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
10 Hemato-biochemical parameters in healthy and hepatic
insufficiency dogs on the day of presentation (Mean±SE)
93
11 Hemato-biochemical parameters in healthy and hepatic
insufficiency dogs on the day of presentation (Mean±SE)
97
12 Total serum bile acids concentrations in dogs with hepatic
insufficiency (n=25)
103
13 Gross examination of urine in dogs with hepatic
insufficiency (n=76)
109
14 Chemical analysis of urine in dogs with hepatic
insufficiency (n=76)
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
following treatment 144
23 Haematological changes in dogs with chronic hepatitis/hepatosis
following treatment 147
24 Biochemical changes in dogs with chronic hepatitis/hepatosis
following treatment 148
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
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)
9
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.
10
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,
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).
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.
30
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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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.
63
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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
66
Fig. 8: Previous history of dogs with hepatic insufficiency (n=140)
67
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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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
*Significant at 5% (P<0.05); ** Significant at 1% (P<0.01)
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
*Significant at 5% (P<0.05); ** Significant at 1% (P<0.01)
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
*Significant at 5% (P<0.05); ** Significant at 1% (P<0.01)
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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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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
Negative 35 (46.06%)
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%)
Negative 56 (73.68%)
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 %)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs
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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
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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%)
Figures in parenthesis indicate percentage. (n) refers to number of dogs
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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.
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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
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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
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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
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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/
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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)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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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)
Figures in parenthesis indicate percentage. (n) refers to number of dogs.
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).
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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
Conventional treatment Conventional + NAC
Parameter Day N Mean±SE N Mean±SE
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
148
Table 24: Biochemical changes in dogs with chronic hepatitis/hepatosis following treatment
Conventional treatment Conventional + NAC
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
149
4.15.3 Efficacy of conventional therapy versus conventional therapy along with
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).
4.15.4 Efficacy of conventional therapy versus conventional therapy along with
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
155
4.15.5 Efficacy of conventional therapy versus conventional therapy along with
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
Conventional treatment Conventional + NAC
Parameter Day N Mean±SE N Mean±SE
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
157
Table 30: Biochemical changes in dogs with primary hepatopathies following treatment
Parameter Conventional treatment Conventional + NAC
Day N Mean±SE N Mean±SE
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
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.
4.15.6 Efficacy of conventional therapy versus conventional therapy along with
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
Day N Mean±SE N Mean±SE
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
160
Table 32: Biochemical changes in dogs with reactive hepatopathies following treatment
Parameter Conventional treatment Conventional treatment +NAC
Day N Mean±SE N Mean±SE
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
Figures with at least one common superscript in a row do not differ significantly (P≤0.05)
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