healthy control dogs (Table

9, 10, 11). Hypocholesterolemia is associated with a long-lasting liver disease. The

reason for this is the drop in the production or absorption from the intestines or higher

conversion to bile acids. The most frequent liver disorder associated with

hypocholesterolemia is the PSS, in which increased conversion to bile acids is the

primary mechanism (Leveille-Webster 2000). However, PSS was not diagnosed in the

present study as it a rare condition and also probably because of the limited number of

dogs among the breeds which are prone to this condition. Hypercholesterolemia is

commonly associated with cholestatic disease (Hall 1985). In addition, increased

production of cholesterol might occur with retention of lecithin in bile.

The most common forms of hepatitis are non-specific reactive hepatitis, acute

hepatitis, and chronic hepatitis. Non-specific reactive hepatitis is a reaction against

102

endotoxin as a result of sepsis or an increased gastrointestinal absorption (Rothuizen

and Van den Ingh 1998). Reactive hepatopathies are characterized by nonspecific

hepatocellular degeneration or necrotic changes without evidence of significant

chronic progressive inflammation. Again, these changes are usually secondary to

manifestations of a primary non-hepatic disease. The reason that the liver often

undergoes these changes evolves from the fact that the liver is involved in many

metabolic and detoxification functions. Endogenous toxins, anoxia, metabolic

changes, nutritional changes and endogenous stress related glucocorticoid release are

examples of conditions responsible for the majority of these changes (Twedt 2013).

In the present study, among the 140 dog diagnosed with liver dysfunction,

only two dogs were diagnosed with cholelithiasis and therefore were not subjected to

statistical analysis. The haemato-biochemical parameters of these two dogs and their

values are depicted in Table 11). Similar observations are reported by other workers

(Church and Matthiesen 1988; Ward 2006). The frequency of cholelithiasis in dogs is

low (Church and Matthiesen 1988; Guilford 1990; Neer 1992; Kirpensteijn et al 1993;

Strombeck and Voros 2001). Choleliths are subclinical and not detected antemortem as

they rarely result in clinical signs. Certain substances, such as bile pigments,

mucoproteins, bacteria, and refluxed intestinal contents, can act as a nidus for

microscopic calculi, which expand into larger calculi known as gallstones (Strombeck

and Guilford 1990). Possible aetiologies for biliary calculi include trauma, biliary

stasis, microbial and parasitic biliary infections, and diet alterations (Kirpensteijn et al

1993; Strombeck and Guilford 1990.

Pre-prandial total serum bile acid concentration was analysed in 25 serum

samples collected randomly from dogs with different categories of hepatic disease

(Table 12).



103

Table 12: Total serum bile acids concentrations in dogs with hepatic insufficiency

(n=25)

(Normal range =15-25 μmol/L)

Hepatic disease TSBAs concentration Total

Acute hepatitis/hepatosis (n=9) <15μmol/L

>15μmol/L

4 (16%)

5 (20%)

Chronic hepatitis/hepatosis (n=5) <15μmol/L

>15μmol/L

3 (12%)

2 (8%)

Liver abscess (n=2) >15μmol/L 2 (8%)

Hepatic neoplasia (n=2) >15μmol/L 2 (8%)

Cholangiohepatitis (n=2) <15μmol/L

>15μmol/L

1 (4%)

1 (4%)

Chronic active hepatitis (n=2) Equivocal range 2 (8%)

Liver cirrhosis (n=2) <15μmol/L 2 (8%)

Cholecystitis (n=1) Equivocal range 1 (4%)


A serum bile acids concentration greater than 25 μmol/L was considered

positive for hepatobiliary dysfunction, whereas serum bile acids concentration less

than 15 μmol/L was considered as negative. Dogs with serum bile acid values

between 15-25 μmol/L were in an equivocal (grey) zone.

Total serum bile acid concentration of 25 fasting serum samples from the dogs

suffering from hepatic disorders ranged from <3.20 - 375.40 μmol/L (mean ± SE,

75.68 ± 22.98 μmol/L). Of these, 10 (40%) samples revealed TSBAs concentration

below the reference range (< 3.2 μmol/L), 3 (12%) samples were within the equivocal

range (15-25 μmol/L) and remaining 12 (48%) samples above the reference range (>

15 μmol/L) (Table 12). Of the nine (36%) serum samples harvested from acute

hepatitis/hepatosis cases, 4 (44.44%) samples revealed TSBAs concentration below

the normal range and 5 (55.55%) were above the higher range. Out of 5 (20%)

samples collected from chronic hepatitis/hepatosis cases, 3 (60%) were below the

grey zone and 2 (40%) above the reference range. Samples collected from dogs with

liver abscess (2, 8%) and dogs with hepatic neoplasia (2, 8%) revealed TSBAs

104

concentrations higher than the reference values. Serum samples (2) from dogs with

cholangiohepatitis revealed reading value below the lower limit (1) and above the

higher range (1). Chronic active hepatitis cases (2, 8%) showed border line reading

(inconclusive). Dogs with liver cirrhosis (2, 8%) showed TSBAs values less than 3.2

μmol/L. One (4%) case with cholecystitis showed TSBAs values within the equivocal

zone.

Elevated serum bile acids concentration is usually associated with hepatic

dysfunction. Common causes of elevated serum bile acids concentrations include:

PSS, hepatic cirrhosis and hepatocellular disease caused by diffuse inflammation or

necrosis (Turgut et al 1997, Gerritzen-Bruning 2006).

In fasting pets, portal bile acids concentration is usually low, so serum bile

acids may be normal despite impaired hepatic function. Since patients with hepatic

dysfunction may have normal or elevated fasting serum bile acids concentration, it

was therefore not possible to predict hepatobiliary disease on the basis of bile acid

determinations. Further investigation of liver function is required to investigate why

bile acid concentrations were decreased or within the grey zone in these dogs.

According to Center et al (1991), the patients with hepatic dysfunction may have

normal or elevated fasting serum bile acids concentrations with an elevated

postprandial bile acids concentration. Focal hepatic diseases, such as certain forms of

hepatic neoplasia, may not impair hepatic clearance of bile acids sufficiently to cause

a measurable increase in serum levels. Thus serious, even terminal, diseases involving

the liver may be associated with normal serum bile acids concentrations. Sevelius

(1995) stated that moderate to marked elevation of ALT, moderate elevation of ALP

in combination with normal SBA and serum albumin concentrations were indicative

of chronic nonspecific hepatitis which is in line with the observations found in the this

study. Center et al (1991) also stated that it is possible for the SBA concentration to

be within the normal range (false negative) and transient decrease in bile flow may

105

lower the concentration. A single, random, markedly elevated serum bile acids

concentration makes liver dysfunction very likely, however, the sensitivity of

detecting hepatic disease with only one sample is significantly lower than using the

two sample bile acids stimulation test.

The specificity of SBA as an indicator of hepatobiliary disease in dogs and

cats was also reported by Center (1990). However, serum bile acids concentrations

can fluctuate markedly hour-to-hour and day-to-day in the same pet, therefore, serial

monitoring of bile acids is of no value in evaluating the activity or progression of liver

disease.

A normal serum bile acids concentration without a food challenge cannot be

relied upon to rule out the presence of liver disease. Multiple conditions other than

hepatic disease can also alter bile acid metabolism sufficiently to increase or decrease

the serum bile acids concentrations, and interfere with the accuracy of the bile acids

test (Richter 2004, Chapman and Hostutler 2013). These include: pancreatitis, which

may obstruct the common bile duct; gastrointestinal motility changes, which alter the

delivery of bile acids to the ileum for absorption; and severe inflammatory bowel

disease or lymphosarcoma, which impair the absorption of bile acids. Haemolysis

and/or lipaemia of the blood sample will interfere with spectrophotometric assay used

for measurement of serum bile acids and markedly complicate end-point

determination.

Recently, the necessity of the pre-test 12-hour fast has been questioned. Some

clinicians have argued that a random pre-prandial sample and two-hour post-prandial

sampling is sufficient, and the test is positive if either value is elevated. This is based

on the fact that a small percentage of cats and dogs have higher fasting than post-

prandial bile acids concentrations (Center 1993). This may be due to gallbladder

contraction during fasting, intestinal malabsorption associated with disease or motility

changes and bacterial overgrowth causing intestinal bile acids metabolism.

106

Although bile acids are excellent indicator of hepatic function, the magnitude

of the increase is not specific to an underlying particular diagnosis or prognosis. Bile

acids typically are not often elevated with nonhepatic disease, antiepileptic therapy, or

glucocorticoid administration. Our study revealed that out of 25 serum samples tested,

only 12 (46.15%) were confirmed to be positive for hepatic dysfunction, 4 (15.38%)

were within the equivocal zone and the rest 10 (38.46%) below the reference range.

Confirmation of hepatic dysfunctions was based on combined data from elevation of

specific liver function tests, haematology, medical imaging and pathological findings.

4.6 URINE ANALYSIS

Urine analysis provides rapid and valuable information about the urinary tract

and other body systems including liver. A complete urine analysis (including dipstick,

specific gravity, and sediment examination) is often required even if one component

part shows no abnormalities (Kumar et al 2012).

4.6.1 Gross examination of urine in dogs with hepatic insufficiency

Normal healthy dogs invariably have acidic urine pH in nature. However,

urine pH can vary with diet. Urine was observed in all dogs, but complete urinalysis

was done only for 76 cases. The urinary pH of 76 dogs in the present study was within

the range of 5.5 to 8.5. Seventy dogs (92.11%) fell in the category of pH 5.5-7 and 6

dogs (7.89%) had pH > 7 as shown in Table 13.

Out of 140 dogs, the colour of the urine was normal (straw coloured) in 73

(52.14%) dogs, dark yellowish in 54 (38.57) dogs (Fig. 26 A) and light (pale)

yellowish in 5 (3.57%) dogs. Transparent and reddish yellow urine each was observed

in 3 (2.14%) dogs. Brownish coloured and greenish coloured urine (Fig. 26 B) each

was observe in 1 (0.7%) case. The pale coloured urine samples mostly contributes to a

low specific gravity of <1.015. According to Forrester and Brandt (1994), the patients

with dark red or brown urine may have haematuria, haemoglobinuria or

myoglobinuria.

107

Out of 140 dogs, the urine of 3 (2.14%) dogs had ammoniac smell and 3

(2.14%) dogs had a sweet fruity odour which was found to be due to presence of

ketone bodies, whereas the urine of 122 (87.14%) dogs had normal urineferous odour.

Dogs which had revealed ammoniac odour were suffering from severe renal

impairment, whereas dogs with sweet fruity odour had diabetic ketoacidosis (DKA).

Macroscopically, the urine of 3 (2.14%) dogs had turbidity whereas turbidity was

absent in 137 (97.86%) dogs (Table 13). Turbidity of the urine could be attributed to

the presence of crystals, casts or proteinuria.

Urine specific gravity of the normal healthy dogs varies from 1.015-1.030. In

the present study, the urine specific gravity was measured in 76 dogs and found to be

less than 1.015 in 9 (11.84%) dogs, more than 1.030 in 2 (2.63%) dogs and rest of the

65 (85.5%) dogs had urine specific gravity within normal range of 1.015-1.030

(Table 13).

A low urine specific gravity is common in patient with liver disease due to

polyuria and polydipsia (Bexfield and Watson 2006; Chapman and Hostutler 2013).

However, renal impairment associated with liver dysfunction also can contribute to

the low urine specific gravity.

4.6.2 Chemical analysis of urine in dogs with hepatic dysfunction

Complete chemical analysis of urine was performed on 76 fresh urine samples

collected from dogs revealing signs of liver damage like jaundice and ascites beside

elevation in liver enzymes (ALT and GGT) and abnormal changes in hepatic

architecture based on ultrasonographic examination. Proteinuria was observed in

majority of cases with varying degree. The degree of proteinuria was trace in 15

(19.74%), 1+ in 10 (13.16%), 2+ in 14 (18.42%) and 3+ in 15 (19.74%) dogs (Table

14). Forrester (1997) reported that proteinuria was observed in CHF, genital

diseases, lower urinary tract infections (UTIs) and renal diseases.

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In present study, glucose level was found in traces in 14 (18.42%) dogs, 1+ in

5 (6.58%), 2+ in 1 (1.32%) and 3+ in 3 (3.95%). Rest of dogs 53 (69.74%) showed

negative glucosuria (Table12). Diabetes mellitus may contribute to the presence of

glucose in urine. During chemical analysis of urine for the presence of blood 35

(46.05%) dogs were found negative; blood cells to the degree of 1+ were seen in 14

(18.42%) dogs, 2+ in 8 (10.53%) and 3+ in 13 (17.11%) dogs. Presence of blood in

urine is mainly due to haematuria or haemoglobinuria. In the present study bilirubin

was found to be 1+ in the urine of 8 (10.53%) dogs which was associated with

icterus, 2+ in 12 (15.79%) and 3+ in 19 (25%) dogs. Bilirubinuria greater than 2+ in

a urine dipstick in a dog, and any bilirubinuria in cats, should raise the index of

suspicion for underlying hepatic disease (Chapman and Hostutler 2013). Increased

bilirubinuria due to overspill is found in dogs with hyperbilirubinemia. Small

quantities (traces) of conjugated bilirubin can be seen in the urine of normal dogs

(particularly male dogs) due to the low renal threshold for bilirubin (Bexfield and

Watson 2012). The causes of increased bilirubinuria are therefore, an indicator of

excessive extravascular haemolysis or hepatobiliary disease (Santilli and Gerboni

2003). Nelson and Couto (1998) and Leib and Monroe (1997) reported that the

common finding in urinalysis consistent with hepatobiliary diseases includes

bilirubinuria. In the present study, bilirubinuria was never seen in traces. It was

invariably in excess concentration and accompanied with high elevation of the

specific liver enzymes and abnormal changes in the architecture of hepatic

parenchyma on USG examination which support the hepatic origin of

hyperbilirubinuria.

Leucocytes were found in traces in 2 (2.63%) dogs. Ketonuria was present in

7 (9.21%) dogs. Nitrites were absent in all the cases. Measurement of urobilinogen

by dipstick analysis has traditionally been used to assess the patency of the

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extrahepatic biliary system. Urobilinogen is normally found in urine. Increased

amounts are associated with hyperbilirubinuria and with complete bile duct

obstruction. If complete bile duct obstruction is present, urobilinogen should be

absent from the urine (very uncommon in dogs). In the present study, value of

urobilinogen varied within the range of 0.1 to 8 mg/dL. The concentration of

urobilinogen was 0.1 mg/dL in 4 (5.3%) urine samples, 0.2 mg/dL in 51 (67.1%) and

1 mg/dL in 15 (19.7%) cases. Rest of dogs showed urobilinogen values of more than

one. The values were 2 mg/dL in 4 (5.3%) dogs, 4 as well as 8 mg/dL each was

observed in 1 (1.31%) urine sample (Table 14). Urobilinogen increases in the urine

following haemolysis and the dogs suffering from hepatitis can develop clinical and

laboratory evidence of renal tubular dysfunction (Langlois et al 2013).

Table 13: Gross examination of urine in dogs with hepatic insufficiency (n=76)

Gross examination of urine (n=76) Total

pH 5.5-7 70 (92.11%)

>7 6 (7.89%)

Straw coloured 73 (52.14%)

Dark yellow 54 (38.8)

Colour Pale yellow 5 (3.6%)

Transparent 3 (2.1%)

Reddish yellow 3 (2.1%)

Brownish 1 (0.7%)

Greenish 1(0.7%)

Ammoniac 3 (2.14%)

Odour Fruity sweet 3 (2.14%)

Nil 134 (95.71%)

Turbidity Present 3 (3.9%)

Absent 73 (96.05%)

<1.015 9 (11.8%)

Specific gravity 1.015-1.030 65 (85.5%)

>1.030 2 (2.6%)


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Table 14: Chemical analysis of urine in dogs with hepatic insufficiency (n=76)

Parameter Result Total

Glucose (mg/dL)

Negative 53 (69.73%)

Trace 14 (18.42%)

1+ 5 (6.57%)

2+ 1 (1.31%)

3+ 3 (3.94%)

Blood


1+ 14 (18.42%)

2+ 8 (10.52%)

3+ 13 (17.10%)

Protein (mg/dL)

Trace 15 (19.73%)

1+ 10 (1.31%)

2+ 14 (18.42%)

3+ 15 (19.73%)

Bilirubin (mg/dL)

Negative 37 (48.7%)

1+ 8 (10.5%)

2+ 12 (15.8%)

3+ 19 (25%)

Leucocytes

Trace 2 (2.63%)

1 8 (10.5%)

2 4 (5.3%)

3 8 (10.5%)


Ketone bodies (mg/dL)

Present 7 (9.21%)

Absent 69 (90.79%)

Nitrites (mg/dL) Absent 76 (100%)

Urobilinogen (mg/dL)

0.1 4 (5.3%)

0.2 51 (67.1%)

1 15 (19.7%)

2 4 (5.3%)

4 1 (1.3 %)

8 1 (1.3 %)


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4.6.3 Microscopic findings of urine in dogs with hepatic insufficiency

Urine analysis provides rapid and valuable information about urinary tract and

other body systems including liver, but microscopic examination of the urine is more

reliable and accurate. Certain urine abnormalities may be present in animals with liver

disease therefore, a well performed microscopic examination of urine can provide

rapid and valuable information about urinary tract and other body systems including

liver (Bexfield and Watson 2006; Kumar et al 2012). Normally, urine of a healthy dog

does not contain erythrocytes, although the presence of 1-2 RBC/HPF is usually not

considered abnormal. In the urine of a healthy dog, up to five RBCs or pus cells per

high power field (HPF) are considered to be normal. In the present study, out of 76

urine samples evaluated, 21 (27.63%) dogs had plenty of RBCs and pus cells count

per HPF (Table15). The presence of pyuria and haematuria in the urine sediment

indicates renal inflammation, haemorrhage or some infection in any part of urinary

tract. Urothelial cells were observed in the urine of 24 (31.58%) dogs (Table15),

calcium carbonate crystals in 1 (1.32%) dog and calcium oxalate crystals in 2 (2.63%)

dogs. Oxalate crystals were associated with acute renal failure cases and hepatic insult

was confirmed. Bilirubinuria was found in 19 (25%) dogs among tested samples, but

bilirubin crystals (Fig. 27) were observed only in 10 (13.16%) dogs with

hyperbilirubinuria. Bilirubinuria greater than 2+ in a urine dipstick in a dog should

raise the index of suspicion for underlying hepatic disease (Chapman and Hostutler

2013). Granular casts were observed in 7 (9.21%) dogs and waxy casts in 4 (5.26%)

dogs. Proteinuria and bacteruria were detected in 6 (7.89%) and 4 (5.26%) dogs

respectively (Table 15). The granular casts might be due to acute renal failure

associated with acute hepatic failure, whereas waxy casts are sometimes associated with

chronic renal disease. Bacteria are commonly seen in the cases of cystitis and they can

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also be seen in cases of pyelonephritis (Acierno and Senior 2010). However, urinary

tract infections can precipitate reactive hepatopathies. In the present study, microscopic

examination of urine was more sensitive and reliable than dipstick test for examination

of bilirubinuria which is highly suggestive of liver dysfunction.

Table 15: Microscopic findings of urine in dogs with hepatic dysfunction (n=76)

Findings Number of dogs

RBC (plenty/HPF) 21 (27.63%)

WBC (5-25/HPF) 21 (27.63%)

Urothelial cells 24 (31.58%)

Calcium carbonate crystals 1 (1.32%)

Calcium oxalate crystals 2 (2.63%)

Bilirubin crystals 10 (13.16%)

Bilirubinuria 19 (25%)

Proteinuria 6 (7.89%)

Bacteruria 4 (5.26%)

Granular casts 7 (9.21%)

Waxy casts 4 (5.26%)


4.7 ELECTROLYTE ALTERATIONS

Out of 140 dogs, serum sodium and potassium concentration was measured in

129 (92.14%) dogs. The mean values of serum sodium and potassium were within the

normal reference range on the day of presentation for almost all the cases irrespective

of the disease and its stage (Table 16). However, the fluctuations in these electrolytes

were detected but they did not manifest any specific trend. To our knowledge, there is

no specific study on electrolytes in dogs with liver disease. However, electrolyte

imbalances rarely occur due to vomiting, diarrhoea, anorexia or faulty fluid therapy.

The most common electrolyte abnormality is hypokalaemia, due to renal or

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gastrointestinal losses, reduced intake and secondary hyperaldosteronism (Bunch

2003). However, one case was suspected for hypoadrenocorticism as there were

typical signs of hypoadrenocorticism beside hyponatremia and hyperkalaemia with

sodium: potassium ratio less than 27:1.

Table 16: Electrolyte alterations (n=129)

Type of electrolyte Sodium (mEq/L) Potassium (mEq/L)

Reference range 140-154 3.8-5.6

Acute hepatitis/hepatosis 143.51±2.01 4.15±0.09

Chronic hepatitis/hepatosis 143.34±1.11 4.15±0.11

Cholangiohepatitis 142.78±0.97 4.04±0.15

Liver abscess 142.22±4.37 4.13±0.30

Liver cirrhosis 145.17±4.05 4.47±22.97

Hepatic neoplasia 141.57±2.29 4.06±0.23

Cholecystitis 142±1.55 4.18±0.11

Obscured hepatopathy 139.5±7.01 5.82±1.73

4.8 EXAMINATION OF FAECAL SMEARS

Examination of faecal samples for eggs, larvae, cysts and oocytes revealed

hook worm eggs in 4 cases. Rest of faecal samples were negative which could be

attributed to the periodical and regular deworming.

4.9 PERITONEAL FLUID ANALYSIS

The biological/peritoneal fluid was collected for better scientific diagnosis and

to alleviate the abdominal discomfort of the animal. It is also routine clinical

procedure on emergency as it is useful in the differential diagnosis. Ascites cases

needed emergency treatment if condition was so severe and peritoneocentesis was one

of the remedy.

Peritoneal fluid analysis was conducted on 50 dogs having peritoneal effusion

and was confirmed with liver disease based on combined data from liver function

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tests, haematology and ultrasonography. Out of these 50 dogs, 26 (52%) were males

and 24 (48%) females. The results of peritoneal fluid analysis are summarized in

Table 17. The peritoneal fluid colour varied from clear transparent to slightly amber

colour in majority of cases and totally bloody in some cases (Fig. 28). The colour of

the peritoneal fluid was colourless pure transudate in 27 (54%) dogs, icteric in 9

(18%), serosangunious in 7 (14%), totally bloody (haemoperitoneum) in 4 (8%) and

milk coloured in 3 (6%) cases. Stiner (2008) reported that characteristic features of

transudate are clear, colourless and pure, specific gravity below 1.016 and low protein

concentration (<2.5 g/dL). Transudate that forms as a result of low osmotic pressure

usually have a low protein concentration. Hypoproteinemia and hypoalbuminemia

were noticed due to low osmotic pressure caused by inadequate albumin synthesis in

severe liver disease, excessive protein loss, maldigestion, malabsorption and

starvation (Tontis 2004). Icteric peritoneal fluids were attributed to jaundice as bile

stained fluid may indicate gall bladder problem (Mondal et al 2012). Blood tinged

ascitic fluids can be a sequel of any inflammatory or neoplastic process (Tennant and

Hornbuckle 1980). Aronsohn et al (2009) conducted a retrospective study on 60 dogs

with acute nontraumatic haemoperitoneum and reported that splenic haematoma,

splenic torsion, hemangiosarcoma, hepatocellular carcinoma and carcinomatosis were

among the most common causes. In the present study hepatocellular carcinoma,

secondary hepatic and splenic hemangiosarcoma and adenocarcinoma were confirmed

through FNAB of liver and spleen and also by cytologic examination of peritoneal

fluids. Haemorrhagic ascitic fluids can also be a consequence of severely enlarged

livers due to hepatic congestion (Rothuizen and Mayer 2000) or traumatic tap

(Runyon et al 1988). Milk–coloured peritoneal fluids may indicate disease conditions

such as carcinoma, lymphoma, tuberculosis or infection (Runyon et al 1988). In the

present study, carcinomas, peritoneal fluid infection and canine lymphosarcoma were

also diagnosed.

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Macroscopically, the peritoneal fluids of 5 (10%) dogs had turbidity whereas it

was absent in 41 (82%) dogs. Four (8%) dogs had haemoperitoneum therefore,

turbidity aspect was ignored in these patients. Turbidity and discolouration of ascitic

fluids were associated with neoplasia and/or severe septic peritonitis which were

oftenly accompanied by RBCs, pus cells and polymorphonuclear cells.

Turbid peritoneal fluid most commonly forms due to bacterial peritonitis. However,

not all instances of cloudy peritoneal fluids are due to infection. Cloudy peritoneal

fluids may be due to pathologic increases of either cellular or non-cellular constituents

of peritoneal fluid. Polymorphonuclear leukocytes may be increased due to either

intra- or juxtaperitoneal inflammation or drug-induced chemical peritonitis

(Teitelbaum 2006).

The odour of peritoneal fluids was foul and putrid in a single case (2%) having

severe septic peritonitis with overwhelming bacteria whereas peritoneal fluids of 49

(98%) dogs had normal odour. Foul smell of peritoneal fluids was attributed to the

sepsis as no contamination of peritoneal fluid with urine and gut contents was

found/or induced during peritoneal fluid collection and examination.

The mean±SE value of ascitic fluids total protein was 0.59±0.13 g/dL (range,

0.02-3.9 g/dL). These observations are in concurrence with Dill-Macky (1995) who

reported that peritoneal fluids was either transudate or modified transudate in dogs

with chronic hepatitis and with Crowe (1984) and Cornelius (1992) who reported low

protein (less than 2.5gm/dL) and few cells (less than 1.0 x 103/L) in hepatic diseases.

Johnson (2000) also stated that most of primary hepatic disorders and

hypoproteinemia ascitic fluids were accompanied by <2.5 g/dL of protein content.

Moreover, chronic portal hypertension causes low protein ascitic fluid (<2.5 g/dL)

(Green 1979). A well performed microscopic examination of peritoneal fluids can

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provide information nearly equivalent to a biopsy in many cases with abdominal

effusion including hepatic diseases.

Out of the fifty dogs which were subjected to the peritoneal fluids

examination, 14 (28%) samples did not show cytopathological changes as only

transudate ascitic fluids were detected. Ten (20%) samples revealed different degrees

of peritonitis, metastatic neoplasias were confirmed in 9 (18%) cases and 1 (2%) case

was suspected for carcinoma. Rest (16, 32%) of the cases revealed nonspecific

findings such as occasional RBCs, pus cells, mesothelial cells and transudate.

Mondal et al (2012) reviewed that ascites of non-inflammatory origin due to

inadequate cardiac function may depict red blood cells, neutrophils, mesothelial cells

and macrophage in the absence of bacteria. An overwhelming infection with bacteria,

fibrinopurulent exudate with less immune response suggestive of severe sepsis as a

result of chronic active hepatitis/fibrinopurulent hepatitis (Fig. 29) was observed in 1

(2%) case and bacterial culture of peritoneal fluid revealed severe infection with

Staphylococcus aureus. Chronic active peritonitis possibly perihepatitis with

haemorrhage and sepsis was diagnosed in 4 (8%) cases as many RBCs, markedly

degenerated neutrophils, few activated macrophages and mesothelial cells with small

clumped bacteria were seen in the background of the smears (Fig. 30). Mild to

moderate suppurative peritonitis was diagnosed in 1 (2%) case as many degenerated

neutrophils and lymphocytes were observed. Severe suppurative peritonitis with

massive neutrophilia and mostly degenerated neutrophils was observed in 4 (8%),

whereas low grade peritonitis with a few markedly degenerated neutrophils

sometimes showing markedly toxic changes and low protein content was also

diagnosed in 4 (8). Mondal et al (2012) reviewed that an increase in total white cell

counts of the fluid including a disproportionate number of polymorph nuclear cells

indicates acute inflammation which may have an infectious origin or else be sterile,

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whereas an increase in mononuclear phagocytes from the peritoneum is an indication

of chronic peritonitis. The authors also stated that the inflammation is considered

suppurative if neutrophils are predominant, and septic if they are degenerative.

Bacteria found as phagocytosed inclusions of leukocytes or by culture of fluid,

indicate an infective peritonitis which may arise by haematogenous spread in which

case infection is likely to be specific one.

Peritoneal fluid analysis was also fruitful in the diagnosis of metastatic

neoplasias. Metastatic hepatocellular carcinoma was diagnosed in 3 samples (Fig. 31

A, B, C, D and Fig. 32 A & B), whereas metastatic adenocarcinoma was noticed in 2

(4%) samples (Fig. 33 A & B). Metastatic hemangiosarcoma was detected in 4 (8%)

samples (Fig. 34 A, B, C, D). Numerous intact to moderately degenerated neutrophils

along with few abnormal and pleomorphic cells resembling neoplastic hepatocytes

and suspected for primary/secondary hepatic carcinoma with high protein

concentration, numerous RBCs and few mesothelial cells was observed in 1 (2%)

cases (Fig.35). USG examination of these cases suggested neoplastic lesion, therefore,

peritoneal fluid cytology was considered to be the most sensitive and specific method

in establishing the neoplastic aetiology of ascites. These observations are in

agreement with Glińska et al (2006) who conducted cytological examination of

peritoneal cavity fluid in 25 dogs with ascites for diagnosis of neoplasia, out of which,

neoplasia was diagnosed in 5 dogs (20%) where USG suggested neoplastic lesions in

2 (8%) dogs.

Canine neoplasia has a high metastatic rate, ranging from 61% for the

hepatocellular carcinomas to 93% for the carcinoids (Patnaik et al 1980).

Degenerative toxic neutrophils suggest probability of infection being present. An

increase in number of mesothelial cells with the distinctive presence of actively

dividing mitotic figure suggests neoplasm (Mondal et al 2012). Markedly degenerated

neutrophils along with many macrophages, few mesothelial cells and fibrocytes and

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severe highly protein contents were seen in one case with chronic active hepatitis due

to old abscess. Rutgers et al (1993) diagnosed idiopathic hepatic fibrosis in dogs

manifesting ascites, anorexia, weight loss and hepatic encephalopathy. A few

markedly degenerated neutrophils and several RBCs, low protein content, fluid shows

certain leakage of GIT content were seen in one case probably due to GIT penetration

during abdominocentesis.

Table 17: Peritoneal fluid analysis of dogs with hepatic insufficiency (n=50)

Parameter Patient result Total

Colour

Colourless (transparent) 27 (54%)

Icteric 9 (18%)

Serosanguinous 7 (14%)

Haemoperitoneum 4 (8%)

Milk colored 3 (6%)

Turbidity

Clear 41 (82%)

Turbid 5 (10%)

Blood 4 (8%)

Odour NAD 49 (98%)

Foul 1 (2.04%)

Protein (g/dL) 0.59 ±0.13 (range, 0.02-3.9) -

Diagnosis

NAD 14 (28.57%)

Peritonitis (with or without sepsis) 10 (20.41%)

Hepatocellular carcinoma 3 (6%)

Adenocarcinoma 2 (4%)

Hemangiosarcoma 4 (8%)

Suspected carcinoma 1 (2%)

Unremarkable 16 (32%)


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4.10 ELECTROCARDIOGRAPHIC CHANGES IN DOGS WITH HEPATIC

INSUFFICIENCY

Electrocardiogram (ECG) provides critical information on a number of

changes in the electrophysiological function, in particular cardiac rhythmicity,

conduction, depolarization and repolarization, which cannot be assessed by other

methods and which have no morphological correlates visible at histopathological

examination. ECG analysis allows early detection of adverse effects on the cardiac

function, establishment of their time of onset and monitoring of their evolution over

time (Detweiler 1981).

In the present study, ECG was performed on 50 dogs showing either abdominal

effusion and/ or having abnormal heart and lung sounds with or without cardiomegaly

on chest x-ray in order to rule out some possible cardiac problems. The majority of

cases showed no abnormalities on ECG except for tachycardia or sinus arrhythmia.

Right ventricular hypertrophy and left ventricular hypertrophy with tachycardia was

observed in one case each. Atrial fibrillation was also diagnosed in a single case in

which echocardiography revealed right atrial enlargement. Altered PR-interval was

observed in a single case. Bradycardia was seen in 2 cases. Reduced PR-interval and

QRS interval amplitude was observed in one case. One dog with hyperkalaemia and

hyponatraemia was suspected for hypoadrenocorticism showed peaking of T-wave.

4.11 RADIOGRAPHIC STUDIES IN DOGS WITH HEPATIC

INSUFFICIENCY

Survey abdominal radiographs (lateral and ventrodorsal view) are useful to

evaluate the morphologic abnormalities in size, shape, position and density

(mineralization/ radiolucencies) of the liver and presence of abdominal effusion.

However, lack of abdominal contrast and insensitivity to detect subtle changes limits

the precision of abdominal radiography.

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In the present study, 96 dogs with signs of hepatic dysfunctions particularly

abdominal distension and jaundice with elevated liver enzymes were subjected to

plain abdominal radiograph, out of which 29 (30.21%) dogs had acute

hepatitis/hepatosis, whereas 67 (69.79%) were chronic cases. In the acute cases,

hepatomegaly was observed in 9 (31.03%) dogs as liver was extending behind the

ribcage with sharp margins (Fig. 36); one of these cases revealed enlargement of both

liver and spleen (Fig. 37). Rest of acute hepatitis/hepatosis cases (19, 68.96%) did not

show significant abnormalities in hepatic size, contour and architecture. Dogs with

acute hepatic disease did not show ascites on lateral abdominal radiograph, except,

one case which revealed mild ascites due to other complication in GIT. Increased

proportion of caudoventral liver margins beyond the costal arch in dogs with

hepatomegaly was also reported by Pechman (1993). Failure of observing ascites in

acute cases of liver disease could be attributed to the poor sensitivity of radiography to

detect very small amounts of peritoneal fluids. In general, survey radiography was not

much useful to diagnose dogs with acute hepatitis/hepatosis. Accurate radiographic

changes in hepatic size has been claimed to be difficult (Godshalk et al 1988, Barr

1992). According to Partington and Biller (1995), although hepatitis chiefly cause

changes in the hepatic parenchyma, it could not be evaluated by standard lateral and

VD radiographs that reveal the liver position, margination, size and opacity.

A majority of chronic hepatic diseases showed gross hepatic enlargement with

rounding of liver margins (Fig. 38) on survey radiograph and in some cases with

severe hepatomegaly stomach was pushed caudally (Fig. 39). Severe diffuse

hepatomegaly causes a substantial portion of caudoventral liver margin to project

beyond the costal arch, indicating clear increase in liver size and rounding of caudal

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liver edges on lateral radiographs (Root 1974; O‟Brien 1978). Homogeneous fluid

opacity with loss of intra-abdominal tissue opacity i.e., ground glass appearance of

abdomen with loss of serosal details suggestive of ascites (Fig. 40) was frequently a

consistent feature of chronic hepatic disease, peritonitis or haemoperitoneum , and

hence was inconclusive. Johnson (2000) also observed that evaluation of liver size on

survey radiograph film could be difficult in the presence of ascites.

Hepatomegaly was observed in 19 (28.35%) cases of chronic hepatopathies in

which ascites was not interfering with the serosal details. One case was diagnosed

with cranial abdominal mass (showing radioopaque non uniform opacity with irregular

margins measuring about 11.7cm x 5.3cm in cranial ventral region caudal to the liver

and ventral to the pylorus) encroaching the liver and was confirmed by FNAC guided

USG as liposarcoma (Fig. 41.), one case showed hepatomegaly with rounded liver

margins and displacing intestines caudally on lateral abdominal radiograph was

confirmed by FNAB-guided USG as hepatocellular carcinoma. Symmetric

hepatomegaly has been ascribed to hepatic neoplasia and an asymmetric cranial

abdominal mass causing gastric displacement was found to be the most common

radiographic appearance of primary nonvascular nonhematopoietic hepatic neoplasm

(Hammer and Sikkema 1995).

Although hepatic tumours do cause hepatomegaly but it seems that they rarely

cause significant changes in hepatic architecture. Two cases with primary

hepatocellular carcinoma confirmed with FNAB revealed multiple nodular densities in

cranial and caudal lung lobes on lateral chest radiograph suggestive of metastasis (Fig.

42 a & b). In one of these two cases, metastasis caused loss of interstitial details ((Fig.

42 b). Choleliths, gall bladder sludge, cholecystitis and cholangiohepatitis could not be

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diagnosed through survey radiography. This could be attributed to the small size and

lack of mineralization. Most choleliths in dogs are radioluscent and are composed

primarily of bilirubin (Nakayama 1969). O‟Brien (1978) and Pechman (1998) also had

the same opinion that gall bladder could not be visualized as a separate entity on plain

radiograph as it has the same radiographic soft tissue density as the liver. Right

ventricular enlargement produced increased craniosternal contact of heart. Beside

hepatic diseases, other changes were occasionally detected in other organs such as

uroliths, splenomegaly, vertebral spondylosis in aged dogs and mild interstitial lung

patterns. Generally, abdominal radiograph was not useful for investigation of hepatic

changes in dogs with severe peritoneal effusion as liver and other organs were totally

masked by the fluid densities. Similarly, it was difficult to evaluate the entire liver as

much of the liver was silhouetted by the diaphragm, stomach and right kidney which

is in agreement with Konde and Pugh (1996). Abdominal radiography was also not

useful in dogs with acute hepatitis/hepatosis that did not develop hepatomegaly yet

due to the lack of marked hepatic changes.

The comparison of radiographic features in acute versus chronic hepatic

disease revealed that some features were more consistent for acute hepatic disease and

some for chronic hepatic disease. Hepatomegaly with sharp liver margins was always

concurrent with acute hepatitis while rounding of hepatic margins was a consistent

feature of chronic liver diseases. Ground glass appearance of the abdomen indicate

peritoneal effusion (ascites; haemoperitoneum) or peritonitis and mostly associated

with primary or secondary chronic liver diseases.

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4.12 ABDOMINAL ULTRASOUND

Ultrasonography is an excellent non-invasive way to evaluate liver

parenchyma. It is particularly useful in differentiating focal from diffuse disease,

cystic from solid masses and obstructive from non-obstructive icterus (Kumar et al

2012). In the present study, out of 140 dogs with hepatic dysfunction, 119 (85%) were

subjected to USG-examination; out of which 8 (6.72%) showed non-significant

abnormalities in hepatic parenchyma.

Twenty-seven cases (22.69%) were diagnosed with acute hepatitis/hepatosis

based on combined data from case history, clinical signs, serum biochemistry profile,

urinalysis and USG examination. In these cases, there was rapid onset of clinical signs

with severe illness and 4 to 5 fold or more elevation in liver enzymes with good body

conditions, borderline serum albumin level and absence of peritoneal fluid effusion.

Out of the 27 cases, 6 (22.22%) dogs showed normoechoic liver with uniform

echotexture and normal liver size. Liver was congested (Fig. 43) in 24 (88.88%) dogs

and hypoechoic as compared to spleen in 18 (66.66%) dogs (Fig. 44). Sharp liver

margins were seen in 26 (96.29%) dogs (Fig. 45). Hypoechogenic echotexture could

be ascribed to increased blood supply to liver due to inflammation or due to uniform

cell infiltration leading to swelling of hepatocytes, which due to its less attenuation to

ultrasonographic beam than normal hepatic parenchyma, appeared hypoechoic

(Partington and Biller 1995; Selcer 1995; Bhadwal 1997). Center (1994) also ascribed

this decrease in echogenicity to hepatocellular swelling, peripheral edema, hepatic

inflammation or congestion or hepatic sinusoidal bed distension.

Hyperechoic echotexture of hepatic parenchyma was observed in 2 (7.41%)

dogs and mixed echotexture in 1 (3.70%) case. Increased echogenicity rendered liver

to be hyperechoic as compared to spleen. In a single case (3.70%), a small quantity of

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free anechoic fluid in the peritoneal cavity (suggestive of ascites) was not enough to

be detected on palpation and survey radiography was observed during abdominal

ultrasound.

Normal liver size was seen in 10 (37.04%) of acute hepatitis scanned dogs,

whereas variable degree of hepatomegaly was detected in 17 (62.96%) adult dogs as

the liver was extending behind the costal arch which is in agreement with Pechman

(1993). Assessment of liver size was subjective and dependent upon user experience.

The left kidney was displaced caudally; which suggested hepatomegaly. One case

with acute hepatitis showed hypoechoic mass which possibly originated from the liver

and was poorly visible due to the presence of lots of gas.

Gall bladder was normal in 25 (92.59%) dogs with or without distension, but 2

(7.40%) dogs showed gall bladder sludge (Fig. 46). Spleen was normal in 25

(92.59%) cases, enlarged with uniform echotexture in 2 (7.40%) dogs and showed

multiple focal hypoechoic areas suggestive of congestion in 1 (3.70%) case. Two

(7.40%) cases showed small amount of debris in urinary bladder without evidence of

cystitis (i.e., no thickening of bladder wall). Hyperechoic renal cortex and medullary

rim sign was observed in both kidneys of 1 (3.70%) case. Mild loss of

corticomedullary junction differentiation of both kidneys with kidney size

approximately 5.1 cm was also observed in a single (3.70%) dog.

Ultrasonogram of dogs with chronic hepatitis/hepatosis (33, 27.73%) revealed

normal liver size with normal hepatic echotexture in 7 (21.21%) dogs and sharp liver

margins in 3 (9.09%) cases. Grossly enlarged liver (Fig. 47 & 48 A) was observed in

26 (78.78%) cases and variable degree of hepatic congestion (Fig. 48 B) was observed

in 11 (33.33%) cases. Alteration in the size of the liver can be due to a number of

diseases in dogs, among them, hepatocellular swelling, hepatic inflammation or

125

congestion and hepatocellular infiltration (Partington and Biller 1995, Yeager and

Mohammed 1992). Hyperechoic hepatic parenchyma (Fig. 47 & 48) was noted in 22

(66.67%) cases and hypoechoic echotexture in 3 (9.09%) dogs. Mixed hepatic

echotexture was seen in 6 (18.18%) cases, whereas normoechogenicity was observed

in 2 (6.06%) cases. Partington and Biller (1995) reported that increase in liver

echogenicity is an outcome of long term diseases such as long term cholangiohepatitis

and lymphosarcoma. The majority of chronic hepatitis/hepatosis cases (30, 90.91%)

revealed rounded liver margins (Fig. 47), whereas sharp liver borders were detected

only in 3 (9.09%) dogs. These observations are in agreement with Root (1974) and

O‟Brien (1978) who reported that diffuse hepatomegaly causes a substantial portion

of caudoventral liver margins to project beyond the costal arch and rounding of caudal

liver edges. Free anechoic abdominal fluids separating the liver lobes (Fig. 47) and

lying between liver and diaphragm was seen in 17 (51.51%) cases. Massive

accumulations of fluids cause separation of intra-abdominal organs and creating an

appearance of floating intestines, undulating and frilled smooth liver margins. Gall

bladder was found normal in all cases but sometimes distended with bile which could

be attributed to anorexia over long periods of time (Partington and Biller 1995).

Spleen was normal in 30 (90.09%) dogs and enlarged with normoechoic and

uniform echotexture in 3 (9.09%) cases. Partington and Biller (1995) also stated that

long term passive congestion most commonly from right heart diseases caused

hepatomegaly, liver hypoechogenicity, dilatation of hepatic veins and caudal vena

cava and splenomegaly. In the present study, right side CHF was also among the

causes of reactive hepatopathies (4 cases). Other USG changes associated with

chronic hepatitis/hepatosis are hypoechoic renal cortex with thickening of renal

capsule with concretions in 2 (3.03%) dog, fluid filled intestine, medullary rim sign

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and hyperechoic cortex each was detected in a single (3.03%) case. Renal impairment

can cause reactive hepatopathy.

Ultrasonogram was very useful in differentiating acute from chronic liver

disease as some features were more consistent for acute and some for chronic cases.

Overall, hepatomegaly, hepatic congestion, hypoechoic echotexture and sharp liver

margins were more consistent findings of acute liver disease; whereas, hepatomegaly,

hyperechogenicity, rounding of liver margins, ascites and undulating liver margins

were more consistent with the chronic liver disease (Table 18, Fig. 49).

Table 18: Ultrasonographic features in acute versus chronic hepatitis/hepatosis

Comparison Feature Acute hepatitis/

hepatosis

Chronic

hepatitis/

hepatosis

Liver size

Normal liver size 10 (37.04%) 7 (21.21%)

Hepatomegaly 17 (62.96%) 26 (78.78%)

Hepatic congestion Dilated blood vessels 24 (88.88%) 11 (33.33%)

Liver margins Sharpe 26 (96.29%) 3 (9.09%)

Rounded 1 (3.70%) 30 (90.91%)

Echogenicity

Normoechoic 6 (22.22%) 2 (6.06%)

Hypoechoic 18 (66.66%) 3 (9.09%)

Hyperechoic 2 (7.41%) 22 (66.66%)

Mixed echotexture 1 (3.70%) 6 (18.18%)

Ascites Anechoic peritoneal

fluids 1 (3.70%) 17 (51.51%)


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Fig. 49: Ultrasonographic features in acute versus chronic hepatitis/hepatosis

Among the scanned dogs, nine dogs had cholangiohepatitis. Size of liver in

these dogs was normal in two cases and mildly to moderately enlarged in seven cases.

Hepatic congestion and gall bladder wall thickening with or without distension was

seen in all the cases. However, double walled GB suggestive of GB oedema (Fig. 50)

was seen in one case. Generalized hyperechogenicity of hepatic parenchyma was seen

in seven cases. Partington and Biller (1995) reported that increase in liver

echogenicity is a consequence of disease like long term cholangiohepatitis and

lymphosarcoma. Thickening of gall bladder wall can be seen with cholangiohepatitis,

and cholecystitis (Nyland and Park 1983; Lamb 1991). Other observations associate

with cholangiohepatitis cases were splenomegaly in one case. Extremely distended

urinary bladder covering most of the abdomen and distended urinary bladder with

multiple small cystoliths each was observed in one case. Hydronephrosis in both

kidneys with renal cortex have focal hyperechoic area suggestive of pyelonephritis

and hypoechoic right kidney each was observed in a single case.

128

Ultrasonographic changes in dogs with cholecystitis revealed a normal

echogenicity of hepatic parenchyma with GB wall was symmetrically thickened and

sometimes double layered hyperechoic wall. The bile was anechoic, and thickening of

gall bladder wall (Fig. 51) was seen in all cases. These observations are in agreement

with other workers (Sander 1980; Mittelstaetd 1987; Spaulding 1993; Stieger and Url

2001; Vijayakumar et al 2001; Assi and Slimani 2009). Gall bladder wall may appear

as layered due to visualization of both outer an inner layers, with presence of

abdominal fluid or peripheral margins of edema in inflammatory conditions (Nyland

and Park 1983). Recently, sensitivity and specificity of diffused thickening of gall

bladder wall as an indicator of cholecystitis (inflammatory process) has become

questionable as peritoneal effusion has been found to cause pseudothickening of gall

bladder wall (Spaulding 1993). Gall bladder wall thickening can be associated with

myriad of diseases such as hepatitis, cholecystitis, cystic mucosal hyperplasia, ICH,

hypoproteinemia, pancreatitis, any form of peritoneal fluid, chronic bile duct

obstruction and most commonly with cholangiohepatitis (Jubb et al 1993, Slatter

1993, Partington and biller 1995, Selcer 1995). True thickening of gall bladder wall

has been ascribed to the increased vascular permeability and cellular infiltration.

Other abnormalities associated with cholecystitis were grossly enlarged spleen with

normoechoic echotexture in one case and enlarged congested spleen in another case.

Distended urinary bladder with echogenic materials and gas bubbles and debris

suggestive of cystitis was seen in two cases; cystoliths and collapsed bladder with

some hyperechoic foci each was observed in a single case. Free anechoic fluids in the

abdomen suggestive of ascites were seen in 3 cases.

Ultrasonogram of two cases with cholelithiasis revealed grossly enlarged and

congested liver with hyperechoic echotexture. Concretions and sludge (cellular

129

debris) along with gall bladder thickening was observed in one case (Fig. 52) and

large gall stone was detected in one case (Fig. 53). Choleliths were hyperechoic,

gravity dependent and did not produce acoustic shadow in one case, but the other case

showed a large gall bladder stone with an acoustic shadow (Fig. 53). The high

attenuation of gall stones results in the formation of an acoustic shadow on the

ultrasound scan. Inadequacies in the dynamic range of available TV display units

necessitate the use of compression-amplification signal processing which may

preclude perception of such a shadow and seriously interfere with diagnostic accuracy

(Taylor et al 1979). Although ultrasound has been demonstrated to have an accuracy

(>95%) for the identification of gallstones, stones that are too small, (usually <1mm

to cast a posterior shadow soft stones) lacking strong internal echoes (Laing 1998), or

gallstones impacted in the gallbladder neck or in the cystic duct that may not be as

readily detectable on ultrasonographic examination as they silhouette with the

surrounding echogenic bowel gas or intraperitoneal fat (Laing and Jeffrey 1983). The

thickening of gall bladder wall appeared as hypoechoic region with echogenic lines

creating a „halo‟ sign. Repositioning the animal and observing the mobility of the

echogenic materials determined a cursory appraisal of viscosity of the bile. The cause

for strands of echogenic materials though is nonspecific but more likely to be

associated with inflammatory processes (Spaulding 1993). Apart from cholelithiasis,

US examination also revealed grossly enlarged normoechoic spleen extending to mid

abdomen.

Ultrasonogram of liver in dogs with suppurative hepatitis/liver abscess

revealed normoechoic hepatic parenchyma in two cases, but FNAC of liver

confirmed suppurative process as many degenerated neutrophils with toxic changes

were observed either singly or in aggregation in the smear. Liver was hyperechoic as

130

compared to spleen in one case (Fig. 54) and multiple hyperechoic areas in the right

liver lobe measuring about 0.81x1.32 cm diameter, with a lot of ascitic fluid (Fig. 55

A & B). A mildly enlarged liver with a small hypoechoic nodule measuring 0.4 cm in

the right liver lobe was detected in one case and mixed echotexture in right hepatic

lobe was observed in one case. One dog with liver abscess showed a large anechoic

pocket in the left liver lobe (Fig. 56 A) measuring about 10 cm and about 400 ml of

sanguinopurulent fluids (Fig. 56 B) was drained from it under USG guidance (Fig. 56

C). The drained fluid was confirmed as an abscess through cytologic examination.

Multiple hypoechoic nodules measuring about 1.5x1.9 cm were seen in the caudal

and right lobes of the liver in one case (Fig. 57 A, B, C) and two hypoechoic nodules

measuring 0.78 cm and 0.55 cm in the middle lobe closer to the gall bladder in one

case. Hepatic abscesses usually develop as a result of septic embolization from an

abdominal site of bacterial infection and the left hepatic lobe usually affected (Hess

and Bunsh 2000). Hepatic abscess may have variable ultrasonographic features

depending upon its cellular compositions and duration (knode et al 1986; Partington

and Biller 1995; Bunsh 2000) and immunocompromized animals are at great risk.

Beside liver abscesses, other lesions where seen in some cases such as multiple

hypoechoic nodules in the mesentery suggestive of abscesses/metastasis, enlargement

of ileac lymph nodes, intussusception, hyperechoic kidneys, multiple concretions/

debris in urinary bladder, and anechoic pockets in the right lobe of prostate.

Ultrasonogram of 6 cases with liver cirrhosis revealed diffuse increase in

echogenicity of the liver as compared to spleen, so-called “bright liver” in all the

cases (Fig. 58). Beside this, mild congestion was seen in two cases. In all the cases

there was rounding of the liver margins. In five cases, irregularity of margins was

also detected (Fig. 59 A & B). Multiple hypoechoic cavitations/lesions with multiple

131

small nodules on the surface of liver parenchyma were observed in one case (Fig. 59

A & B). In four of the cases suffering from liver cirrhosis, distension of gall bladder

with thickened wall was also observed (Fig. 59 C). Three cases of liver cirrhosis

revealed ascites (Fig. 59 A, B, C & D). Microhepatica which is a common finding of

hepatic cirrhosis was observed in five cases. These findings are in concurrence with

Biller et al (1992) who reported that potential ultrasonographic findings with cirrhosis

included irregular liver margins, focal lesions which represented regenerating

nodules, increased parenchymal echogenicity due to increased fibrous tissue.

Moreover, the hyperechogenicity of hepatic parenchyma reported in the present

study, was also reported in both human and dogs with liver cirrhosis (Cartee 1981;

Lamb 1990). Microhepatica is a frequent finding of hepatic fibrosis or cirrhosis due

to replacement of parenchymal tissue by fibrous tissue (Partington and Biller 1995;

Johnson 2000; Hill et al 2000; McGrotty et al 2003). Normoechoic grossly enlarged

spleen was detected in a single case. Splenomegaly can occur in cirrhosis as a result

of portal hypertension. Prehepatic changes were also observed in hepatic diseases

(Partington and Biller 1995; Johnson 2000). Silva et al (2007) reported that ascites

was the most common clinical finding in dogs with hepatic cirrhosis. Ascites is

always the end stage of hepatic disease and is mostly due to presinusoidal

hypertension which develops as result of idiopathic hepatic fibrosis or canine

hepatitis (James et al 2008). In the present study, aetiologies of liver cirrhosis could

not be trace. The association of liver cirrhosis with toxic principles, parasitism, CHF

and other lesions which impairing blood flow to the liver has been proposed earlier

by Popper 1977.

Ultrasonogram of four dogs with obscured hepatopathy did not reveal any

abnormalities in hepatic parenchyma in all the cases, except; in one young (3 month

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old) dog a slightly enlarged liver with gall bladder wall thickening and containing

some debris (Fig. 60) was seen. Slightly enlarged liver in young animals could be

attributed to the small age. However, these dogs showed poikilocytosis on blood

smears and hypoalbuminaemia and hypoproteinaemia on serum biochemistry

analysis. No other changes could be detected and therefore were classified as

obscured hepatopathy. However, normal ultrasound findings do not rule out liver

disease, whereas abnormal findings may not be pathognomonic (Koyama 2004).

Ultrasonogram of liver with hepatic neoplasia revealed variable features.

Adenocarcinoma caused hepatomegaly with heterogeneous echotexture and few

anechoic cavitations in one case (Fig. 61 A & B) and tumour mass involving both

liver and spleen in one case (Fig. 62 A & B). One case of adenocarcinoma revealed

mixed echotexture in right liver lobe with a hypoechoic irregular mass originating

from spleen suggestive of nodular hyperplasia (Fig. 63 A & B). Two cases each

showed two hypoechoic focal nodules of different sizes in the liver (Fig. 64).

Moreover, enlargement of mesenteric lymph nodes suggestive of metastasis was

observed in two cases. One case of canine lymphosarcoma revealed reactive

hepatomegaly with normal hepatic echotexture, but fine needle aspiration cytology of

the lymph node confirmed lymphosarcoma. Metastatic neoplasias are common in

dogs and can originate from multiple organ systems like spleen, pancreas, mammary

gland, adrenal glands, lungs, bones, thyroid glands and GIT. Primary hepatic

neoplasms are not common in dogs but most commonly metastasized from other

organs (Crow 1985, Hammer and Sikkema 1995).

Hepatic hemangiosarcoma showed a large hypoechoic mass close to the right

liver lobe; possibly originated from the right caudate liver lobe in one case. Three

dogs with hepatic hemangiosarcoma showed multiple hypoechoic nodules of different

133

size in the liver. The nodules were involving all the hepatic lobes with the liver looks

like to be eaten up in one case and involving the right and left lobes in one case. One

case with hepatic hemangiosarcoma showed normal hepatic echotexture with

hypoechoic nodules measuring 4.4 cm in left lobes on the medial aspect (Fig. 65).

Hepatic hemangiosarcoma diagnosed in the present study was always metastasized

from spleen as the tumour mass was bigger in size than that in the liver. Johnson

(2000) stated that most common metastatic tumours are hemangiosarcoma, pancreatic

carcinoma and fibrosarcoma.

Hepatocellular carcinoma showed large irregular mass in the mid abdomen in

proximity of middle and right lobes of liver (Fig. 66), mixed hepatic echotexture with

rounded margins of the middle lobes in one case (Fig. 67) and multiple hypoechoic

nodules measuring about 1 cm and below in different lobes in one case (Fig. 68).

These findings were in line with Feeney et al (1984) and Lamb (1991) who reported

that the primary hepatic tumours could appear as large moderately circumscribed,

infiltrating masses building beyond liver margins with mixed echogenicity or as

solitary or multiple focal lesions of altered echogenicity. Other abnormalities

associated with hepatic neoplasia irrespective of the type were gall bladder wall

thickening in one case, distended gall bladder with some cellular debris in one case,

splenic neoplasia in five cases (suggestive of metastasis), and splenomegaly in one

case. Multiple concretions in urinary bladder was observed in a one case,

hydronephrosis in a single case, calcification of the kidneys with possible nephroliths

and multiple small anechoic areas in renal cortex of right kidney suggesting possible

metastasis/cystic lesion in one case. Enlargement of mesenteric lymph nodes was

observed in two cases, lots of echogenic free fluids in abdomen (haemoperitoneum/

134

peritonitis) in 3 cases, ascites in one case and multiple cystic ovarian remnants in one

case. The sensitivity of ultrasonography was useful in visualizing the peritoneal

effusion even when the fluid volume was too little and could not be appreciated by

abdominal palpation/ ballottement and X-ray. These observations are in line with Yeh

et al (1977) who revealed that ultrasound could detect up to 100 ml of ascitic fluid in

abdomen that was otherwise not appreciable on radiography. Sensitivity of the

ultrasound for detecting free fluids in peritoneal cavity was also confirmed in

previous studies (Matton and Nyland 1995; Kamonrat 2001).

Ultrasonogram of hepatic lipidosis showed grossly enlarged liver with

hyperechoic hepatic parenchyma in all the cases. One case showed multiple

hypoechoic nodules measuring 0.81 to 2.2cm in liver lobes suggestive of

abscess/neoplasia, but FNAC of the lesion revealed large focal hepatic necrosis (Fig.

69). Fatty liver was associated with hepatic congestion in one case and grossly

enlarged spleen with normal echotexture in one case. High echogenicity could be

attributed to increases in the number and intensity of the internal echoes where the

liver appears white on ultrasonograms and is difficult to differentiate from

surrounding tissue (Tharwat 2012). This could be attributed to the increased adipose

mass associated with increased adipocyte cell size. However, fatty liver observed in

the present study was secondary to diabetes mellitus.

In conclusion, US examination of liver was in line with the results of

haematology, biochemistry, urinalysis, x-ray, and fine needle aspiration in

differentiating acute from chronic hepatic disease and suspecting liver abscess/

neoplasia, liver fibrosis and cirrhosis, and other diseases in the liver that could not be

expected without US scanning.

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4.13 FINE NEEDLE ASPIRATION BIOPSY/ CYTOLOGY

Though ultrasonography has become an essential imaging tool for identifying

abnormalities of the liver parenchyma, biliary tract, and vascular system and in many

cases has replaced radiography as the initial imaging procedure in screening for liver

disease, normal ultrasound findings do not rule out liver disease, whereas abnormal

findings may not be pathognomonic (Koyama 2004). Therefore, fine-needle aspiration

or ultrasound-guided biopsy of the liver is required to obtain cytologic or histologic

samples for a specific diagnosis. Fine needle aspiration specimen usually does not

require sedation and is rarely associated with haemorrhage; thus, it is frequently

chosen for animals that are poor anaesthetic risks or have coagulopathies (Weiss and

Moritz 2002). In the present study, USG guided FNAC/FNAB was performed in 31

dogs and examined by an expert pathologist. Out of 31 samples 7 (22.58%) were not

fruitful as only pure blood could be seen during microscopic evaluation due to failure

of harvesting the hepatocytes. Failure of harvesting the hepatocytes was in majority of

cases attributed to the exaggerated amount of peritoneal fluids which rendered the

liver floating and highly movable, thus interfered with piercing of the liver. Hepatic

neoplasia was diagnosed in 9 (29.03%) cases. The high incidence of neoplasia

reported in the present study could be attributed to the chance as neoplasms of the

liver and biliary tracts are uncommon in domestic animals with frequency in the dog

varies from 0.6% to 1.3% of all neoplasms (Patnaik et al 1980) or could be attributed

to the increased and misuse of pesticides in Punjab. LeBaron et al (2014) reported

pesticide induced rodent hepatotoxicity when administered at high dietary

doses; specially, hepatocellular adenomas and carcinoma were increased. Of the nine

cases with hepatic neoplasia, hepatocellular carcinoma was diagnosed in 4 (12.90%)

dogs (Fig.70 A & B, 71), adenocarcinoma in 3 (9.68%) cases (Fig. 72, 73) and

136

hemangiosarcoma in 2 (6.45%). One case (3.23%) revealed a large cranial abdominal

mass encroaching the liver during USG examination confirmed to be a liposarcoma

and showed massive fatty change and necrosis with prominent nuclei and contains

multiple fat vacuoles (Fig.74 A & B). Neoplasias usually show nuclear pleomorphism

and vacuolated cells. One case of hepatocellular carcinoma showed central necrosis

(cavitation with necrotic debris) of the liver leading to marked compression and

descrushing of hepatic parenchyma along with marked derailment of hepatic function.

Two cases with hepatocellular carcinoma were suspected during US scanning and

later were confirmed by peritoneal fluids cytology as metastatic neoplasia, but FNAB

of liver failed to confirm the diagnosis due to failure of harvesting hepatocytes.

Cytologic evaluation is limited by the lack of architectural relations that can be

visualized in biopsy specimens. The ability to define architectural alterations within

small clusters of hepatocytes in cytologic specimens requires considerable skill and

experience, but the type of disease suspected is also important. Diseases like

suppurative hepatitis, hepatic lipidosis and malignant lymphoma are readily diagnosed

cytologically, whereas hepatocellular adenomas, hyperplastic nodules, fibrosis, and

chronic inflammation are more difficult to identify cytologically (Stockhaus and

Teske 1997). The relative diagnostic value of cytology versus histopathology for

evaluation of liver disease is controversial. Suppurative hepatitis/liver abscess was

diagnosed in 9 (30.0 %) cases as numerous neutrophils and mononuclear cells were

dominating in the smear (Fig.75). One case of suppurative hepatitis was secondary to

hepatocellular carcinoma. Bile duct hyperplasia, focal suppurative hepatitis with

hepatocellular regeneration of hepatocyte was seen with one case of adenocarcinoma

carcinoma (Fig. 76). Suppurative hepatitis along with vacular degeneration/fatty

degeneration in hepatocytes and necrotic debris with some foci showing bile duct

137

proliferation was reported in one case, possibly there was fibrosis and

cholangiohepatitis. One aspiration of liver revealed an old abscess with

fibrinopurulent exudate inside and many neutrophils (Fig. 77). One case of liver

cirrhosis with abscess (as USG examination revealed hyperechoic small liver with

irregular margins and multiple hypoechoic regeneration nodules) showed

unremarkable changes as only fatty changes, lymphocytes and neutrophils were

observed (Fig. 78). Fatty change irrespective of the cause was observed in 8 (25.81%)

dogs. One dog with hepatic lipidosis revealed hepatocellular degeneration, severe

fatty changes and necrosis with loss of nuclei and in some places, mild mononuclear

cells infiltration, RBCs and neutrophils (Fig. 79). A fatty change with suppuration and

generation suggestive of hepatitis was also seen in one case. One case of chronic

hepatitis revealed domination of lymphocytes along with few macrophages,

neutrophils and hyperplasia of hepatocytes (Fig. 80). One case of cholecystitis was

diagnosed based on the presence of biliary epithelial proliferation (Fig 81).

Table 19: Fine needle aspiration cytology/biopsy of the liver (n=31)

Sl. No Diagnosis Total

1. Failure of diagnosis 7 (22.58%)

2. Neoplasia

Hepatocellular carcinoma 4 (12.90%)

Adenocarcinoma 3 (9.68%)

Hemangiosarcoma 2 (6.45%)

Liposarcoma 1 (3.23%)

3. Suppurative hepatitis/liver abscess 9 (30.0 %)

4. Fatty change irrespective of the cause 8 (26.66%)

5. Cholecystitis 1 (3.23)


138

4.14 AETIOLOGY OF HEPATIC INSUFFICIENCY

Based on combined data from case history, clinical signs, haemato-

biochemical analysis, medical imaging, pathological findings and urinalysis,

possible causes of hepatic insufficiency encountered in the present study were

determined whenever possible and are presented in Table 20. Out of 140 dogs, 73

(52.14%) did not get a certain aetiology, although they were definitely found to have

had some kind of liver disease. Sherding (1985) reported that acute hepatic failure is

characterized by a sudden catastrophic compromise of hepatic failure and in many

cases, the inciting cause is not determined. Poldervaart et al (2009) also reviewed in

a retrospective study on primary hepatitis in dogs that aetiology of most cases of

canine hepatitis remains unknown.

Neoplasia constituted 15 (10.71%) cases with the majority metastasized from

spleen. Hammer and Sikkema (1995) reported that primary hepatic neoplasms were

not common in dogs and cats and metastasis to liver was much more common

accounting for 7-36% of dogs having cancer. Reactive hepatopathies due to Babesia

gibsoni infection was detected in 11 (7.86%) dogs and by E. canis in 5 (3.57%)

cases. High incidence of infection with haemoprotozoan parasites is due to the

endemicity of the vector ticks (Rhipicephalus) in and around Punjab. Immune

mediated haemolytic anaemia with elevation of hepatic enzymes has been associated

with babesiosis (Irizarry-Rovira et al 2001). Hepatomegaly and elevation of liver

enzymes has also been associated with E. canis (Kumar and Varshney 2006;

Niwetpathomwat et al 2006). Liver abscess constituted 6.42% of the cases (9/140).

Causes of liver abscess were always unknown due to failure of culturing the abscess

obtained through FNAC. Drug induced hepatopathy due to ivermectin overdosage

was detected in one (0.71%) case. Clinically apparent liver injury has been reported

139

after a single dose therapy with ivermectin and was characterized by a hepatocellular

pattern of serum enzyme elevations without jaundice (Veit et al 2006). Similarly,

prolonged administration of glucocorticoids (prednisolone for 2 months) was

reported in a single (0.71%) case with chronic idiopathic dermatitis. Johnson (2000)

reported that steroid administration produces vacular hepatopathy while

keratinization was noticed in dermatitis. Right sided CHF as a secondary cause of

hepatopathy was detected in 4 (2.86%) cases. Alvarez and Mukherjee (2011)

concluded that the primary pathophysiology involved in hepatic dysfunction from

heart failure is either passive congestion from increased filling pressures or low

cardiac output and the consequences of impaired perfusion. They also stated that

passive hepatic congestion due to increased central venous pressure may cause

elevations of liver enzymes and both direct and indirect serum bilirubin. Impaired

perfusion from decreased cardiac output may be associated with acute hepatocellular

necrosis with marked elevations in serum aminotransferases. Cardiogenic ischemic

hepatitis „„shock liver‟‟ may ensue following an episode of profound hypotension in

patients with acute heart failure. Diabetic mellitus, viral hepatitis and sepsis each

contributed 2.14% (3/140). Diabetes mellitus is a common cause of reactive

hepatopathy. Among viral hepatitis, 2 cases were infected with canine distemper

virus (CDV) and one case had canine adenovirus 1 (CAD-1) infection. Viral

hepatitis was rarely encountered because of regular and proper vaccination

programme that most of dog owners follow. Septicemic endometritis, suppurative

peritonitis and chronic active peritonitis was the underlying cause in one case each.

Immune mediated hemolytic anaemia and mixed causes (chronic active peritonitis

and right sided CHF) were detected in 2 (1.43%) cases each. A suspected case of

hypoadrenocorticism and chronic active peritonitis diagnosed in one case.

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Table 20: Causes of hepatic insufficiency (n=140)


SI. No Aetiology Total

1 Idiopathic 74 (52.86%)

2 Neoplasia 15 (10.71%)

3 Babesia gibsoni 11 (7.86%)

4 Liver abscess 9 (6.42%)

5 Ehrlichia canis 5 (3.57%)

6 Ivermectin overdosage 4 (2.86%)

7 Right side CHF 4 (2.86%)

8 Cholelithiasis 2 (1.43%)

9 Diabetic mellitus 3 (2.14%)

10 Viral (ICH & CD) 3 (2.14%)

11 Sepsis 3 (2.14%)

12 IMHA 2 (1.43%)

13 Mixed 2 (1.43%)

14 Hypoadrenocorticism (suspected) 1 (0.71%)

15 Chronic active peritonitis 1 (0.71%)

16 Prolonged administration of glucocorticoids 1 (0.71%)

Total 140

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4.15 THERAPEUTIC MANANGEMENT OF HEPATIC INSUFFICIENCY

IN DOGS

The primary goal of present study was to evaluate the clinical use of N-

acetylcysteine (NAC) in management of various hepatic diseases in dogs. N-

acetylcysteine is a readily available, inexpensive amino-acid derivative with four

decades of scientific validation which makes it a convenient drug and good choice.

According to Center et al (2002) low levels of the intracellular antioxidant glutathione

(GSH) values are common in necroinflammatory liver disorders, extrahepatic bile

duct occlusion, and hepatic lipidosis. N-acetylcysteine has a unique role in treatment

and prevention of many common diseases, both acute and chronic as it replenishes

levels of GSH supply and mitigates oxidative damage (De Flora et al 2001). N-

acetylcysteine also play a pivotal role in gene expression modifications which may

also help reduce the acute oxidant-provoked inflammatory response following

exercise, making vigorous activity safer and even more beneficial (Kerksick and

Willoughby 2005). N-acetylcysteine has also been widely used clinically in human

medicine for treating several diseases with marked clinical improvement observed

(Julius 2010), but there is scarce of data on its use in canine medicine. Stravitz (2008)

suggested that the administration of N-acetylcysteine should be considered in patients

with early-stage hepatic encephalopathy regardless of aetiology.

Dogs with different categories of hepatic insufficiency were divided in two

groups. One group was given conventional treatment along with NAC and the other

group was given only conventional (symptomatic) treatment. The response to the

treatment was evaluated on the basis of haematological and biochemical parameters

before and after treatment. Dogs with hepatic neoplasia and liver cirrhosis were given

supportive treatments and discarded from assessments as they had poor prognosis,

whereas dogs with cholelithiasis were referred to the department of surgery and

radiology.

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4.15.1 Efficacy of conventional therapy versus conventional therapy along with

N-acetylcysteine in dogs with acute hepatitis/hepatosis

The haemato-biochemical picture of dogs suffering from acute

hepatitis/hepatosis and their response to treatment are presented in Table 21 and 22. A

total of 37 dogs were suffering from acute hepatitis/hepatosis, out of which 22 dogs

were treated with conventional treatment and 15 with conventional treatment along

with NAC. Out of the 22 dogs which were treated with only conventional therapy, 17

(77.27%) died and the remaining 5 (22.73%) were clinically cured. Of the 15 dogs in

which NAC was added to their treatment programme, 10 (66.66%) were completely

cured within a period of 30 days and the remaining 5 (33.33%) died. During the

course of treatment, there was increase in the mean values of Hb, TEC, lymphocytes,

PCV, MCH, MCHC, platelets count, total proteins and albumin on day 8, 16 and 30

as compared to day 0, whereas mean value of TLC, neutrophils, eosinophils, BUN,

creatinine, globulins, A/G ratios, total bilirubin, ALT, AST, ALP and GGT which

were high at day 0 decreased by day 30. In general, blood glucose and cholesterol

values were fluctuated but within the normal range. However, NAC group showed

more significance in response to the treatment as compared to conventional group.

Significant (P<0.05) decrease in mean values of TLC, neutrophils, ALT and ALP

were seen in NAC group but not in the conventional one. Though increase in the

haematological indices was recorded in both groups following treatment, there was

increasing trends in NAC group as compared to conventional one but there was no

significant difference noticed. However, only MCHC were significantly increased in

NAC group indicating blood regeneration but not in conventional group.

Conventional group showed no significant increase in haematological values except in

mean TEC but still less than that observed in NAC group. Hepatic disease is often

treatable and has a predictable prognosis when a definitive diagnosis is made and

proper therapy is given (Watson 2004).

143

Table 21: Haematological changes in dogs with acute hepatitis/hepatosis following treatment

Conventional treatment Conventional + NAC

Parameter Day N Mean±SE N Mean±SE

Hb (g/dL)

0 22 10.42±0.98 15 9.18±1.12

8 9 9.59±1.17 13 9.76±1.09

16 6 9.37±1.07 11 10.51±1.08

30 5 11.96±0.46 10 12.49±0.22

TEC (106/µL)

0 22 5.13±0.48ab

15 4.21±0.54

8 9 4.32±0.41a 13 4.90±0.57

16 6 4.23±0.92a 11 5.19±0.56

30 5 5.98±0.24 b 10 6.21±0.12

TLC (103/µL)

0 22 20.80±2.85 15 25.70±4.98b

8 9 18.60±3.46 13 15.50±1.93ab

16 6 15.40±2.44 11 12.10±0.8ab

30 5 10.50±0.78 10 10.30±0.73a

N (%)

0 22 89.00±2.00b 15 87.73±2.37

b

8 9 86.78±3.36b 13 81.36±1.71

b

16 6 81.83±2.71ab

11 81.29±2.02 b

30 5 73.88±0.90 a 10 73.20±0.74

a

L (%)

0 22 10.82±1.97a 15 11.07±2.16

a

8 9 12.89±3.15ab

13 16.73±2.06ab

16 6 17.50±2.39ab

11 18.29±1.87bc

30 5 22.00±4.42b 10 23.62±1.40

c

M (%)

0 22 0.00±0.00 15 0.00±0.00

8 9 0.00±0.00 13 0.73±0.49

16 6 0.00±0.00 11 0.00±0.00

30 5 0.00±0.00 10 0.00±0.00

E (%)

0 22 0.18±0.18 15 1.20±0.70

8 9 0.33±0.33 13 1.18±0.77

16 6 0.67±0.67 11 1.14±0.77

30 5 0.80±0.80 10 0.62±0.63

PCV (%)

0 22 31.56±2.56ab

15 29.45±3.2ab

8 9 27.35±3.05a 13 29.87±3.70

ab

16 6 27.69±3.19ab

11 32.07±2.26a

30 5 36.34±1.00b 10 36.49±1.19

b

MCV (fL)

0 22 51.50±2.29 15 44.81±2.57ab

8 9 52.41±1.76 13 49.57±3.04ab

16 6 62.36±7.74 11 51.93±0.91a

30 5 59.45±4.48 10 55.34±1.99b

MCH (pg)

0 22 21.52±0.97 15 19.77±0.57

8 9 20.80±1.07 13 20.66±0.70

16 6 20.82±0.57 11 21.40±0.00

30 5 23.45±3.60 10 22.44±1.03

MCHC (g/dL)

0 22 38.41±0.97 15 36.58±1.46a

8 9 35.77±3.56 13 39.01±1.72ab

16 6 36.38±1.99 11 40.64±1.47ab

30 5 38.56±1.52 10 43.25±0.80b

Platelet (105/µL)

0 22 1.99±0.39 15 2.08±0.29

8 9 2.33±0.47 13 2.28±0.20

16 6 1.800±0.25 11 2.33±0.52

30 5 2.52±0.97 10 2.59±0.58

Figures with at least one common superscript in a row do not differ significantly (P≤0.05)

144

Table 22: Biochemical changes in dogs with acute hepatitis/hepatosis following treatment



GLU (mg/dL)

0 22 97.55±6.45 15 116.40±16.42

8 8 91.62±4.98 13 98.36±7.53

16 5 100.60±8.42 11 81.50±4.06

30 4 106.00±11.00 10 92.00±6.68

BUN (mg/dL)

0 22 56.00±14.70 15 61.13±20.14

8 8 16.75±4.96 12 23.73±6.10

16 5 18.60±5.87 10 22.29±7.93

30 3 11.33±0.67 8 13.33±0.62

Creatinine (mg/dL)

0 22 1.77±0.36 15 2.55±1.00

8 8 1.18±0.40 12 0.86±0.17

16 5 0.98±0.28 10 0.96±0.36

30 3 0.60±0.10 8 0.58±0.03

Total proteins (g/dL)

0 22 5.84±0.27 15 5.32±0.37

8 9 5.29±0.26 13 5.02±0.36

16 6 5.90±0.33 11 5.14±0.35

30 5 6.30±0.26 10 6.11±0.28

ALB (g/dL)

0 22 2.79±0.20 15 2.26±0.28

8 9 2.47±0.28 13 2.14±0.20

16 6 2.82±0.25 11 2.23±0.23

30 5 2.60±0.13 10 2.72±0.15

Globulin (g/dL)

0 22 2.99±0.17ab

15 3.39±0.19

8 9 2.82±0.21a 13 3.01±0.19

16 6 3.08±0.21ab

11 2.91±0.15

30 5 3.70±0.18b 10 2.88±0.21

A/Gratio

0 22 0.95±0.10 15 0.81±0.05

8 9 0.94±0.14 13 0.76±0.07

16 6 0.93±0.10 11 0.75±0.09

30 5 0.71±0.04 10 0.75±0.06

TB (mg/dL)

0 22 4.80±1.73 15 4.98±2.46

8 9 1.58±0.73 13 0.91±0.28

16 5 1.14±0.77 9 0.71±0.07

30 3 0.77±0.37 9 0.49±0.06

ALT (U/L)

0 22 303.32±60.20 15 347.87±68.20 b

8 9 237.44±63.85 13 164.00±31.03a

16 6 182.00±75.35 11 100.14±17.49a

30 5 75.00±24.13 10 63.62±2.55a

AST (U/L)

0 20 239.40±70.03 15 198.27±58.68

8 9 119.78±39.26 11 93.18±36.05

16 5 42.20±10.06 9 71.14±8.40

30 5 41.80±8.26 8 50.14±4.42

ALP (U/L)

0 21 809.05±104.59c 15 671.40±104.27

b

8 9 761.89±143.92bc

13 293.73±73.09a

16 6 335.67±90.01ab

10 169.86±47.30a

30 5 116.60±12.35a 9 107.75±7.77

a

GGT (U/L)

0 21 140.81±72.15 15 38.27±17.77

8 7 25.86±14.36 13 12.27±3.19

16 6 21.00±13.27 10 18.43±5.17

30 4 8.75±1.29 9 3.75±1.55

Cholesterol

(mg/dL)

0 19 198.32±32.64 14 170.43±24.08

8 7 210.00±44.41 11 153.22±27.45

16 4 241.25±36.99 9 125.60±12.84

30 4 146.50±20.50 8 122.33±2.39


145

4.15.2 Efficacy of conventional therapy versus conventional therapy along

with N-acetylcysteine in dogs with chronic hepatitis/hepatosis

The hemato-biochemical picture of dogs suffering from chronic

hepatitis/hepatosis and their response to treatment are presented in Table 23 and 24. A

total of 41 dogs were suffering from chronic hepatitis/hepatosis, out of which 27 dogs

were treated with conventional treatment and 14 with conventional treatment along

with NAC. Of the 27 dogs which received conventional treatment, 22 (81.48%) died

and the remaining 5 (18.52%) clinically recovered within 30 days. Of the 14 dogs

which were given NAC beside the conventional treatment, 10 (71.43%) clinically

recovered within a period of 30 days and the remaining 4 (28.57%) died. However,

both treatment regimens did not result in complete cure (i.e., haematological indices

and liver enzymes were closer to the reference values but did not fall within the

normal range. The group, which recieved NAC beside the conventional therapy,

showed significant (P<0.05) increase in the mean Hb, TEC, PCV and platelets count

values, and significant (P<0.05) decrease in creatinine, albumin, A/G ratios, ALT and

ALP on day 30 as compared to day 0. Similarly, these parameters also improved with

the conventional treatment but did show significant difference on day 30 as compared

to day 0. Blood glucose and cholesterol values fluctuated in both groups during the

treatment but did not show significant difference on day 0 and day 30. The values of

cholesterol and blood glucose in general were within the normal range. TLC values

decreased in both groups by day 30 as compared to day 0 but did not reach level of

significance. Neutrophils and lymphocytes were significantly (P<0.05) reduced in

both groups on day 30. MCV and MCH values were increased in both groups,

however, dogs treated with conventional treatment showed significantly (P<0.05)

146

increased values of MCV and MCH on day 30 as compared to day 0. The mean

MCHC value showed non-significant increase in both groups by day 30. The number

of dogs died of chronic hepatitis/ hepatosis was very high particularly in the group

treated only with conventional treatment as compared to the dogs which received

NAC. Sevelius (1995) reported that the mean survival of dogs with chronic non-

specific hepatitis was 36.4 months as majority of cases progress to liver cirrhosis.

Strombeck et al (1988) and Honeckman (2003) also came to a conclusion that the

prognosis for chronic hepatitis is quite variable. They also stated that dogs with end-

stage disease (hypoalbuminemia, hypoglycaemia, prolonged clotting times, and

bridging fibrosis) have poor prognosis and tend to have shorter survival times and

early diagnosis and intervention is the key to the successful treatment of dogs with

chronic hepatitis. Despite the positive results obtained from incorporation of NAC in

the treatment protocol, there was complete recovery but not cure for survived dogs

with chronic hepatitis/hepatosis. The haematological indices and liver enzymes came

closer to the reference values but never fell within the normal range, except in 4 cases

in which liver function tests and CBC parameters came within the normal range after

treatment with conventional therapy and NAC, though USG examination showed

increased echogenicity of hepatic parenchyma. Watson (2004) stated that dogs with

chronic liver diseases may recover but never cure. Unfortunately, in the course of

chronic liver disease the meticulously regulated regeneration process is imbalanced

resulting in a decreased regenerative capacity (Arends B 2008). However, the hemato-

biochemical results indicate that NAC was able to counteract lipid peroxidation and

enzyme leakage. Effectiveness of NAC is attributed to its membrane stabilizing

ability, antioxidant, anti-inflammatory and hepatoprotective properties.

147

Table 23:Haematological changes in dogs with chronic hepatitis/hepatosis following treatment

Parameter Conventional treatment Conventional + NAC

Day N Mean±SE N Mean±SE

Hb (g/dL)

0 27 8.18±0.71 14 8.89±0.78a

8 13 8.19±0.83 14 8.87±0.76a

16 7 10.07±1.13 12 10.24±0.64ab

30 5 10.30±1.86 10 11.95±0.47b

TEC (106/µL)

0 27 4.22±0.35 14 4.61±0.49 a

8 13 4.28±0.47 14 4.44±0.40 a

16 7 4.98±0.51 12 4.89±0.31ab

30 5 4.72±0.96 10 5.16±0.24 b

TLC (103/µL)

0 27 36.50±7.16 14 18.10±3.62

8 13 20.50±2.87 14 13.80±1.56

16 7 14.90±2.01 12 13.60±1.49

30 5 12.00±3.80 10 12.40±1.30

N (%)

0 27 90.19±1.78b 14 90.14±2.07

b

8 13 85.46±2.98b 14 77.50±4.77

a

16 7 79.57±2.43b 12 80.67±1.28

a

30 5 60.00±13.18a 10 76.80±2.32

a

L (%)

0 27 9.22±1.61a 14 9.71±2.06

a

8 13 12.92±2.33ab

14 17.43±2.66b

16 7 20.29±2.52b 12 18.58±1.29

b

30 5 21.20±4.03b 10 21.40±2.66

b

M (%)

0 27 0.07±0.07 14 0.14±0.14

8 13 0.15±0.15 14 0.29±0.19

16 7 0.00±0.00 12 0.83±0.83

30 5 0.00±0.00 10 0.00±0.00

E (%)

0 27 0.07±0.07 14 0.00±0.00

8 13 3.15±1.50 14 0.93±0.45

16 7 1.83±1.64 12 0.08±0.08

30 5 0.20±0.20 10 0.00±0.00

PCV (%)

0 27 24.78±1.63a 14 25.09±2.60

a

8 13 25.30±2.22ab

14 25.62±2.16a

16 7 31.71±3.0ab

12 29.64±2.84ab

30 5 32.00±6.22b 10 36.27±2.02

b

MCV (fL)

0 27 51.60±1.40a 14 59.23±3.47

8 13 53.28±2.00ab

14 59.79±2.35

16 7 48.16±3.76a 12 57.74±3.60

30 5 59.52±2.12b 10 57.21±3.54

MCH (pg)

0 27 17.55±1.67a 14 21.08±0.55

8 13 19.50±0.48ab

14 20.42±0.63

16 7 19.69±0.43ab

12 19.74±0.23

30 5 20.36±0.60b 10 19.96±0.41

MCHC (g/dL)

0 27 35.13±1.54 14 34.50±1.99

8 13 35.38±1.27 14 34.45±1.21

16 7 36.55±1.77 12 36.00±18.16

30 5 38.49±1.43 10 39.07±3.09

Platelet (105/µL)

0 27 184±0.31 14 1.46±0.43 a

8 13 1.68±0.44 14 2.38±0.95ab

16 7 1.69±0.29 12 2.69±0.86ab

30 5 3.13±0.70 10 5.90±2.93b


148

Table 24: Biochemical changes in dogs with chronic hepatitis/hepatosis following treatment


Parameter Day N mean±SE N mean±SE

GLU (mg/dL)

0 27 120.04±15.69 14 152.36±42.37

8 13 98.38±4.94 13 159.00±55.45

16 5 86.40±6.06 9 142.11±46.45

30 4 80.00±11.81 5 157.60±71.84

BUN (mg/dL)

0 27 55.37±13.56 14 39.92±11.61

8 11 20.18±3.58 13 20.00±5.03

16 5 12.00±3.11 8 16.12±3.54

30 3 16.33±3.48 4 13.50±2.26

Creatinine (mg/dL)

0 27 1.60±0.24 14 1.56±0.31b

8 13 1.38±0.22 13 1.06±0.09ab

16 5 0.82±0.12 8 0.80±0.07a

30 4 0.88±0.13 4 0.57±0.03a

Total proteins

(g/dL)

0 27 4.70±0.22 14 5.19±0.21

8 13 4.71±0.27 14 5.11±0.23

16 7 5.30±0.26 12 5.43±0.21

30 5 5.50±0.30 10 5.76±0.20

ALB (g/dL)

0 27 1.71±0.15 14 1.87±0.17a

8 13 1.75±0.18 14 2.26±0.18ab

16 7 2.08±0.23 12 2.49±0.17b

30 5 2.40±0.09 10 2.67±0.15b

Globulin (g/dL)

0 27 3.00±0.12 14 3.32±0.11

8 13 2.95±0.16 14 2.84±0.16

16 7 3.39±0.38 12 3.15±0.21

30 5 3.10±0.26 10 3.09±0.14

A/Gratio

0 27 0.58±0.04 14 0.57±0.05a

8 13 0.60±0.06 14 0.84±0.09b

16 7 0.60±0.13 12 0.78±0.09ab

30 5 0.79±0.07 10 0.88±0.07b

TB(mg/dL)

0 27 5.62±1.60 14 1.46±0.37

8 13 1.22±0.40 12 0.94±0.22

16 7 0.78±0.25 9 0.70±0.12

30 5 0.70±0.09 6 0.52±0.04

ALT (U/L)

0 27 173.48±33.97 14 212.00±62.84b

8 13 167.00±44.26 14 126.07±30.57ab

16 7 71.29±13.05 12 74.58±9.17a

30 5 77.40±24.00 10 62.40±6.43a

AST (U/L)

0 27 173.67±29.43 13 229.46±111.84

8 13 94.08±12.22 14 84.86±9.46

16 7 74.00±5.11 11 80.55±9.19

30 5 63.00±13.84 10 61.10±6.94

ALP (U/L)

0 27 461.89±75.32 13 589.92±131.89ab

8 13 420.31±103.75 14 465.64±116.91ab

16 7 174.29±59.93 10 232.50±36.61a

30 5 275.20±140.88 8 188.38±30.22a

GGT (U/L)

0 27 30.52±7.40 14 35.29±12.29

8 13 15.54±3.60 12 30.71±12.46

16 5 13.40±2.87 9 14.89±4.13

30 4 6.25±2.78 8 11.75±2.10

Cholesterol (mg/dL)

0 26 107.04±12.66 14 118.57±10.52

8 11 117.00±10.06 9 133.92±9.43

16 6 117.17±16.46 7 151.30±18.37

30 4 99.50±27.25 7 163.67±26.12


149


N-acetylcysteine in dogs with cholangiohepatitis

A total of 9 dogs were suffering from cholangiohepatitis, out of which 7

dogs received only conventional treatment and the remaining 2 were given NAC

beside the conventional treatment. All 7 dogs which were treated with

conventional treatment were suffering from chronic cholangiohepatitis and the

remaining 2 dogs which which recieved NAC had acute cholangiohepatitis. All

seven dogs with chronic cholangiohepatitis died during the course of treatment.

One dog with acute cholangiohepatitis died within one week and one cured after

one month. There was no significant difference in the mean values of haemato-

biochemical parameters in dogs with chronic cholangiohepatitis during the course

of treatment (Table 25). The prognosis is variable and depends on the severity of

the disease. Some pets may require therapy for many months to years while others

return to normal in a few days. The disease sometimes recurs after recovery.

However, chronic cases of liver disease never cure (Watson 2004) and some cases

may require cholecystectomy (O'Neill et al 2006).


N-acetylcysteine in dogs with cholecystitis

A total of 16 dogs were suffering from cholecystitis, out of which 9 (56.25%)

dogs were subjected to conventional treatment and remaining 7 (43.75%) were treated

with conventional treatment along with NAC. The hemato-biochemical picture of

dogs suffering from cholecystitis and treated with conventional treatment is presented

in Table 26. No significant differences in the hemato-biochemical parameters were

seen between day 0 and day 8 for all parameters.

150

Table 25: Haemato-biochemical changes in dogs with cholangiohepatitis

following conventional treatment

Parameter Day 0 Day 8

N Mean±SE Mean±SE

Hb (g/dL) 7 7.89±1.471 8.23±2.85

TEC (106/µL) 7 4.878±0.68 4.23±1.47

TLC (103/µL) 7 18.80±4.56 9.67±4.66

N (%) 7 89.43±2.26 85.67±3.76

L (%) 7 19.43±10.926 13±3.215

M (%) 7 2.57±2.26 0±0

E (%) 7 0.57±0.57 1.00±0.58

PCV (%) 7 21.36±3.98 25.22±9.45

MCV ((fL) 7 52.66±2.34 66.28±15.70

MCH (pg) 7 17.19±2.29 21.83±2.43

MCHC (g/dL) 7 37.24±1.78 35.03±4.79

Platelets (105/ µL) 7 1.32±0.15 2.25±0.14

GLU (mg/dL) 6 127.83±26.54 100±9.71

BUN (mg/dL) 7 30.57±6.64 32.33±6.44

Creatinine (mg/dL) 7 1.44±0.32 1.77±0.15

Total proteins (g/dL) 7 4.96±0.36 5.53±0.76

ALB (g/dL) 7 2.03±0.24 2.13±0.50

Globulin (g/dL) 7 2.93±0.25 3.40±0.70

A/Gratio 7 0.72±0.11 0.69±0.23

TB (mg/dL) 7 3.91±2.37 1.55±0.65

ALT (U/L) 7 169.71±49.19 150±97

AST (U/L) 7 127.14±24.97 128.50±81.50

ALP (U/L) 7 439.57±178.47 166±53

GGT (U/L) 6 37.33±17.61 27.5±19.50

Cholesterol 6 136.83±21.98 89±23.26


151

Table 26: Haemato-biochemical changes in dogs with cholecystitis following

conventional treatment

Parameter Day 0 Day 8

N Mean±SE N Mean±SE

Hb (g/dL) 9 7.82±1.19 5 7.08±2.06

TEC (106/µL) 9 3.99±0.64 5 3.65±1.08

TLC (103/µL) 9 24.60±5.93 5 23.20±9.49

N (%) 9 87.33±2.21 5 87.60±3.92

L (%) 9 12.00±1.86 5 12.40±3.92

M (%) 9 0.00±0.00 5 0.00±0.00

E (%) 9 0.67±0.67 5 0.00±0.00

PCV (%) 9 20.21±2.73 5 21.25±4.45

MCV (fL) 9 50.88±2.05 5 50.83±2.86

MCH (pg) 9 20.34±0.64 5 20.30±1.02

MCHC (g/dL) 9 40.29±1.40 5 36.75±2.95

Platelets (105/µL) 9 0.96±0.49 5 0.91±0.28

GLU (mg/dL) 9 119.33±13.54 5 100.40±12.81

BUN (mg/dL) 9 51.11±21.77 5 51.80±34.41

Creatinine (mg/dL) 9 1.59±0.56 5 1.30±0.57

Total proteins (g/dL) 9 4.54±0.34 5 4.86±0.37

ALB (g/dL) 9 1.50±0.16 5 1.92±0.30

Globulin (g/dL) 9 3.04±0.27 5 2.94±0.42

A/G ratio 9 0.52±0.08 5 0.75±0.20

TB (mg/dL) 9 4.42±1.50 5 1.60±0.62

ALT (U/L) 9 79.22±15.86 5 70.00±15.20

AST (U/L) 9 147.44±40.74 5 120.60±19.49

ALP (U/L) 9 574.56±166.78 5 245.60±97.39

GGT (U/L) 9 34.78±15.16 5 22.60±12.44

Cholesterol 9 121.56±17.32 5 122.00±19.36

No significant difference was observed between the day of presentation and 8thday

152

All the nine dogs died after a period of 1 weak. However, these dogs were

suffering from chronic cholecystitis. Center (2009) stated that untreated necrotizing

cholecystitis culminates in gall bladder rupture and bile peritonitis which is fatal.

Of the 7 dogs which were treated with conventional treatment plus NAC, 3

(42.86%) cured, 2 (28.57%) clinically recovered, 1 (14.29%) was euthanized due to

old age and 1(14.29%) was referred to the department of surgery and radiology as it

was complicated with splenic hemangiosarcoma.

The hemato-biochemical picture of dogs suffering from cholecystitis and

subjected to conventional treatment plus NAC is presented in Table 27 and 28. During

the course of treatment, there were increase in the mean values of Hb, TEC,

lymphocytes, eosinophils, PCV, MCH, MCHC and platelets count from day 0 through

day 30 but it did not reach level of significance. This is because anaemia associated

with the acute cases of cholecystitis was mild to moderate. Mean values of TLC and

neutrophils which were high at day 0 decreased by day 30 but did not reach

significance level as compared to day 0. Similarly, the mean values of glucose, BUN,

creatinine, total bilirubin, ALT, AST, ALP and GGT decreased by day 30. Among

these parameters only ALP revealed significant (P<0.05) decline on day 30 as

compared to day 0. Mean values of total proteins and albumin were significantly

(P<0.05) increased on day 30 as compared to day 0, whereas mean values of globulins

and A/G ratios increased but did not reach the level of significance. Mean value of

cholesterol fluctuated between day 0 and day 30 but did not differ significantly.

153

Table 27: Haematological changes in dogs with cholecystitis following treatment

with conventional treatment + NAC

Parameter Day N Mean±SE

Hb (g/dL) 0 7 10.03±2.06

8 7 9.37±1.25

16 4 8.47±0.32

30 4 11.48±0.55

TEC (106/µL) 0 7 5.15±1.07

8 7 4.92±0.54

16 4 4.75±0.66

30 4 4.92±0.28

TLC (103/µL) 0 7 20.20±6.69

8 7 19.00±5.85

16 4 17.40±6.83

30 4 14.50±3.01

N (%) 0 7 80.67±5.48ab

8 7 85.00±1.41a

16 4 79.25±2.4ab

30 4 72.25±2.32b

L (%) 0 7 18.33±4.60

8 7 17.00±2.61

16 4 17.00±4.80

30 4 25.50±1.50

E (%) 0 7 1.00± 1.00

8 7 0.00±0.00

16 4 0.25±0.25

30 4 1.75±0.85

PCV (%) 0 7 31.06±3.93

8 7 27.11±3.16

16 4 25.70±4.62

30 4 33.28±1.87

MCV (fL) 0 7 51.34±1.92

8 7 59.61±4.76

16 4 56.84±4.93

30 4 56.29±0.93

MCH (pg) 0 7 20.46±0.71

8 7 20.10±0.32

16 4 19.30±0.07

30 4 20.30±0.64

MCHC (g/dL) 0 7 39.30±1.55

8 7 34.92±2.80

16 4 34.81±3.28

30 4 36.15±1.61

Platelet (105/µL) 0 7 2.06±0.68

8 7 2.70±0.12

16 4 2.91±0.24

30 4 3.44±0.90


154

Table 28: Biochemical changes in dogs with cholecystitis following conventional

treatment + NAC Parameter Day N mean±SE

GLU (mg/dL)

0 7 102.83±13.08

8 7 91.00±10.86

16 4 79.50±4.50

30 4 76.33±1.33

BUN (mg/dL)

0 7 18.17±3.86

8 7 13.86±2.41

16 2 13.50±0.50

30 4 17.75±3.47

Creatinine (mg/dL)

0 7 0.90±0.16

8 7 0.98±0.18

16 4 0.67±0.12

30 4 0.60±0.04

Total proteins (g/dL)

0 7 4.17±0.28a

8 7 4.87±0.26ab

16 4 4.75±0.26ab

30 4 5.45±0.13b

ALB (g/dL)

0 7 1.40±0.17a

8 7 1.83±0.24ab

16 4 2.20±0.20bc

30 4 2.50±0.09 c

Globulin (g/dL)

0 7 2.77±0.36

8 7 3.03±0.29

16 4 2.55±0.29

30 4 2.95±0.06

A/Gratio

0 7 0.63±0.21

8 7 0.67±0.15

16 4 0.92±0.18

30 4 0.85±0.03

TB(mg/dL)

0 7 1.65±0.59

8 7 0.92±0.43

16 4 0.67±0.42

30 4 0.38±0.11

ALT (U/L)

0 7 88.00±41.55

8 7 84.00±36.81

16 4 69.75±41.08

30 4 45.25±17.69

AST (U/L)

0 7 142.00±70.97

8 7 85.17±27.46

16 4 91.00±28.00

30 4 71.75±16.52

ALP (U/L)

0 7 382.17±80.38b

8 7 252.83±65.96ab

16 4 221.75±70.62ab

30 4 128.00±34.20a

GGT (U/L)

0 7 65.00±31.73

8 7 42.67±14.01

16 4 19.00±5.15

30 4 12.25±4.15

Cholesterol (mg/dL)

0 7 128.00±26.21

8 6 162.00±22.91

16 4 111.00±28.36

30 4 160.75±45.29


155


N-acetylcysteine in dogs with primary hepatopathies

Ninety-five dogs were suffering from primary hepatopathies (excluding

hepatic cirrhosis and neoplasia) out of which 57 (60%) dogs were treated with

conventional treatment and remaining 38 (40%) were given conventional treatment

plus NAC. Of the 57 dogs on conventional treatment, 46 (80.7%) died and remaining

11 (19.3%) showed apparent clinical recovery with improvement in appetite and

physical activity. Of 38 dogs which received NAC, 18 (47.37%) died and remaining

20 (52.63%) were either cured or clinically recovered depending on the severity and

stage of the hepatic disease and the presence of other complications. The

haematological picture of dogs that suffered from primary hepatopathies and their

response to treatment is presented in Table 29.

The group of dogs which received NAC beside the conventional therapy

showed better results as compared to the group on conventional treatment solely.

During the course of treatment, dogs received NAC showed significant (p<0.05)

increase in the mean values of Hb, TEC, lymphocytes, PCV, and MCH on day 30 as

compared to day 0. The mean values of MCHC and platelet count were also increased

through day 30 but did not reach level of significance as compared to day 0. There

were decline in the mean values of TLC and MCV. Monocytes and eosinophils were

rarely seen and did not differ significantly from control values. Significant (P<0.05)

decline in mean TLC value was observed on day 8, 16 and 30 and in neutrophils on

day 16 and 30 as compared to day 0.

The biochemical picture of dogs suffering from primary hepatopathies and

treated with conventional treatment plus NAC is presented in Table 30.

156

Table 29: Haematological changes in dogs with primary hepatopathies following

treatment



Hb (g/dL)

0 57 8.97±0.53ab

38 9.12±0.56a

8 24 7.83±0.68a 28 9.49±0.51

a

16 18 9.75±1.06ab

23 9.93±0.56a

30 11 10.99±1.14b 20 11.73±0.49

b

TEC (106/µL)

0 57 4.50±0.25ab

38 4.93±0.30a

8 24 4.09±0.37a 28 4.96±0.29

a

16 18 5.04±0.55ab

23 4.35±0.30ab

30 11 5.66±0.59b 20 5.98±0.25

b

TLC (103/µL)

0 57 28.60±3.35b 38 27.80±2.84

b

8 24 21.30±3.18ab

28 17.60±1.71a

16 18 13.80±2.16ab

23 16.30±1.78a

30 10 10.60±1.87a 20 13.60±1.27

a

N (%)

0 57 90.54±0.86c 38 88.82±1.60

c

8 24 85.96±2.21ab

28 85.33±1.62bc

16 18 80.00±3.10b 23 81.74±1.34

ab

30 11 69.09±6.53a 20 77.05±1.35

a

L (%)

0 57 8.95±0.82a 38 10.24±1.43

a

8 24 12.50±1.82a 28 14.07±1.71

ab

16 18 19.75±2.97b 23 17.79±1.61

bc

30 11 18.73±3.19b 20 19.55±1.70

c

M (%)

0 57 0.07±0.05 38 0.08±0.06

8 24 0.17±0.12 28 0.37±0.21

16 18 0.00±0.00 23 0.00±0.00

30 11 0.00±0.00 20 0.00±0.00

E (%)

0 57 0.35±0.15 38 0.84±0.33

8 24 1.42±0.82 28 0.67±0.33

16 18 1.71±1.41 23 0.95±0.49

30 11 0.27±0.20 20 0.85±0.46

PCV (%)

0 57 24.76±1.57a 38 27.61±1.69

a

8 24 21.37±1.72a 28 27.88±1.71

ab

16 18 25.81±2.85ab

23 27.88±1.66ab

30 11 33.57±3.59b 20 32.85±1.86

b

MCV (fL)

0 57 52.11±1.29a 38 57.20±2.22

b

8 24 52.58±1.73a 28 55.07±2.14

ab

16 18 52.04±3.13a 23 48.79±3.08

a

30 10 61.11±1.56b 20 55.29±1.72

ab

MCH (pg)

0 57 20.03±0.32 38 19.03±0.44a

8 23 19.90±0.43 28 19.97±0.31ab

16 18 20.09±1.20 23 20.24±0.25b

30 11 20.53±0.52 20 20.43±0.37b

MCHC (g/dL)

0 57 39.52±1.08 38 36.88±1.53

8 24 37.34±0.99 28 38.12±1.31

16 18 39.43±1.99 23 38.28±1.34

30 11 34.30±0.77 20 38.59±1.59

Platelet

(105/µL)

0 57 2.02±0.27 38 2.13±0.30

8 24 1.60±0.29 28 2.32±0.33

16 18 2.04±0.10 23 2.45±0.14

30 11 3.30±0.57 20 3.09±0.68


157

Table 30: Biochemical changes in dogs with primary hepatopathies following treatment

Parameter Conventional treatment Conventional + NAC


GLU (mg/dL)

0 54 106.33±7.31 37 116.19±10.20

8 23 101.30±4.96 26 119.15±14.76

16 5 96.80±5.39 11 142.45±35.45

30 7 87.57±8.71 10 102.70±6.15

BUN (mg/dL)

0 55 47.88±7.92 38 38.61±8.15

8 21 18.95±2.33 26 25.40±4.82

16 4 18.50±4.21 19 24.00±7.83

30 5 13.60±2.14 11 14.56±2.35

Creatinine (mg/dL)

0 56 1.96±0.33 38 1.84±0.41

8 23 1.17±0.15 26 1.26±0.17

16 5 1.00±0.21 19 1.35±0.36

30 7 1.04±0.26 11 0.65±0.05

Total proteins

(g/dL)

0 57 5.22±0.26 38 4.88±0.19a

8 24 4.92±0.22 28 5.12±0.18ab

16 18 5.62±0.36 23 5.23±0.22ab

30 11 5.90±0.28 20 5.60±0.15b

ALB (g/dL)

0 57 2.10±0.14 38 1.85±0.13a

8 24 1.97±0.18 28 2.09±0.13ab

16 18 2.25±0.25 23 2.41±0.18bc

30 11 2.35±0.18 20 2.53±0.10c

Globulin (g/dL)

0 57 2.99±0.09 38 3.01±0.12

8 24 2.95±0.14 28 3.03±0.13

16 18 2.53±0.57 23 2.72±0.19

30 11 3.76±0.38 20 3.08±0.09

A/Gratio

0 57 0.71±0.05 38 0.64±0.05

8 24 0.71±0.08 28 0.74±0.06

16 18 0.50±0.12 23 0.85±0.11

30 11 0.64±0.09 20 0.83±0.04

TB (mg/dL)

0 55 3.86±0.74 37 3.47±1.04

8 24 1.42±0.34 27 1.04±0.23

16 9 0.40±0.08 14 0.64±0.13

30 6 0.60±0.09 10 0.45±0.06

ALT (U/L)

0 57 184.77±27.04 38 198.47±38.99b

8 24 147.29±29.58 28 120.48±18.63ab

16 18 85.00±10.77 20 87.15±11.95a

30 11 68.91±14.68 20 55.55±5.44a

AST (U/L)

0 54 213.57±42.83 38 178.65±45.28b

8 22 86.64±12.89 27 76.74±8.12ab

16 13 56.86±8.46 19 91.87±14.78ab

30 11 52.91±7.05 17 62.94±6.63a

ALP (U/L)

0 56 569.48±62.22 38 530.84±67.69b

8 24 388.75±72.52 28 345.72±63.76ab

16 14 161.50±43.97 21 244.56±41.67a

30 11 191.55±64.55 18 162.67±27.38a

GGT (U/L)

0 53 75.79±20.32 35 73.66±24.70

8 22 15.86±3.53 27 38.41±13.67

16 5 4.60±1.25 13 21.15±6.53

30 8 6.12±1.54 13 15.92±2.86

Cholesterol(mg/dL)

0 51 127.57±8.80a 34 134.53±12.12

8 20 122.40±11.22a 26 139.31±11.22

16 3 233.67±57.38b 12 151.58±17.41

30 7 144.29±27.91a 6 145.67±26.10


158

There were decrease in the mean values of BUN, creatinine, total bilirubin,

ALT, AST, ALP, and GGT. Among these parameters, significant (P<0.05) decease

was observed in the mean values of ALT and ALP on day 16 and 30 and in AST on

day 30 as compared to day 0. A non-significant decrease was also observed in mean

value of GGT on day 8, 16 and 30. Mean values of total proteins and albumin showed

significant (P<0.05) increase on day 30 as compared to day 0. Mean values of

globulins and A/G ratios also revealed increase in the mean values but did not reach

level of significance. Mean values of glucose and cholesterol showed fluctuation

between day 0 and 30 but within the normal range.

The group of dogs which received only conventional treatment also revealed

improvements in the values of haemato-biochemical parameters (Table 29 and Table

30) in survived animals. However, percentage of survived animals was higher in

group of dogs which received NAC which also indicated better improvement in

haemato-biochemical profile.


N-acetylcysteine in dogs with reactive hepatopathies

Thirty-nine dogs were suffering from reactive hepatopathies (like CRF, CHF,

peritonitis, babesiosis and ehrlichiosis), out of which 28 (71.79%) dogs were

subjected to conventional treatment and remaining 11 (28.21%) were treated with

conventional treatment plus NAC. Of the 28 dogs on conventional therapy, 22

(78.57%) died and remaining 6 (21.43%) clinically recovered. Of the eleven dogs that

received NAC, 3 (27.27%) died and remaining 8 (72.72%) were either cured or

clinically recovered. The hemato-biochemical picture of dogs suffering from reactive

hepatopathies and their response to treatment is presented in Table 31 and Table 32.

159

Table 31: Haematological changes in dogs with reactive hepatopathies following

treatment

Parameter Conventional Conventional treatment + NAC


Hb (g/dL)

0 28 9.54±0.86 11 8.55±1.17a

8 12 8.87±1.07 10 8.93±1.12ab

16 7 7.80±0.87 9 9.98±0.79ab

30 6 10.48±1.39 8 11.70±0.54b

TEC

(106/µL)

0 28 5.00±0.42 11 4.30±0.54a

8 12 4.22±0.48 10 4.39±0.58a

16 7 4.23±0.39 9 4.90±0.39ab

30 6 5.40±0.72 8 5.98±0.28b

TLC

(103/µL)

0 28 25.50±4.59 11 19.00±5.73

8 12 15.70±1.64 10 11.70±1.89

16 7 18.30±3.35 9 10.00±1.45

30 6 11.80±0.77 8 11.10±1.36

N (%)

0 28 88.68±2.02 11 87.09±2.25b

8 12 86.42±2.33 10 74.20±5.77a

16 7 85.14±2.99 9 81.22±1.30ab

30 6 80.50±3.30 8 74.88±2.97a

L (%)

0 28 13.25±3.21 11 17.27±4.83

8 12 12.83±2.22 10 19.40±2.05

16 7 14.43±2.84 9 17.67±1.21

30 6 20.50±3.98 8 23.12±2.79

M (%)

0 28 0.57±0.57 11 0.00±0.00

8 12 0.00±0.00 10 0.20±0.20

16 7 0.00±0.00 9 1.11±1.11

30 6 0.00±0.00 8 0.00±0.00

E (%)

0 28 0.00±0.00 11 0.00±0.00

8 12 2.42±1.01 10 0.80±0.61

16 7 0.71±0.57 9 0.00±0.00

30 6 0.67±0.67 8 0.50±0.50

PCV (%)

0 28 26.08±2.45ab

11 24.18±2.67a

8 12 25.50±3.07ab

10 25.31±3.33a

16 7 24.08±2.27a 9 27.82±3.23

a

30 6 36.18±2.70b 8 34.68±1.52

a

MCV (fL)

0 28 52.17±2.91 11 56.59±4.78

8 12 53.82±2.61 10 58.29±2.37

16 7 55.13±4.10 9 58.44±4.85

30 6 59.64±7.06 8 60.17±2.77

MCH (pg)

0 28 19.52±0.66 11 20.80±0.99

8 12 22.41±2.81 10 20.13±0.74

16 7 19.31±1.25 9 19.99±0.50

30 6 24.77±6.17 8 18.94±0.91

MCHC

(g/dL)

0 28 42.95±2.59b 11 35.44±2.84

8 12 34.80±1.63ab

10 35.65±1.19

16 7 33.46±2.74ab

9 60.79±24.24

30 6 31.13±4.70a 8 33.38±1.41

Platelet

(105/µL)

0 20 1.50±0.34 11 1.83±0.62

8 12 1.60±0.67 10 1.92±0.37

16 7 1.82±0.27 9 2.01±1.01

30 6 1.84±0.32 8 2.35±0.54


160

Table 32: Biochemical changes in dogs with reactive hepatopathies following treatment

Parameter Conventional treatment Conventional treatment +NAC


GLU (mg/dL) 0 28 140.18±22.45 11 148.45±52.97

8 12 94.25±5.09 10 169.40±71.92

16 5 93.20±6.31 8 194.00±106.01

30 4 81.50±6.67 8 190.00±84.86

BUN (mg/dL) 0 28 57.69±13.55 10 51.50±17.77

8 12 37.67±14.40 10 13.70±3.38

16 7 40.43±16.76 5 17.60±5.44

30 6 16.17±3.70 0 18.23±0.25

Creatinine (mg/dL) 0 28 2.36±0.54 11 1.85±0.46

8 12 1.53±0.32 9 0.74±0.09

16 6 1.58±0.45 6 0.72±0.08

30 5 1.06±0.30 1 0.50±0.02

Total proteins (g/dL) 0 28 5.05±0.25 11 4.05±4.01

8 12 5.16±0.25 10 5.07±0.31

16 7 5.33±0.18 9 5.30±0.30

30 6 5.34±0.24 8 5.92±0.29

ALB (g/dL) 0 28 2.08±0.29 11 1.80±0.19a

8 12 2.18±0.17 10 2.08±0.16ab

16 7 2.24±0.16 9 2.11±0.19ab

30 6 2.34±0.17 8 2.59±0.16b

Globulin (g/dL) 0 28 3.00±0.14 11 7.25±3.98

8 12 2.81±0.18 10 2.99±0.30

16 7 3.37±0.35 9 3.42±0.31

30 6 3.15±0.13 8 3.34±0.21

A:Gratio 0 28 0.81±0.06 11 0.49±0.06a

8 11 0.82±0.08 10 0.77±0.11b

16 7 0.60±0.14 9 0.60±0.10ab

30 6 0.70±0.07 8 0.79±0.07b

Total bilirubin

(mg/dL)

0 28 5.59±1.65 11 1.25±0.41

8 12 1.37±0.47 9 0.91±0.28

16 6 1.80±0.79 7 0.83±0.14

30 6 1.30±0.58 6 0.57±0.04

ALT (U/L) 0 28 203.3±43.66 11 304.64±77.74b

8 12 160.33±48.35 10 150.10±34.42a

16 7 191.29±73.60 9 94.44±15.57a

30 6 98.80±40.06 8 68.88±9.15a

AST (U/L) 0 26 186.00±29.14 9 185.44±96.71

8 11 116.82±26.81 9 109.78±44.53

16 7 50.00±13.00 8 172.33±30.05

30 6 47.50±11.03 7 73.50±4.77b

ALP (U/L) 0 28 672.50±102.88 11 675.50±158.15ab

8 12 441.67±130.72 10 568.20±138.01a

16 7 503.00±188.23 8 270.88±52.38a

30 6 191.83±58.53 6 203.14±32.88

GGT (U/L) 0 26 25.35±6.14 11 21.55±4.53

8 10 20.50±10.18 10 25.90±7.77

16 5 30.80±14.61 8 18.50±5.54

30 5 11.60±5.84 6 17.33±4.10

Cholesterol(mg/dL) 0 26 183.38±28.17 10 151.70±21.74

8 8 166.12±42.93 6 171.50±28.09

16 4 205.50±57.21 3 224.67±29.54

30 5 185.80±27.18 2 232.00±43.00


161

Dogs treated with conventional treatment showed a non-significant increase in

the mean values of Hb, TEC, lymphocytes, PCV, MCV, MCH, and platelets count

which were observed on day 8, 16 and 30 as compared to day 0. A significant

(P<0.05) decrease in the mean values of MCHC was observed on day 30 as compared

to day 0. There was also a non-significant decrease in the mean values of TLC,

neutrophils and eosinophils on day 30 as compared to day 0. Mean values of glucose,

BUN, creatinine, A/G ratios, TB, ALT, AST, ALP and GGT were decreased on day 8,

16 and 30. The non-significant decrease in the values of these parameters could be

attributed to the slight/moderate elevations of initial values on the day of presentation.

Mean values of total proteins and albumin were also non-significantly increased

probably for the same reason as values of these parameters did not decline

dramatically when liver became involved.

The group of dogs which received NAC revealed significant increase in Hb,

TEC and ALB values on day 30 as compared to day 0. Significant decrease in

neutrophilic count, ALT and ALP was seen on day 8, 16 and 30 as compared to day 0,

whereas AST values revealed significant decrease only on day 30. TLC, MCH,

MCHC, BUN, creatinine, globulins, total bilirubin and GGT values were non-

significantly decreased by day 30 as compared to day 0 whereas lymphocytes, PCV,

platelet count, GLU, TP, A/G ratio and cholesterol were non-significantly increased.

4.16 Prognosis

The prognosis of hepatitis in dogs is influenced by the clinical signs of disease

at the time the dog is diagnosed, as well as by the extent of liver damage that then

exists. Early diagnosed and properly treated hepatitis may be associated with

prolonged survival times. Unfortunately, most of dogs with hepatic insufficiency are

presented in quite advanced stage after development of noticeable clinical signs where

162

most of the hepatic function has been lost. The prognosis for long-term survival in

such cases is guarded to grave. Median survival time was longer in dogs with acute

hepatitis than in dogs with chronic hepatitis as liver has huge regenerative capacity in

the early stages of disease.

4.17 POST MORTEM FINDINGS IN DOGS WITH HEPATIC INSUFFICIENCY

Post mortem investigation was carried out on six dogs that died during the

course of treatment. Typical lesions were recorded in all the 6 dogs. All organs were

investigated thoroughly and histologic findings were described as morphologic

diagnoses using light microscopy (Table 33 A & B).

Case 1 (DO2-1310):

This case was diagnosed with hepatic cirrhosis antemortem. Necropsy of this

case revealed generalized congestion of vital organs (liver, kidneys, spleen, pancreas,

small intestines, diaphragm and heart) (Fig. 82 B). Gall bladder was distended with

the wall thickened (Fig. 82 A), and large mass originating from the body of spleen

measuring about 2 cm in diameter was also seen (Fig. 82 B). Urinary bladder was

collapsed. There was also mild gastritis along with haemorrhagic enteritis (Fig. 82 B).

Lung lobes revealed marbling appearance (Fig. 82 B) and kidneys showed bluish

discolouration in some areas. Moderate amount of haemoperitoneum was observed in

the peritoneal cavity and abdominal viscera were bile tinged. Abdominal and thoracic

lymph nodes were normal. Histopathology of liver showed early hepatic cirrhosis,

post necrotic collapse with loss of hepatic architecture, fibrosis and pseudolobulations

(Fig. 83 A). Kidneys revealed chronic membrano-proliferative glomerulitis, necrosis

of tubular epithelium and thickened Bowman's capsule indicating chronic renal failure

(Fig. 83 B). No pathological changes were seen in the heart. Stomach showed massive

necrosis (Fig. 83 C) and intestines were totally autolytic. Lungs revealed chronic

163

interstitial lung disease probably due to chronic renal failure with several

macrophages were seen in the alveoli (Fig. 83 D). Spleen revealed hemosiderosis,

depletion of lymphocytes and proliferation of reticuloendothelial cells. Pancreas

showed lymphocytic infiltration and fibrous tissue with architecture disrupted and

pseudolobulations indicating chronic pancreatitis and pancreatic failure (Fig. 84 E).

Haemorrhages and leakage of enzymes with fat necrosis were also seen in the

pancreas (Fig. 84 F).

Case 2 (DO2-1771):

This case was diagnosed with chronic hepatitis. Necropsy of this case showed

grossly enlarged and congested liver with distended and wall thickened gall bladder

(Fig. 85 A & B). Spleen was grossly enlarged with a few areas of focal infarcts (Fig.

85 A & B). Kidneys appeared to be congested (Fig. 85 A). Gastric mucosa was mildly

inflamed and edematous (Fig. 85 C) along with mild haemorrhagic enteritis (Fig. 85

D). Mesenteric lymph nodes were hemorrhagic. Coronary artery was also dilated.

Lungs were congested and showed both red and gray hepatisation (Fig. 85 E).

Pancreas was congested and carcass was slightly icteric. Histologically, liver showed

marked congestion and multifocal areas of chronic hepatitis (Fig. 86 A & B). Spleen

showed decrease in white pulp and relative increase in red pulp with haemorrhage

(Fig. 86 C). Lungs revealed necrotizing suppurative bronchopneumonia must

probably aspiration pneumonia. Edema with severe congestion and necrosis to the

epithelium with a lot of exudates in bronchioles suspected for aspiration pneumonia

(Fig. 86 D) were also seen. Stomach revealed mild chronic superficial gastritis

(fibroblasts with a few inflammatory cells), stomach wall edema and dilated ducts

with debris (Fig. 86 E). Intestine revealed chronic superficial enteritis (Fig. 87 F).

164

Kidneys showed mild focal interstitial nephritis with degeneration, necrosis and

sloughing of tubular epithelium (Fig. 87 G) and pancreas was congested (Fig. 87 H).

Case 3 (DO3-2584):

This case diagnosed with chronic hepatitis. Grossly, the PM showed grossly

enlarged and congested liver and spleen. Carcass and mucus membranes were

jaundiced and anaemic with some serosanguineous fluids in the peritoneal cavity (Fig.

88 A & B). There were also petechiation on the mucus membranes of the gum and

pericardium (Fig. 88 A, B & C arrowed). No other gross abnormalities were detected.

Histology of liver revealed chronic hepatitis dominated by lymphocytes along with

few macrophages and neutrophils, focal proliferation of hepatocytes (hyperplasia of

hepatocytes) and fatty change (Fig. 89 A & B). It also revealed multifocal chronic

hepatitis with granuloma formation. Multiple granulomas were seen in the liver

(suspected for Salmonella infection) composed mainly of macrophages and lymphoid

cells (Fig. 90 arrowed). Rest of the liver showed hepatocellular degenerations

including vacular degeneration and marked fatty change, hepatocellular necrosis and

intrahepatic biliary obstructions, dilated sinusoids, defective hepatocyte architecture

and hyperplasia of hepatocytes indicating chronic situation. Chronic cholecystitis with

hyperplasia of overlying epithelia with intrahepatic obstruction were also seen (Fig.

91 A & B). Kidneys revealed mild focal interstitial nephritis with degeneration,

necrosis. Lungs showed mild to moderate interstitial pneumonia with emphysema,

peribronchiolitis, activated macrophages, chronic inflammation and mild oedema

(Fig. 92). Stomach and small intestines both showed mild superficial inflammation.

Spleen revealed relative decrease in white pulp and increase in red pulp indicating

immunosuppression. Pancreas showed mild pancreatic congestion. No abnormalities

were observed in heart.

165

Case 4 (DO4-3618):

This case diagnosed with chronic active hepatitis. Grossly, the PM revealed

grossly enlarged and congested liver, pale spleen and jaundice (Fig. 93). Impression

smear of liver revealed chronic active hepatitis of diffuse type with some cells may be

immune mediated. Histopathology of liver revealed focal chronic hepatitis with

severe chronic congestion, anaemia, and accumulation of bile due to obstruction,

disruption of architecture, atrophy of cords, and degeneration and necrosis of

hepatocytes (Fig. 94). Kidneys showed chronic renal failure characterized by chronic

interstitial nephritis, metastatic calcifications, tubular necrosis and loss of tubular

epithelia (Fig. 95). Metastatic calcifications in the lungs, severe congestion, oedema

and haemorrhages (interstitial lung disease) due to chronic renal failure were also seen

(Fig. 96). Stomach showed superficial chronic gastritis with necrosis, slight erosions

and ulcerations. Intestines showed superficial chronic hyperplasia with mild enteritis

and sloughing, loss of villus epithelium and excess deposition of goblet cells. Spleen

revealed decrease in white pulp and increase in red pulp indicating

immunosuppression. Pancreas revealed mild pancreatic congestion and fat necrosis.

These observations indicate multiple organ system failure.

Case 5 (DO8-8290):

This case diagnosed with hepatic lipidosis secondary to diabetes mellitus.

Grossly, PM revealed hepatomegaly with fatty change, friable liver with distended

GB, jaundice, pancreatitis, swelling of mesenteric LNs and generalized congestion of

the carcass. Urinary bladder was distended with urine and engorged (Fig. 97).

Impression smear of the liver revealed massive fatty change with disruption and

necrosis of hepatocytes and sinusoidal congestion (Fig. 98 A & B). Histopathology

revealed massive vacular degeneration/fatty change and necrosis in the liver (Fig. 99),

166

mild focal chronic interstitial nephritis with infiltration of monocytes, marked diffuse

nephrosis indicative of ARF (Fig. 100 A & B), membranoproliferative glomerulitis

i.e., proliferation of fibroblasts, and shrinkage of glomerular tufts (Fig. 100 C).

Glomerular casts interlaced in bilirubin and hyaline casts in the renal tubular lumen

(Fig. 100 D). These observed findings indicating multiple organ system failure.

Case 6 (DO4-4787):

Histology of liver revealed chronic hepatic congestion, dilatation of sinusoids

and atrophy of hepatocytes (Fig. 101).

167

Table 33A: Post mortem changes in dogs with hepatic failure (n=6).

Cases Liver Kidney Lung Stomach Intestine Heart Spleen Pancreas

1. PM

Case 1

(DO2-1310)

Liver

cirrhosis

1. Early hepatic cirrhosis

2. Post necrotic collapse

3. Loss of hepatic

architecture

4. Fibrosis

5. Pseudolobulations

1. Chronic membrano-

proliferative-

glomerulitis

2. Necrosis of tubular

epithelium

3. Thickened Bowman's

capsule.

1. Chronic interstitial lung

disease

2. Several macrophages in

alveoli

Massive necrosis Autolysis NAD 1. Hemosiderosis

2. Depletion of

lymphocytes &

proliferation of RE cells

1. Lymphocytic

infiltration &

fibrous tissue

2. Architecture

disrupted &

pseudolobulations

indicating chronic

pancreatitis &

pancreatic failure

3. Hemorrhages

4. Leakage of

enzymes

5. Fat necrosis

2. PM

Case 2

(DO2-1771)

Chronic

hepatitis

1. Marked congestion

2. Multifocal areas of chronic

hepatitis

1. Mild focal interstitial

nephritis with

degeneration &

necrosis

2. Sloughing of tubular

epithelium

1. Necrotizing suppurative

bronchopneumonia

2. Edema with severe

congestion

3. Epithelial necrosis

4. A lot of exudates in

bronchioles suspected

for aspiration

pneumonia

1. Mild chronic

superficial gastritis

2. Fibroblasts with

a few inflammatory

cells

3. Stomach wall

edem& dilated

ducts with debris

1. Chronic

superficial

enteritis

NAD

1. Decrease in white pulp&

relative increase in red

pulp

2. Haemorrhage (blood

filled cavities)

1. Congestion

3. PM

Case 3

(DO3-2584)

Chronic

hepatitis

1. Multifocal chronic

hepatitis with granuloma

formation

2. Vacular degeneration &

marked fatty change

3. Hepatocellular necrosis

4. Intrahepatic biliary

obstructions & dilated

sinusoids

5. Defective hepatocyte

architecture & hyperplasia

of hepatocytes

6. Chronic cholecystitis with

hyperplasia of overlying

epithelia

1. Mild focal interstitial

nephritis with

degeneration, necrosis

1. Mild to moderate

interstitial pneumonia

with emphysema

2. Peribronchiolitis

3. Activated macrophages

4. Chronic inflammation

& mild edema

1. Mild superficial

gastritis

1. Mild

superficial

enteritis

NAD Relative decrease in white

pulp& increase in red pulp

indicating

immunosuppression

Mild pancreatic

congestion

168

Table 33B: Post mortem changes in dogs with hepatic failure

Cases Liver Kidney Lung Stomach Intestine Heart Spleen Pancreas

4. PM

Case 4

(DO4-3618)

Chronic

active

hepatitis

1. Focal chronic hepatitis

2. Severe chronic

congestion

3. Anemia

4. Accumulation of bile

due to obstruction

5. Disruption of

architecture

6. Atrophy of cords

7. Degeneration and

necrosis of hepatocytes

1. CRF characterized by

chronic interstitial

nephritis

2. Metastatic

calcifications

3.Tubular necrosis

4. Loss of tubular

epithelium

1. Metastatic

calcifications in

the lungs

2. Severe

congestion &

edema

3. Hemorrhages

(interstitial lung

diseasdue to

CRF)

1. Superficial

chronic gastritis

with necrosis

2. Slight erosions

& ulcerations

1. Superficial chronic

hyperplasia with

mild enteritis and

sloughing

2. Loss of villus

epithelium & excess

deposition of goblet

cells

___

1. Hemorrhage

2. Decrease in white

pulp & increase in

red pulp

1. Mild congestion

2. Fat necrosis

5. PM

Case 5

(DO8-8290)

Hepatic

lipidosis

1. Massive vacular

degeneration/

fatty change

1. Mild focal chronic

interstitial nephritis

2. Infiltration of

monocytes

3. Marked diffuse

nephrosis

4.Membranoproliferative

glomerulitis

5. Shrinkage of

glomerular tufts

6. Glomerular casts

interlaced in bilirubin

7. Hyaline casts in the

renal tubular lumen

6. PM

Case 6

(DO4-4787)

Chronic

hepatitis

1. Chronic hepatic

congestion

2. Dilatation of sinusoids

3. Atrophy of

hepatocytes.

___

___

___

___

___

___

___

CHAPTER V

SUMMARY AND CONCLUSIONS

A study on “Clinico-Pathological and Therapeutic Studies on Hepatic

Insufficiency in Dogs” was conducted on 140 dogs with the objectives of diagnosing

and categorizing various types of hepatopathies and monitoring the therapeutic

response in the clinical conditions. After recording the history and clinical signs,

blood samples were analysed for haematological indices, blood biochemistry and

haemoprotozoan parasites. The urine, peritoneal fluids and faecal samples were

examined for any abnormality. Electrocardiography, radiography and ultrasonography

and USG-guided fine needle aspiration were performed whenever required and

possible. Efforts were made to ascertain the causes of hepatic insufficiency and dogs

with hepatic insufficiency were treated symptomatically and palliatively. One group

of dogs was given N-acetylcysteine tablets in addition to the conventional therapy to

determine whether it can improve the diseased condition.

The cause of hepatic insufficiency was found to be idiopathic and could not be

traced in 74 (52.86%) cases. Primary and metastatic hepatic neoplasias were seen in

15 (10.71%) cases. Babesia gibsoni and E. canis as secondary causes of hepatic

insufficiency were diagnosed in 11 (7.86%) and 5 (3.57%) cases, respectively. Liver

abscess formed 9 (6.42%) cases. Ivermectin overdosage and right sided CHF each

caused hepatic insufficiency in 4 (2.86%) cases. Cholelithiasis was seen in 2 (1.43%)

cases. Diabetes mellitus, viral hepatitis and sepsis were detected in 3 (2.14%) cases

each. IMHA and mixed causes each were seen in 2 (1.43%). Hypoadrenocorticism,

chronic active peritonitis and prolonged administration of glucocorticoids formed

0.71% each.

170

In the present study it was observed that seventy per cent of cases (98) were

suffering from primary hepatopathies whereas thirty per cent (42) constituted reactive

hepatopathies. Out of all the cases, of hepatic dysfunction, chronic hepatitis/hepatosis

formed the largest group (30%) followed by acute hepatitis/hepatosis (26.43%). It was

followed by cholecystitis (11.43%), hepatic neoplasias (10.71%), cholangiohepatitis

and liver abscess (6.43%) each, liver cirrhosis (4.29%), obscured hepatopathy

(2.86%) and cholelithiasis (1.43%). Hepatic diseases were maximum (44.29%) in the

young age group (0-4 years), followed by middle age (4-8 years) group (35%) and

minimum in geriatric (> 8 years) dogs (20.71%). All dog breeds were prone to hepatic

insufficiency. However, in the present study Labrador breed showed the maximum

involvement (71, 50.71%) due to the higher population of this breed in and around

Punjab. Male dogs with hepatic insufficiency were more frequent than female dogs

because of higher ratio of males kept by dogs‟ owners as compared to females.

Distribution of clinical signs in different liver dysfunctions revealed

complete anorexia (45.7%), inappetence (10.7%), pale mucus membranes (37.1%),

mucus membranes congestion (17.1%), vomiting (45.7%), jaundice (32.2%),

diarrhoea (11.4%), constipation (2.1%), melena (43.57%), haematochezia (3.6%),

acholic faeces (2.9%), oliguria (33.1%), polyuria and polydipsia (23.6), pollakiuria

(5%), urinary incontinence and urine retention (0.7%) each, dark yellowish urine

(38.8), haematuria (2.1%), brownish coloured urine as well as greenish coloured

urine (0.7%) each, PU and PD ((36.4), hepatodynia (3.6%), skin bruises (6.4%),

petechiation and ecchymoses (9.28%), epistaxis (0.7%), dyspnoea (7.1%),

coughing and peripheral oedema (5%) each, ascites (35.8%), haemoperitoneum

(5%), corneal opacity i.e., blue eye syndrome (1.4%), exercise intolerance (3.5%),

halitosis (32.9%), sweet fruit mouth odour (92.1%) and oral ulceration (3.57%).

171

Some clinical signs were found to be more consistent for some particular liver

dysfunctions as evident by acute versus chronic hepatitis/hepatosis. Ascites, pale

mucus membranes, weight loss and depression were common signs of chronic

hepatitis/hepatosis. Dogs with acute hepatitis/hepatosis and cholangiohepatitis were

more consistent in signs of anorexia, fever, vomiting, jaundice dehydration,

abdominal pain on palpation and hepatomegaly.

The overall haemato-biochemical changes of dogs with hepatic dysfunction

revealed anaemia, neutrophilic leukocytosis, prolonged clotting time, azotaemia,

hypoproteinemia, hyperbilirubinemia and rise of serum liver enzymes (ALT, AST,

ALP and GGT). The cases of liver dysfunctions with jaundice depicted higher levels

of ALT, GGT and bilirubin as compared to that showing no jaundice. Similarly, acute

hepatitis could be suspected over chronic hepatitis as the former resulted in a

significantly greater rise in ALT, AST and bilirubin as compared to chronic

hepatitis/hepatosis. The acute hepatic insufficiency had higher albumin level than

globulins level as compared to the chronic insufficiency. Pre-prandial TSBAs test was

highly specific but did not reveal tendency toward sensitivity. In general, levels of

glucose, cholesterol and electrolytes were fluctuated but did not reach level of

significance in majority of cases. Obscured hepatopathy revealed poikilocytosis and

hypoproteinemia without significant changes in liver function tests.

Hepatobiliary diseases mostly associated with thrombocytopenia, though

normal platelet count as well as thrombocytosis could be seen. In addition, at least one

abnormal clotting profile was seen in all tested samples. In general, there was increase

in PT and APTT time and decrease in fibrinogen concentration. However, significant

(p<0.05) increase in APTT and significant decrease in fibrinogen were seen only in

172

cases with liver cirrhosis. Hyperfibrinogenemia was observed in B. gibsoni infected

dogs.

Peritoneal fluid analysis revealed low total protein in the ascitic fluid and was

useful for diagnosis of metastatic neoplasia, peritonitis and sepsis. On urinalysis, high

urobilirubin level greater than +2 in dogs was found to be a good indicator for

suspecting hepatobiliary disease.

Hepatic radiography and ultrasonography were very useful in diagnosing

various hepatopathies, however, with ultrasonography, detailed information pertaining

to the liver dysfunction could be obtained. Hepatomegaly, hepatic congestion i.e.,

dilated hepatic vessels, sharp liver margins and hypoechoic hepatic parenchyma were

found to be very consistent findings of acute hepatitis/hepatosis, while chronic

hepatitis/hepatosis was manifested by hepatomegaly, rounded liver margins,

hyperechoic echotexture and ascites. At the end stage of chronic hepatitis (cirrhosis),

irregular liver margins, diffused increase in echogenicity (bright liver), microhepatica,

nodules of regeneration and ascites were commonly seen. Ultrasonography was also

helpful in differentiating ascitic fluids from haemoperitonium and peritonitis as the

former was characterized by presence of only anechoic fluids in peritoneal cavity

whereas the latter was associated with the presence of echogenic particles in the

peritoneal fluids. In addition, USG-examination was very sensitive in detecting the

very small amount of fluid which could not be detected by physical examination.

Cholangiohepatitis cases showed hepatomegaly with hepatic congestion (acute cases)

or increased hepatic echotexture (chronic cases) along with distension and thickening

of gall bladder wall on ultrasonography, while radiography did not reveal any

changes. Cholecystitis cases revealed dilatation of gall bladder with the wall

thickened on ultrasonography and no information could be taken from x-ray.

173

Hepatic neoplasias and liver abscesses usually reveal hypo or hyperechoic

nodules on ultrasonography arising from liver parenchyma and surface; however,

anechoic nodules could be seen in cases of cystic tumours. In addition, hepatomegaly

with mixed echotexture was also seen in cases of liver abscess and neoplasia.

Ultrasound showed accuracy for the identification of gall stones but acoustic shadow

could not be associated with gall stones in some cases. Presence of peritoneal effusion

causes loss of serosal details which acts as a barrier of diagnosing the alterations in

abdominal organs including liver. Ultrasonographic guided fine needle aspiration

cytology/biopsy of liver was useful in approaching an accurate diagnosis when the

samples were taken from the right location of the lesion.

In the therapeutic management of hepatic insufficiency, incorporation of N-

acetylcysteine to conventional therapy enhanced clinical improvement during the

early stages of hepatic disease and helped in restoring normal haemato-biochemical

values. Histopathology of dogs revealed multiple organ systems failure. Regular

screening of apparently healthy dogs helped in early detection of hepatobiliary

diseases in acute stage.

CONCLUSIONS

Hepatobiliary diseases are prevalent in dogs of all ages but are comparatively

more frequent in the young dogs.

History of dark yellow urine with clinical presentation of jaundice and/or ascites

should be considered as a strong evidence of hepatic insufficiency.

Most of the dogs with hepatic insufficiency are presented in the advanced stages

and ascites is a poor prognostic sign.

Nonregenerative normocytic normochromic anaemia is most common in dogs

with chronic liver disease.

174

Increases in liver enzyme activities are sensitive indicators of hepatobiliary

disease but it needs to be ruled out from other extrahepatic disease.

Serum bile acids test is non-invasive and documents liver dysfunction.

Imaging of the liver, using radiography and ultrasonography is very useful in

evaluation of liver status and classification of hepatopathies. However, ultrasound

provides more data than radiographs for examination of the liver.

Fine needle aspiration of liver gives an accurate diagnosis provided that aspiration

is taken from the right location of the lesion using 23G needle.

Incorporation of N-acetylcysteine in the therapeutic management of hepatic

disease during the early stages enhances clinical improvement and helps restoring

normal haemato-biochemical values.

Regular screening of apparently healthy dogs can be helpful in early detection of

hepatobiliary diseases.

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i

ANNEXURE I

Case Report

Case No:…………………………………… Date: ………/………/…………

Owner's name & address

Name:……………………………………………………………………………………………………

Address…………………………………………………………………………………………………

Mobile No……………………………………………

Animal Spp…………, Breed…………………, Weight (kg)….…….., Age…………. Sex: M / F

Vaccination status: Vaccinated (regular, irregular), not vaccinated, Unknown

Deworming Status: Dewormed (regular, irregular), not dewormed, Unknown

Current condition: Active, Weak, very weak, Strong, Sternal recumbency, Lateral recumbency,

Weight loss, Cachexia

External parasites: Yes / No (Ticks, Fleas)

Patient’s diet (Veg, Non Veg.)

Chicken Yes / No Mutton Yes / No Bedigree Yes / No

Rice Yes / No Pulses Yes / No Curd Yes / No

Milk Yes / No Lasi Yes / No Vegetables Yes / No

Chapatti Yes / No Fruits Yes / No

History of previous illness & treatment:

…………………………………………………………………………………………………………….

.………………………………………………………………………………………………………..…

……………………….………………………………………………………………………….………

…………….……………………………………………………………………………………….……

…………………..………………………………………………………………………………………

…………………………………………………………………………………….…………………….

……………………………………………………………………………………………………………

….………………………………………………………………………………………...........................

Tentative diagnosis:

…………………………………………………………………………………………….………………

ii

Current history:

Feed intake: Normal / Inappetence/ Anorectic / Polyphagia/ Spoon feeding, Since………days

Vomiting: Yes / No Colour ……………., Frequency……………….

Hematemesis: Yes / No Frequency………………

Faeces Colour: (Brown, Bloody, Tarry Black, Yellowish, Acholic), Consistency (Firm, Soft, lose)

Constipation / Diarrhoea

Faecal ova/larvae/cysts/oocysts ………………………………

Haemoprotozoa:………………………………………………..

Urination: Colour…………, Frequency (Normal, Reduced, PU, Pollakiuria, Dysuria, Stranguria)

Water intake: Normal, PD, Reduced

Others specify: ……………………………………………………………………………………………………………

……………………………………………………………………………………………………………

Physical Examination

Rectal Temp.…………….. F H.R / P.R ……………………bpm

R.R ……………/min

Dehydration: Yes / No Percentage: …………. ………..%

MMs Color: (Normal, Icteric, Pale, congested, cyanosed).

Abdominal Palpation & ballottement:……………………. ………LN swelling: Yes / No

CRT ……………………../Sec

Exercise Intolerance: Yes / No, Coughing Yes / No Weight loss: Yes / No

Heart auscultation:

…………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

Lung osculation:

………………………………………………………………………………………………….

…………………………………….……………………………………………………………

………..……………………………………………………………………………………...…

…………………………………………………………………………………………………

iii

Ascites: Yes / No Petechiation / Ecchymoses: Yes / No

Hemoperitonium: Yes / No

Nervous manifestation: Yes / No (Alert, Lethargic, Apathy, Depressed, head pressed,

Aggression, Agitation, Disorientation, Restlessness, Tremors, Ataxia, Staggering,

Dementia, Blindness, Seizures, Circling, Convulsive, Stupor, collapse, Comatose)

Other abnormalities like:

Hepatocutaneous syndrome: Yes / No (Sores on the footpads, Foot pain, Reluctance

to rise, walk, exercise or play, Pruritus of the feet, interdigital erythema, Sores on the ear

flaps, external genitalia, oral cavity, eyes, elbows, lower abdomen.

Skin bruises: Yes / No

Unkempt hair: Yes / No

ECG findings:

...………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

…………………………………………………………………………………………………

X- Ray No ( ) & findings:

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

USG No ( ) & findings:

*Liver:……………………………………………………………………………………………..

………………………………………………………………………………………………………

….………………...…………………………………………………………………………………

………………………………………………………………………………………………………

*Gall bladder:…………………………………………………………………………………..........

…………………………………………………………………………………………..….……...

..........................................................................................................................................................

iv

*Spleen:………………………………………………………………………………………………

…………….……………………………………………………………………………………..…

…..……………………………………………………………………………………………….…

*UB:…………………………………………………………………………………………………

….....................................................................................................................................................

*Kidneys:……………………………………………………………………………………………..

……………………………………………………………………………………………………

……………………...…………………………………………………………………………….

…………………………………....................................................................................................

* Other observations (GIT, Prostate, Pancreas, Adrenals, Uterus)

………………………………………………………………………………………....................

........................................................................................................................................................

…………...……………………………………………………………………………………….

CBC Results Urinalysis Results

Hb (g/dL) DLC (%) Urobilinogen

TLC (103/µL) N Bilirubin

TEC (106/µL) L Glucose

PCV (%) M Protein

MCV(fL) E Ketones

MCH (pg) B SPG

MCHC (g/dL) Blood

Platelet (x 105)

Leucocytes

MPV Ph

Crystals

Nitrate

Interpretation:

CBC:……………………………………………………………………………………………………..

.……...……………………………………………………………………………………………………

……………………………………………………………………………………………………………

……………………………………………………………………………………………………………

……………………………………………………………………………………………………………

v

Urinalysis:……………………………………………………………………………………………….

.……………………………………………………...……………………………………………………

……………………………………………………………………………………………………………

……………………………………………………………………………………………………………

……………………………..…………………………………………………………………………….

Coagulation Parameters: PT….………(s), APTT…..………(s), Fibrinogen ……………(g/dL)

Serum Biochemistry Result Result Result Result

Glucose (g/dL)

BUN (g/dL)

Creatinine (mg/dL)

TP (g/dL)

Albumin (g/dL)

A/G ratio

TSBAs Conc. (mg/dL)

Bilirubin (mg/dL)

ALT (U/L)

AST (U/L)

ALP (U/L)

GGT (U/L)

Cholesterol (mg/dL)

Sodium (MEq/L)

Potassium (MEq/L)

PBTSBA:………………………( μmol/L)

Abdominocentesis (Peritoneal fluid analysis)

Gross examination: Transparent, Clear, Turbid, Creamy, Icteric, Serosanguinous,

Haemorrhagic

vi

Cytological examination

………………………………………………………………………………………………..…

………………………………………………………………………………………………..…

………………………………………………………………………………………………….

Microbiological examination (Culture)………………………………………………………

Total protein………….g/dL

Pathology:

FNAC/FNAB

Necropsy & histopathology

Interpretation:…………………………………………………………………………………………

……………………………………………………………………………………………………………

.………………..…………………………………………………………………………………………

Therapeutic Protocol:

Rx:

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

………………………………………………………………………………………………………

Outcome:

…………………………………………………………………………………………………………

…………………………………………………………………………………………………………

…………………………………………………………………………………………………………

…………………………………………………………………………………………………………

…………………………………………………………………………………………………………

…………………………………………………………………………………………………………

…………………………………………………………………………………………………………

VITA

Name of the student : Murad A. M. Hiblu

Fathers‟ name : Ali Mohamed Elhiblu

Mother‟s name : Mrs. Hawa Mohamed Alshushan

Nationality : Libyan

Date of birth : 18th

August, 1973

Permanent home address : Tripoli, Zanata, Libya

Educational qualification

Bachelor‟s degree : B.V.Sc.

University : Alfateh University, College of veterinary

medicine, Tripoli-Libya

Year of Award : 1997

OCPA : 76.62 %

Master degree : Master in Laboratory Animal Sciences

University : Ghent University, Belgium

Year of award : 2006

OCPA : 685/1000

Master degree : Master of Applied Microbial Systematics

University : Ghent University, Belgium

Year of award : 2008

OCPA : 616/1000

Ph.D. degree : Ph.D. in Veterinary Medicine

OCPA : 8.36/10.00

CLINICO-PATHOLOGICAL AND THERAPEUTIC STUDIES ON … · CERTFICATE – II This is to certify that...

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