MUHAMMAD AHSAN ALI RANAprr.hec.gov.pk/jspui/bitstream/123456789/2915/1/Muhammad...2016/05/26 · Maria Ahsan who at times could not get my full attention during the study period but

EPIDEMIOLOGY, SEROLOGY AND THERAPEUTIC

TRIALS OF FASCIOLOSIS IN CROSSBRED COWS

THE THESES SUBMITTED TO

THE UNIVERSITY OF THE PUNJAB

LAHORE IN FULFILLMENT OF THE REQUIREMENTS FOR

THE DEGREE OF

DOCTOR OF PHILOSOPHY IN ZOOLOGY

BY

MUHAMMAD AHSAN ALI RANA

DEPARTMENT OF ZOOLOGY

UNIVERSITY OF THE PUNJAB,

LAHORE, PAKISTAN.

DECEMBER, 2015

DEDICATION

Dedicated to my beloved (late) mother, family members,

Particularly my father, wife and daughter, who always encouraged and

prayed

for me in achieving the aspiration of life.

CERTIFICATE

This is to certify that the research work described in this thesis is the original work of the

author and has been carried out under our direct supervision. We have personally gone

through all of the data/results/materials, reported in the manuscript, and certify their

correctness/ authenticity. We further certify that the materials included in this thesis have

not been used in part or full in a manuscript already submitted or in the process of

submission in partial/complete fulfillment of the award of any other degree from any

other institution. We also certify that the thesis has been prepared under our supervision

according to the prescribed format and we endorse its evaluation for the award of Ph. D.

degree through the official procedures of the University.

Here, thesis is in pure academic language and it is free from typos and grammatical

errors.

Supervisors

1. Dr. Nabila Roohi

Associate Professor

Department of Zoology,

University of the Punjab,

Lahore.

2. Prof. Dr. Azhar Maqbool

Chairman,

Department of Parasitology,

University of Veterinary and

Animal Sciences

Lahore, Pakistan

DECLARATION CERTIFICATE

It is hereby declared that I am submitting my Ph.D Thesis entitled “Epidemiology,

Serology and Therapeutic trials of Fasciolosis in Crossbred Cows”. I have conducted

my research work under the supervision of Dr. Nabila Roohi, Associate Professor,

Department of Zoology and Professor Dr. Azhar Maqbool, Chairman, Department of

Parasitology, University of Veterinary and Animal Sciences, Lahore.

(Muhammad Ahsan Ali Rana)

Ph.D. Scholar

ACKNOWLEDGEMENT

All the praises to God Almighty who is the creator of heaven and earth and is

most benevolent, merciful and compassionate who gave me courage and potential to

pursue this goal.

I would like to express my deepest and heartfelt gratitude to my supervisor Dr.

Nabila Roohi for facilitating me with her valuable guidance and affable ambience to

fulfill the desired tasks and experimental work. Her way of teaching eased me in

understanding even the toughest concepts of latest research work. I appreciate the

meticulous approach and guidance rendered by Professor Dr Azhar Maqbool, my co-

supervisor in the accomplishment of research and thesis work. His cheerful personality

had been a constant source of inspiration and encouragement for me during this course of

study.

I express my heartfelt gratitude to Dr. Javed Iqbal Qazi, Chairman Department of

Zoology University of Punjab Lahore for his support and provision of all the privilege in

the completion of this research work.

My sincere thanks also goes to Major General Imtiaz Hussain Sherazi, HI(M),

Sitara-i-Eisaar, and Major General Jamil Rehmat Vance HI(M), who provided me an

opportunity to join PhD, and encouraged me to fulfill this uphill task as without their

precious support it would not be possible to conduct this research..

I am thankful to Brigadier Asad Ullah, Brigadier Rana Faheem Sakhawat Ali and

Lieutenant Colonel Muhammad Ali Raza who encouraged me and provided all the

assistance required to complete my long study. Colonel Muhammad Amjad Khan is a

gem of person who was always helpful and assisted all the times with cheering face

during the course of my study. My thanks to Mr Maqsood Hussain, laboratory supervisor

of Physiology & Endocrinology lab Punjab University Lahore.

I would like to express my heartfelt gratitude to Muhammad Anwar Senior Clerk

and Ghulam Murtaza Junior Clerk to combat the tense moments of compiling the research

work. I have no words to express my love and gratitude to my father who always prayed

for my success in the life. I am thankful to my wife, whose tireless and prayers has

enabled me to accomplish this tedious task. Her continuous support resulted in timely

completion of my study. I display my heartfelt loving passions to my lovely daughter

Maria Ahsan who at times could not get my full attention during the study period but

always remained cheerful. , I would like to thank my brothers and sisters for supporting

me spiritually throughout writing this thesis and my life in general.

My mother, may God bless her soul, has always remained a source of inspiration

for me and still I find him around me.

As a serving Army officer, performing my official duties and at the same time

enjoying the status of a university student had been a wonderful experience. Regards for

all of my lab fellows and I wish them all the successes in their future endeavors.

Lieutenant Colonel Muhammad Ahsan Ali Rana

CONTENTS

Chapter

No

Title Page

No

- List of Tables i

- List of Figures vi

- List of Abbreviations xi

-` Abstract xi

1 Prevalence of fasciolosis in crossbred cows 1

1.1 Summary 1

1.2 Introduction 1

1.3 Materials and methods 5

1.4 Results 8

1.5 Discussion 50

1.6 Concluding remarks 53

2 Prevalence and ecology of various genra of snails in Punjab 54

2.1 Summary 54

2.2 introduction 54


2.4 Results 57

2.5 Discussion 76


3 Prevalence of fasciolosis in relation to meteorological parameters

80

3.1 Summary 80

3.2 Introduction 80


3.4 Results 83

3.5 Discussion 98


4 Therapeutic trails with herbal and allopathic drugs against

fasciolosis in crossbred cows

101

4.1 Summary 101

4.2 Introduction 101


4.4 Results 105

4.5 Discussion 138


5 Serum enzymes and electrolyte analysis in crossbred cows

infected with fasciolosis

142

5.1 Summary 142

5.2 Introduction 142


5.4 Results 144

5.5 Discussion 151


5.7 Control programme 152

6 References 153

- Appendix – A 183

- Appendix – B 186

- Appendix – C 190

- Appendix – D 191

- Appendix – E 194

i

LIST OF TABLES

Table

No

Titles Page

No

1.1 Prevalence of fasciolosis in slaughtered crossbred cows of Lahore

district 22

1.2 Prevalence of fasciolosis in crossbred cows at farms of Lahore district 23

1.3 Prevalence of fasciolosis in crossbred cows at veterinary hospitals of

Lahore district 24

1.4 Prevalence of fasciolosis in household crossbred cows of Lahore district 25

1.5 Prevalence of fasciolosis in slaughtered crossbred cows of Sialkot

district 26

1.6 Prevalence of fasciolosis in crossbred cows at farms of Sialkot district 27

1.7 Prevalence of fasciolosis in crossbred cows at veterinary hospital of

Sialkot district 28

1.8 Prevalence of fasciolosis in household crossbred cows of Sialkot district 29

1.9 Prevalence of fasciolosis in slaughtered crossbred cows of Attock district 30

1.10 Prevalence of fasciolosis in crossbred cows at farms of Attock district 31


Attock district 32

1.12 Prevalence of fasciolosis in household crossbred cows of Attock district 33

1.13 Prevalence of fasciolosis in slaughtered crossbred cows of Multan

district 34

1.14 Prevalence of fasciolosis in crossbred cows at farms of Multan district 35


Multan district 36

1.16 Prevalence of fasciolosis in household crossbred cows of Multan district 37

1.17 Prevalence of fasciolosis in slaughtered crossbred cows in four district 38

1.18 Prevalence of fasciolosis in crossbred cows at farms in four districts 39

1.19 Prevalence of fasciolosis in crossbred cows at veterinary hospital in four

districts 40

1.20 Prevalence of fasciolosis in household crossbred cows in four districts 41

1.21 Season wise prevalence of fasciolosis in crossbred cows in four districts 42

1.22 Sex wise prevalence of fasciolosis in crossbred cows in four districts 43

ii

1.23 Age wise prevalence of fasciolosis in crossbred cows in four districts 44

1.24 Prevalence of fasciolosis in crossbred cows in four district 45

1.25 Prevalence of fasciolosis in lactating and non-lactating crossbred cows at

farms in four districts 46

2.1 Month-wise prevalence (%) of infection of different snails at Lahore

district from November 2012 - October 2013 61

2.2 Month-wise prevalence (%) of infection of different snails at Sialkot


2.3 Month-wise prevalence (%) of infection of different snails at Attock


2.4 Month-wise prevalence (%) of infection of different snails at Multan


2.5 Season-wise prevalence (%) of infection of different snails at Lahore


2.6 Season- wise prevalence (%) of infection of different snails at Sialkot


2.7 Season- wise prevalence (%) of infection of different snails at Attock


2.8 Season-wise prevalence (%) of infection of different snails at Multan


2.9 District-wise prevalence of different snails in four districts 69

2.10 Season-wise prevalence of different snails at four districts 70

3.1 Average month-wise temp, humidity, rainfall and pan evaporation at

Lahore from November 2012 to October 2013 85


Sialkot from November 2012 to October 2013 86


Attock from November 2012 to October 2013 87


Multan from November 2012 to October 2013 88

3.5 Prevalence of fasciolosis infection with reference to environmental

temperature in four districts 89

3.6 Maximum and minimum prevalence of fasciolosis infection in relation to 90

iii

temp in four districts

3.7 Prevalence of fasciolosis in relation to relative humidity in four districts 91

3.8 Maximum / minimum prevalence of fasciolosis infection in relation to

relative humidity in four districts 92

3.9 Prevalence of fasciolosis infection in relation to total rain fall in four

districts 93

3.10 Maximum / minimum prevalence of fasciolosis infection in relation to

rainfall in four districts 94

3.11 Prevalence of fasciolosis infection with reference to pan evaporation

(mm) in four districts 95

3.12 Maximum and minimum prevalence of fasciolosis infection with

reference to pan evaporation (mm) in four districts 96

3.13 Overall maximum / minimum prevalence of fasciolosis infection in

relation to meteorological factors in Punjab 97

4.1 Egg per gram (faeces) on different days in crossbred cows at dose rate of

80 mg / kg treated by N. sativa (Gp-A1) 112






80 mg / kg treated by Fumaria parviflora (Gp-B1) 115


100 mg / kg treated by Fumaria parviflora (Gp-B2 116


120 mg / kg treated by Fumaria parviflora (Gp-B3) 117


80 mg / kg treated by Flemingia macrophylla (Gp-C1) 118


100 mg / kg treated by Flemingia macrophylla (Gp-C2 119

4.9 Egg per gram (faeces) on different days in crossbred cows at dose rate

of 120 mg / kg treated by Flemingia macrophylla (Gp-C3) 120

4.10 Egg per gram (faeces) on different days in crossbred cows at dose rate of 121

iv

10 mg / kg treated by Triclabendazole (Gp-D

4.11 Egg per gram (faeces) at different days in fasciolosis positive untreated

cows control group (Gp-E) 122

4.12 Efficacy % at dose rate of 80 mg / kg treated by Nigella sativa (Gp-A1) 123

4.13 Efficacy % at dose rate of 100 mg / kg treated by Nigella sativa (Gp-A2 124

4.14 Efficacy % at dose rate of 120 mg / kg treated by Nigella sativa (Gp-A3) 125

4.15 Efficacy % at dose rate of 80 mg / kg treated by Fumaria parviflora

(Gp-B1) 126


(Gp-B2 127


(Gp-B3) 128

4.18 Efficacy % at dose rate of 80 mg / kg treated by Flemingia macrophylla

(Gp-C1) 129


(Gp-C2) 130


(Gp-C3) 131

4.21 Efficacy % at dose rate of 10 mg / kg treated by triclabendazole (Gp-D) 132

4.22 Corporative efficiency of different herbal and allopathic drugs among

each other and with control group against Fasciolosis 133

4.23 Milk yield at different dose levels on pre and post treatment with Nigella

sativa

135

4.24 Milk yield at different dose levels on pre and post treatment with

Fumaria parviflora 135

4.25 Milk yield at different dose levels on pre and post treatment with

Flemingia macrophylla 136

4.26 Milk yield at 10 mg/kg on pre and post treatment with triclabendazole

(Gp-D)

136

4.27 Milk yield pre and post treatment with three herbal and one allopathic

drugs at different dose levels

137

5.1 Serum enzyme activities in infected cases of fasciolosis on pre and post

treatment with triclabendazole 10 mg/kg body weight

146

v

5.2 Serum electrolytes activities in infected cases of fasciolosis on pre and

post treatment with triclabendazole10 mg/kg body weight

148

vi

LIST OF FIGURES

Figure

No

Titles Page

No

1.1 Map showing epidemiological study area of fasciolosis in four

districts of Punjab province 20

1.2 Life cycle of Fasciola gigantica in crossbred cows (final host) 21

1.3 Overall season wise prevalence of fasciolosis in crossbred cows in

four districts from November 2012 to October 2013 47

1.4 Overall district wise prevalence of fasciolosis in crossbred cows in


1.5 Overall sex wise prevalence of fasciolosis in crossbred cows in four

districts from November 2012 to October 2013 48

1.6 Overall age-wise prevalence of fasciolosis in crossbred cows in four

districts from November 2012 to October 2013 48

1.7 Overall month wise prevalence of fasciolosis in crossbred cows in


2.1 Showing month-wise prevalence of different snails at Lahore district 71

2.2 Showing season-wise prevalence of different snails at Lahore district 71

2.3 Showing month-wise prevalence of different snails at Sialkot district 72

2.4 Showing season-wise prevalence of different snails at Sialkot district 72

2.5 Showing month-wise prevalence of different snails at Attock district 73

2.6 Showing season-wise prevalence of different snails at Attock district 73

2.7 Showing month-wise prevalence of different snails at Multan district 74

2.8 Showing season-wise prevalence of different snails at Multan district 74

2.9 Showing district-wise overall prevalence of different snails in Punjab

province 75

2.10 Showing season-wise overall prevalence of different snails in Punjab

province 75

3.1 Graph showing maximum and minimum prevalence of fasciolosis in

relation to environmental temp in four districts 90


relation to environmental humidity in four districts 92

3.3 Graph showing maximum and minimum prevalence of fasciolosis in 94

vii

relation to rainfall in four districts


relation to pan evaporation (mm) in four districts 96

4.1 EPG reduction of control group without any treatment 122

4.2 Graphical representation of Nigella sativa 80 mg/kg (Gp A1) 123

4.3 Graphical representation of Nigella sativa 100 mg/kg (Gp A2) 124

4.4 Graphical representation of Nigella sativa 120mg/kg body weight (Gp

A-3) 125

4.5 Graphical comparison of Nigella sativa 80,100 and 120 mg/kg with

Triclabendazole 10 mg /kg 125

4.6 Graphical representation of Fumaria parviflora 80 mg/kg (Gp B1) 126



4.9 Graphical representation of Fumaria parviflora 80,100 and 120 mg/kg

with triclabendazole 10 mg /kg 128

4.10 Graphical representation of Flemingia macrophylla80 mg/kg (Gp C1) 129

4.11 Graphical representation of Flemingia macrophylla100 mg/kg (Gp-

C2) 130

4.12 Graphical representation of Flemingia macrophylla 120 mg/kg (Gp-

C3) 131

4.13 Graphical representation of Flemingia macrophylla 80, 100 and 120

mg/kg with triclabendazole 10 mg /kg 131

4.14 Graphical representation of triclabendazole 10mg/kg (Gp- D) 132

4.15 Graphical representation of triclabendazole 10 mg/kg with control

group 132

4.16 Corporative efficiency of different herbal and allopathic drugs among

each other and with control group against fasciolosis 134

5.1 Graph showing values of SGPT before and after treatment 147

5.2 Graph showing values of SGOT before and after treatment 147

5.3 Graph showing values of LDH before and after treatment 147

5.4 Graph showing values of sodium before and after treatment 149

5.5 Graph showing values of potassium before and after treatment 149

viii

5.6 Graph showing values of calcium before and after treatment 150

5.7 Graph showing values of magnesium before and after treatment 150

ix

LIST OF ABBREVIATIONS

A/G Albuminglobuing

ANOVA Analysis of variance

Bella Bellamya

Buli Bulinus

DNA Deoxyribo nuclic acid

ELISA Enzyme linked immunosorbant assay

FEC Faecal egg count

GDP Gross domestic product

Gm/dL Grams per deciliter

Gyr Gyraulus

Hb Haemoglobin

IgG Immunoglobulin g

IGS Intergenic spacer

Indo Indoplanorbis

ITS Internal transcribed spacer

LDH Lectate dehydrogenase

Lym Lymnae

M Million

MCHC Mean corpuscular haemoglobin

MCV Mean corpuscular volume

Meq/L Miliequalent per liter

mg/kg Milligram per kilogram

mg/L Milgram per liter

mm3 Cubic millimeter

mmol/L Millimole per litre

NSAID Non steroidal anti inflammatory drug

Onc Oncomelania

PCV Packed cell volume

Phy Physa

PI Post infection

PP Prepatent period

x

PU Punjab university

RBC Red blood cell

rpm Revolutions per minute

SAP Serum alkaline phosphatase

SEM Standard error mean

SGOT Serum glutamic oxaloacetic transaminase

SGPT Serum glutamic pyruvic transaminase

TLC Total leukocyte count

Temp Temperature

U/L Unit per liter

u/L Units/Litre

uL Mico liter

Um Micro meter

UVAS University of veterinary and animal sciences

WBC White blood cell

xi

ABSTRACT

The present study consists of five chapters.

Chapter 1: Deals in the Epidemiology of Fasciolosis in crossbred cattle. It was

undertaken at slaughter houses, livestock farms, veterinary hospitals and in household

crossbred cows under different climatic conditions in four districts of Punjab Province.

Infection rate 18.75%, 15.17%, 15.67% and 13.50% at slaughter houses, livestock farms,

veterinary hospitals and in household crossbred cows was recorded respectively.

Overall the highest season wise prevalence was recorded 36.50% during autumn

and the lowest was recorded 7.88% during summer season. It was also observed that the

highest infection rates were recorded in older crossbred cows (above two years of age)

than younger (below two years of age). Sex wise prevalence indicated that males were

commonly more affected than females. Prevalence was the highest at Sialkot 17.92%

followed by Lahore 17.67%, Attock 17.25% and Multan 10.25%.

Chapter 2: Deals in the prevalence and ecology of snails. In the present study seven

snail genus belonging to Pulmonata and Prosobranchia classes were collected from four

districts (Lahore, Sialkot, Attock and Multan) of Punjab Province. These genus were

namely Lymnaea, Indoplanorbis, Bulinus, Physa, Gyraulus, Bellamaya and

Oncomelania. District wise prevalence of snails was the highest at Sialkot 16.34%

followed by Attock 14.15%, Lahore 11.53% and Multan 9.33%. Season wise prevalence

of snails was higher in Summer season and Autumn followed by Spring and lowest

during Winter. Factors affecting snails population were also studied under field

conditions including temperature humidity, rainfall and pan evaporation.

Chapter 3: Deals in the role of meteorological data in the prevalence of parasites and

snails. Temperature plays very vital role in the onset of disease. Temperature has affects

on the metabolic processes of both, snails host and parasite, thus interfering with parasite

reproduction within the snail, snail growth and snail survival rate development of various

larval stages of fasciolosis takes place within and outside of snail. At temperature 100C

little development of larval stages of parasites takes place. No cercarial transmission

occurs at 50C. The ideal temperature range is 22 – 25

0C where development within the

snail takes place in an efficient manner similarly humidity range from 55 – 70% is ideal

for the cercariae and snail. Rainfall is very important to complete the life cycle of

xii

Fasciolosis and also it helps in the spread of cercariae from one place to other. The role of

pan evaporation has also been discussed by various workers.

Chapter 4: Deals in the therapeutic trials against Fasciolosis in crossbred cows. For

this purpose a total of one hundred and ten crossbred cows were used in eleven controlled

experiments to compare the efficacy of certain indigenous herbal drugs, including Nigella

sativa seeds, Fumaria parviflora aerial parts, Flemingia macrophylla aerial parts and with

one allopathic drug Triclabendazole against Fasciolosis. Efficacy was quantified by

determining the difference of eggs count per gram faeces (EPG) on pre and post

treatment.

Firstly, with Nigella sativa seeds, on 1st dose of 80, 100 and 120 mg/kg body weight at

18th

day the EPG reduced to 46.34%, 51.35% and 66.67% respectively and after the 2nd

dose at 28th

day the respective reduction in EPG was 82.93%, 89.19% and 97.44%.

Secondly, with Fumaria parviflora on 1st dose of 80, 100 and 120 mg/kg body weight at

18th

day the EPG reduced to 53.66%, 56.10% and 50% respectively and after the 2nd

dose

at 28th


Thirdly, with Flemingia macrophylla on 1st dose of 80, 100 and 120 mg/kg body weight

at 18th

day the EPG reduced to 64.10%, 73.17% and 74.36% respectively and the after the

2nd

dose at 28th


Fourthly, with Triclabendazole suspension on 1st dose of 10 mg/kg body weight at 18

th

day the EPG reduced to 67.50% and on 2nd

dose at 28th

day the EPG reduced up to

97.50%.

Chapter 5: These herbal drugs played a vital role on various blood parameters when

treatment was rendered to infected animals. During infection serum enzymes were

recorded as SGOT (85.37u/L), SGPT (45.40u/L) and LDH (4157.83u/L) which were

decreased to approximately normal values after treatment as SGOT (78.50u/L), SGPT

(43.97u/L) and LDH (4042.47u/L) respectively.

Whereas, some increase in serum electrolyte of infected animals was observed i.e. sodium

(140 mmol/L) and magnesium (0.16 mmol/L) but a little increase in calcium (0.56

mmol/L) and potassium (5.82 mmol/L). When infected animals were treated with these

herbal and allopathic drugs it played a vital role on various blood parameters and reduced

the elevated values of serum electrolytes as sodium (134.43 mmol/L), magnesium (0.15

mmol/L), calcium (0.54 mmol/L) and potassium (5.07 mmol/L).

Chapter 6: References.

1

1.1 SUMMARY

Fascioliasis is one of the parasitic and most pathogenic diseases in domestic

animals which cause heavy economic losses to livestock industry to the tune of several

million rupees annually. Epidemiological study was performed at slaughter houses,

livestock farms, veterinary hospitals and household crossbred cows with different

climatic conditions at four districts of Punjab province. The infection rate 24.83%,

22.08%, 13.58% and 10.00% in crossbred cows in slaughter houses, livestock farms,

veterinary hospitals and household was recorded respectively. Overall the highest season

wise prevalence was recorded 29.88% during autumn and the lowest 12.19% in summer

season. It was also observed that the infection rate was higher in older crossbred cows

(above two years of age) than younger (below two years of age). Whereas male and

female prevalence showed that female were more commonly effected than that of males.

The prevalence was the highest at Sialkot 14.52%, followed by Attock 12.57%, Lahore

10.25% and the lowest at Multan 8.41%.

1.2 INTRODUCTION

The cattle association with mankind is since ancient times. These are very

important productive animals as their milk and meat is an essential constituent of the

human diet. They also serve as a material source for the leather industry and provide

organic fertilizer as soil additive. According to Pakistan Livestock Census the population

of cattle, buffaloes, sheep and goat in Pakistan is about 29.55, 27.33, 26.48 and 53.78

million respectively. Out of the total cattle population, 49% reside in Punjab province

alone [1]. Several factors affect the health and productivity of these animals.

Parasitism is one of the most important problems of livestock sector responsible

for lowering the productivity and accounts for infecting more than 300 million cattle and

buffaloes around the globe [2&3]. The parasite spends complete or a part of its life cycle

CHAPTER 1

PREVALENCE OF FASCIOLIASIS IN CROSSBRED

COWS

2

in or on another organism’s expense. The helminths in cattle are very important parasites.

The characteristic signs and symptoms of endoparasites are diarrhoea, loss of production,

anemia, weakness and emaciation. The disease is very much important due to its broad

distribution and definite hosts [4]. The incidence of helminthes in various species of

animals had been reported ranging from 21.41 to 92% in Pakistan [5&6].

Internal parasitism is a major problem that lowers the productivity of the livestock

throughout the world [3]. It causes acute and chronic infections [7]. The parasites become

lodged in the biliary ducts of the liver after 3-4 months [8]. The mature flukes live in the

bile ducts and immature live in the parenchyma of the liver, very rarely in other organs.

They are flat and mostly resemble a laurel leaf in outline (The adult F. hepatica averaging

5cm in length and 1.5cm wide, whereas, F. gigantica is having 7.5cm in length and 1.2cm

wide, appearing dirty gray to brownish in colour).

Faeces are usually examined by flotation method as used by [9, 10, 11, 12&13].

Eggs are excreted through faeces and require snail as intermediate host that lives mainly

in water (Referred as snail Intermediate host). The parasites pass through different stages

in the snail before attaching themselves in the form of cysts to the ground vegetation.

Thereon it is taken up by the host during grazing. Cyst wall is dissolved in the

gastrointestinal tract of the host and the young fluke emerges. It penetrates and passes

through the intestines into the liver. Spends 6-8 weeks drifting in the liver and then settles

down in the bile duct. In the host the total period of development, i.e. from swallowing of

the cysts to develop into sexually mature parasite is 2 ½ - 3 months.

Most of the above descriptions are also applicable to F. gigantica, another species

of liver fluke, which is restricted to certain areas, mainly in tropical areas. Under certain

circumstances, it may cause severe damage to the liver. The infection in adult cattle

usually takes a chronic form with no obvious clinical signs.

The most common gastrointestinal parasites in cattle are Paramhistomum,

Haemonchus, Ostertagia, Cooperia, Moniezia, Toxocara, Trichuris, Dicrocelium,

Fasciola, Schiztosoma, Cysticercus, Trichostrongylus, [14]. Fascioliasis (liver rot) is

caused by either F. gigantica or F. hepatica [15]. These two species are generally

distributed across the world infecting humans and animals [16,17&18].

Fasciola species are common liver flukes in Pakistan [6]. It is a food borne an

important parasitic disease which is responsible for public health problems and huge

economic losses to the livestock industry. It is responsible for causing huge losses in

livestock productivity that are attributed to poor growth, reduced milk production, loss of

3

weight, liver condemnation at slaughter and death in cases of acute infection [19&20].

Fascioliasis not only gained attention due to its prevalence but economic significance to

animal stock in the world [19&21]. It has also a zoonotic importance.

In different parts of Pakistan, different prevalence records of fascioliasis were

reported in livestock [22, 23&24].

The economic significance and presence of fascioliasis has been known since long

by various scientists [25, 26, 27, 28, 29, 30&31]. The significance of helminthes infection

has been increased many folds in developing countries. Many parasitic infections cause

death even when the measures to control the disease are neglected [14].

Mainly there are two spp of Fasciola namely F. hepatica and F. gigantica,

extensively distributed across the world, which affects the host as humans and animals

[32&33]. The two spp of Fasciola i.e. F. hepatica and F. gigantica are very much similar

morphometrically to each other [34&35]. Previously, morphometric tools were used on

adult flukes and their eggs to identify the spp. Which were recovered from various

domestic animal hosts [36]. The prevalent species responsible for infection are difficult

to identify as the distribution of F. hepatica and F. gigantica spp of Fasciola overlap

[37].

Fascioliasis is caused largely by F.hepatica in moderate climates whereas, F.

gigantica in tropical areas. It is identified in acute cases by sudden death having blood

stained froth at natural orifices [38, 39&40], while in chronic cases the prominent clinical

signs are bottle jaw, jaundice, diarrhea and ascites. Economic losses caused by the

fascioliasis are largely because of mild to heavy mortality, condemned of livers,

expenditure on diagnosis and treatment of diseased animals [41], less meat production,

less milk yield and fertility disorders [42]. F. hepatica has a broad-based distribution

mainly in moderate regions, whereas F. gigantica is almost found in Asia and Africa in

tropical regions. The two fasciolid spp overlap in most of the African and Asian countries

and sometimes in the same countries having environmental necessities of flukes and its

intermediate host (snail) are very much distinct [39].

Heavy production losses were found in the herds having F. hepatica infection of

25 % or above [3]. High prevalence of F. hepatica infection has been reported in dairy

cattle in many countries [43]. Acute fascioliasis causes huge economic losses as directly

or indirectly in terms of anemia due to its ability to suck blood with expected blood loss

of about 0.2 - 0.5 ml /worm/animal/day [24&2] while chronic diseases can reduce the

growth rate, wool production and feed conservation rate. Chronic fascioliasis causes

4

chronic inflammation of bile ducts and the liver that accompanied by general reduction in

productivity, loss of condition and digestive disturbances.

The probable reason for the highest prevalence in winter might be due to the

optimal conditions of environment for growth, development and transmission with

parasitic life cycle stages [44]. The two very important factors that influence the

prevalence of fascioliasis are moisture and temperature which mainly affect the eggs

hatching of Fasciola [45]. The adverse agro-climatic conditions, animal husbandry

practices and management of pasture determine the prevalence and sternness of various

parasitic infections in the region [46&47].

Significant economic losses to cattle reared in Nigeria through mortalities abortion

and ailments like decrease in weight gain, un-thriftiness, reduce milk, infected livers

condemnation and expenditure on treatment of disease animals have been reported by

[48, 49&29]. They have also reported huge financial losses due to liver condemnations at

slaughter houses. Furthermore, [50] declares that fascioliasis is among the zoonotic

diseases that needs liver condemnations and organ condemnations in slaughtered cattle.

The presence of disease due to F. hepatica and F. gigantic at abattoir surveys in some

parts of the country has long been known and prevalence of fascioliasis and its economic

importance have been described by a number of workers [48, 51 &52].

The infection usually takes a chronic course in adult cattle with no obvious

clinical signs. Chronic fascioliasis is the most common form of cattle and is the one

detected during the course of meat inspection [53, 54, 55, 56, 57, 58&20]. Significant

production losses occur in the herds having a prevalence of F. hepatica infection of 25%

or above [3]. High prevalence of F. hepatica infection had been reported in dairy cattle in

many countries [59].

The incidence of fascioliasis is increasing in Pakistan as the water logging (water

table) is increasing in Pakistan. Control of fascioliasis involves a regular use of

appropriate anthelmintic. However, continuous administration of anthelmintic has led to

the development of resistance [60]. Thus therapeutic agents are needed to be replaced the

existing one, because of the increasing resistance in animals [61].

Fascioliasis causes tremendous loss to livestock owners as fresh water snails are

the obligatory intermediate host for a large number of trematode. Therefore, it must be

controlled [62]. Fresh water snails are of considerable veterinary and medical importance

in Pakistan and are found throughout the year except in extreme cold and hot weather

[63].

5

The present epidemiological survey was conducted in different agro climatic zones of

Punjab, Pakistan from November 2012 to October 2013.

Objectives

The proposed study has therefore been planned:

To record the prevalence of fascioliasis, in relation to area, month, age, sex,

season wise in randomly selected animals at slaughter houses, veterinary hospitals,

household and livestock farms in four districts of Punjab.

1.3 MATERIALS AND METHODS

Attached as appendix -A

1.4 RESULTS

Survey of abattoirs

During study period i.e. from November 2012 to October 2013, surveys of

abattoirs were carried out including Lahore, Sialkot, Attock and Multan districts. The

detailed study was carried out as follows:-

Lahore abattoir

Total 300 livers of slaughtered crossbred cows were examined for the presence of

fascioliasis, (by F. gigantica and F. hepatica) out of these 60 found positive and the

infection rate was thus 20%. The maximum number of flukes recorded from livers and

bile ducts was 515.

Prevalence of fascioliasis

Month wise

The peak F. gigantica infectivity was found in September having 56% infection.

Whereas the minimum prevalence, 1% was found in May (Table 1.1).

Season wise

In crossbred cows, the maximum prevalence 52% was found during the autumn.

The lowest prevalence 11% was found in winter (Table 1.1).

6

Age wise

In slaughtered crossbred cows, the infection rate was maximum in adult animals

than younger as shown in Table 1.1.

Sex wise

In males and females the disease pattern was analyzed in crossbred cows reveled

that infection was more in males 19.26% than that of females 18.44% (Table 1.1).

Sialkot abattoir

A total 300 livers of slaughtered crossbred cows were examined for the presence

of F. gigantica out of these 67 found positive and the infection rate was thus 22.33%. The

maximum number of flukes recorded from livers and bile ducts was 610.


Month wise

The peak F. gigantica infection was found during September with an infection

rate of 64%. While the lowest prevalence, 4% was recorded in May (Table 1.5).

Season wise

In crossbred cows, the maximum prevalence 27% was found in autumn. The

minimum prevalence 11% was found in summer (Table 1.5).

Age wise

In slaughtered crossbred cows, the infection rate was higher in adult 42% (over

two years) animals than younger 25% (below two years) (Table 1.5)

Sex wise

An analysis of disease pattern in male and female crossbred cows showed that

infection was higher in males 42% than females 25% (Table 1.5).

7

Attock abattoir


of F. gigantica out of these 63found positive and the infection rate was thus 21%. The



Month wise

The peak F. gigantica infection was found in October having an infection rate of

48%. While the lowest prevalence, 1% was recorded during May (Table 1.9).

Season wise



Age wise


two years) animals than younger 26% (below two years) (Table 1.9).

Sex wise

The pattern of disease in male and female crossbred cows showed that infection

was higher in males 37% than females 26% (Table 1.9).

Multan abattoir


of Fasciola out of these 35 found positive and the infection rate was thus 11.7%. The



Month wise

The peak F. gigantica infection was found in September with an infection rate of

32%. While the lowest prevalence, 4.0% was recorded during January, February, May

and June (Table 1.13).

8

Season wise



Age wise


two years) animals than younger 13% (below two years) (Table 1.13).

Sex wise

The pattern of disease in male and female crossbred cows showed that infection

was higher in males 22% than females 13% (Table 1.13).

Overall prevalence of fascioliasis in abattoirs in crossbred cows at four

districts of Punjab province

The overall prevalence of fascioliasis in slaughtered crossbred cows in four

districts showed that the infection rate was the highest at Sialkot 22.33%, Attock, 21%,

then Lahore 20% and the lowest in Multan 11.67% (Table 1.24 and Fig. 1.4).

The highest (64%) month wise prevalence was recorded in September at Sialkot

and the lowest (4%) in May at Lahore, Sialkot and Attock while 4% was also recorded

during January, February, May and June at Multan (Table1.1, 1.5, 1.9 and 1.13 and Fig.

1.7).

The data on seasonal prevalence of fascioliasis in slaughtered crossbred cows in

four districts indicated that the highest (44%) prevalence was found during autumn,

spring (24.5%) then winter (11.5%) and the lowest in summer 10.5% (Table 1.21 and Fig

1.3).

Age wise prevalence of fascioliasis in slaughtered crossbred cows showed that the

higher prevalence was recorded in adult animals 22.88% than younger 14.26% (Table

1.23 and Fig 1.6).

It was also evident that male animals 19.26% were more commonly affected than

females 18.44% (Table1.22 and Fig 1.5).

When analysis of variance was applied highly significant different (p < 0.001) was

noted during autumn than spring. During spring significant difference (p<0.05) was noted

than that of other seasons so it was concluded that season played major role in the

9

transmission of fascioliasis. No significant difference was noted in male and female

animals. Highly significant difference (p<0.001) was noted in cows over two years of age

than that of under two years of age. District wise prevalence indicated that significant

difference (p<0.05) was noted between Sialkot and Multan but among Sialkot, Lahore

and Attock no significant difference was recorded.

Survey of Cattle farms

Survey of cattle farms in different ecological zones was carried out in four

districts. Every farm was visited on each month from November 2012 to October 2013.

On every visit 25 randomly collected faecal samples were examined. The information of

each livestock farm is given below:-

Cattle farms at Lahore

Three hundred (300) randomly collected faeces samples of crossbred cows were

studied. Out of which 48 were found positive the percentage of prevalence was thus16%

(Table 1.2).


Month wise

In crossbred cows the maximum (44%) prevalence was found during September

while the minimum (4%) in June and December (Table1.2).

Season wise



Age wise

In crossbred cows age wise prevalence of fascioliasis showed that maximum

infection rate was reported in adult (30%) than that of in younger 18% (Table 1.2).

Sex wise

The infection rate was 31% in males higher than that of females 17% (Table 1.2).

10

Cattle farms at Sialkot


evaluated. Out of which 51 were found positive and the percentage of prevalence thus

remained 17% (Table 1.6).


Month wise


while the minimum (4%) in June and Jul (Table1.6).

Season wise



Age wise



Sex wise


Cattle farms at Attock





Month wise

In crossbred cows the maximum (44%) prevalence was found during October

while the minimum (4%) in May and June (Table1.10).

11

Season wise



Age wise



Sex wise


Cattle farms at Multan



remained 9.7% (Table 1.14).


Month wise


while the minimum (4%) in January, February, May, June, July and November (Table

1.14).

Season wise



Age wise



Sex wise


12

Overall prevalence of fascioliasis at farms in crossbred cows at four


Overall prevalence of fascioliasis in crossbred cows at farms in 4-districts of

Punjab province showed that the highest infection was found at Attock 18 %, Sialkot 17

%, Lahore 16 % and the lowest in Multan 9.67% (Table 1.24 and Fig. 1.4).

The maximum (64%) month-wise prevalence was recorded in September at

Sialkot whereas the minimum (4%) in June at Lahore, June, July and December at

Sialkot, May and June at Attock , January, February, May, June, July and December at

Multan (Table 1.2, 1.6, 1.10 and 1.13 and Fig. 1.7).

The data on seasonal prevalence of fascioliasis in crossbred cows at farms in four

districts showed that the maximum (36%) prevalence was found in autumn, spring (24%)

then winter (8.5%) and the minimum in summer 7 % (Table 1.21 and Fig 1.3).

Age wise prevalence of fascioliasis in crossbred cows at farms showed that higher

(23.52%) prevalence was recorded in adult animals than younger 10.07% (Table 1.23 and

Fig 1.6).

It was also evident that male animals 24 % were more commonly affected than

females 8.86 % (Table 1.22 and Fig 1.5).

When analyses of variance were applied significant different (p<0.05) was noted

during autumn than spring. During spring highly significant difference (p<0.001) was

noted than the other season so it was concluded that season played an important role in

the transmission of disease. A highly significant difference was noted in male (p<0.001)

than female animals at farms. Highly significant difference (p<0.001) was also noted in

animals above two years than under two years of age. District wise prevalence indicated

that significant difference (p<0.05) was noted at Sialkot than Multan whereas, Lahore,

Attock and Multan have no significant difference among each other.

Survey of veterinary hospital

During the one year period of investigation, survey of eight veterinary hospitals of

Lahore, Sialkot, Attock and Multan districts was done. A total of 2400 randomly selected

faecal samples were examined i.e.300 faecal samples from each hospital (25 per month

per hospital). The details are given as follows:-

13

Veterinary hospital at Lahore district





Month wise


while the minimum (4%) in December (Table 1.3).

Season wise


minimum prevalence 9% was in during summer (Table 1.3).

Age wise



Sex wise


Veterinary hospital at Sialkot district

A total of 300 crossbred cows were examined out of which 49 (16.3%) were

found positive.


Month wise


while the minimum (4%) in during June (Table 1.7).

Season wise



14

Age wise



Sex wise

The prevalence of fascioliasis was higher in males (25%) than females 13%

(Table 1.7).

Veterinary hospital at Attock district

A total of 300 crossbred cows were examined out of which 46 (15.3%) were

found positive.


Month wise


while the minimum (4%) in January, May, June and December (Table 1.11).

Season wise



Age wise



Sex wise

The prevalence of fascioliasis was higher in males, i.e. 21% than that of females

11% (Table 1.11).

Veterinary hospital at Multan district

A total of 300 crossbred cows were examined out of which 30 (10%) were found

positive.

15


Month wise


while the minimum (0%) in June (Table 1.15).

Season wise



Age wise



Sex wise

The prevalence of Fascioliasis was higher in males, (20%) than females i.e. 6%

(Table 1.15).

Overall prevalence of fascioliasis in veterinary hospitals in crossbred

cows at four districts of Punjab province

The overall prevalence of Fascioliasis in crossbred cows at veterinary hospital in

four districts of Punjab province indicated that the infection was highest in Lahore (21%)

followed by, Sialkot (16%), then Attock (15.33%) and the lowest in Multan (10%)(Table

1.24 and Fig 1.4).

The highest (52%) month wise prevalence was recorded during October at Lahore

while the lowest (4%) during May at Sialkot, January, May, June and November at

Attock, January, May, July and December at Multan (Table 1.3, 1.7, 1.11 and 1.15 and

Fig 1.7).

The data of seasonal prevalence of fascioliasis in crossbred cows at veterinary

hospital in four districts of Punjab province showed that the highest (35%) prevalence

was noted during autumn, spring (25.5%) then winter (8.8%) and the lowest 8% (Table

1.21 and Fig 1.3).

16

Age wise prevalence of fascioliasis in crossbred cows at veterinary hospital

showed that the higher prevalence (21.39%) was recorded in adult animals than younger

11.51% (Table 1.23 and Fig 1.6).

It was also evident that male animals 25.69 % were more commonly affected than

females 9.19 % (Table 1.22 and Fig 1.5).

When analyses of variance was applied highly significant different (p<0.001) was

noted during autumn than spring. Whereas, spring showed the highly significant

(p<0.001) difference than the other two seasons of the year. It was concluded that season

played important role in the transmission of disease. The highly significant difference

(p<0.001) was noted in female than male animals. A highly significant difference was

noted in animals above two years than under two years of age. District wise prevalence

indicated that highly significant difference (p<0.001) was noted between Lahore and

other districts whereas, significant difference (p<0.05) was noted in Sialkot and Attock

than Multan.

Survey of Household crossbred cows

During the period i.e. from November 2012-October 2013, a survey of 1200

crossbred cows kept in houses throughout the four districts including, Sialkot, Attock and

Multan was carried out. Details are as under:-

Household survey at Lahore

A total of 300 faecal samples were examined, out of which 41 (13.7%) were found

positive for eggs of F. gigantica (Table 1.4).


Month wise


while the minimum (4%) in June, July and December (Table 1.4).

Season wise



17

Age wise



Sex wise

Sex wise prevalence was higher in males than females (Table 1.4).

Household survey at Sialkot district

Total of 300 faecal samples were examined, out of which 48 (16.0%) were found



Month wise


while the minimum (4%) in June, July and December (Table 1.8).

Season wise



Age wise



Sex wise


Household survey at Attock district



18


Month wise


while the minimum (4%) in January, May, July and December (Table 1.12).

Season wise



Age wise



Sex wise


Household survey at Multan district




Month wise

In crossbred cows the maximum (24%) prevalence was found during April and

October while the minimum (4%) in January, February, June, July, August and November

(Table 1.16).

Season wise


minimum prevalence 5% was found in winter and summer (Table 1.16).

In crossbred cows, the highest (20%) season wise prevalence was reported during

autumn and the lowest (5%) during winter and summer (Table 1.16).

19

Age wise



Sex wise


Overall prevalence of fascioliasis in household crossbred cows at four


The overall prevalence of fascioliasis in household crossbred cows in four districts

of Punjab province indicated that the infection was highest at Sialkot (16%) followed by

Attock, (14.67%), then Lahore (13.67%) and the lowest in Multan 9.67% (Table 1.24 and

Fig 1.4).

The highest (44%) month wise prevalence was recorded during October at Sialkot

while the lowest (4%) during June, July and December in Lahore, January, June, July and

December at Attock, January, May, July and December at Multan (Table 1.4, 1.8, 1.12

and 1.16 and Fig 1.7).

The data on seasonal prevalence of fascioliasis in household crossbred cows in

four districts showed that the highest (31%) prevalence was found during autumn, spring

(22.5%) then winter (7.8%) and the lowest6%during summer (Table 1.21 and Fig 1.3).

Age wise prevalence of fascioliasis in household crossbred cows showed that

higher (18.70%) prevalence was recorded in adult animals than younger 9.89% (Table

1.23 and Fig 1.6).

It was also evident that male animals 23.36% were more commonly affected than

females 7.41% (Table 1.22 and Fig 1.5).

When analysis of variance was applied highly significant different (p<0.001) was

noted during autumn than winter and summer. Significant difference was noted in spring

(p<0.05) than the winter and summer seasons so it was concluded that seasons played

important role in the transmission of disease. Highly significant difference (p<0.001) was

noted in male than female animals. Highly significant difference (p<0.001) was also

noted in animals above two years than under two years of age. District wise prevalence

20

indicated that significant difference (p<0.05) was noted among Sialkot, Lahore and

Attock and highly significant of all three districts with Multan.

Fig. 1.1: Map showing epidemiological study area of fascioliasis in four districts of

Punjab province.

21

Life cycle

The life cycle of F. gigantica is as under: ova (travelled with faeces) → ova

hatched → to miracidium → miracidium infect intermediate host (snail) → to

sporocyst → to redia → to daughter redia → to cercaria → (comes out of the snail) → to

metacercaria → infect to the host → then adult stage produces ova.

Fig. 1.2: Life cycle of Fasciola gigantica in crossbred cows (final host) 1: Adult

flukes in the liver 2: Egg containing miracidium 3: Rediae 4:

Intermediate host for Fasciola gigantica &are water snails of the genus

Lymnaea5: Cercariae 6: Metacercariae on plants 7: Encystment of the

metacercariae in the rumen of the final host

Metacercariae on grass

Cercaria 5-7 weeks

Snail

Miracidium 10-12 Days

Eggs Shed 8-12 week

After infection

https://en.wikipedia.org/wiki/Miracidium

https://en.wikipedia.org/wiki/Trematode_lifecycle_stages

https://en.wikipedia.org/wiki/Trematode_lifecycle_stages

https://en.wikipedia.org/wiki/Metacercaria

22

Table 1.1: Prevalence of fascioliasis in slaughtered crossbred cows of Lahore district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 4 16.00

Dec 2012 25 2 8.00

Jan 2013 25 2 8.00

Feb 2013 25 3 12.00

Mar 2013 25 7 28.00

Apr 2013 25 3 12.00

May 2013 25 1 4.00

Jun 2013 25 2 8.00

Jul 2013 25 4 16.00

Aug 2013 25 6 24.00

Sep 2013 25 14 56.00

Oct 2013 25 12 48.00

Seasons

Winter 100 11 11.00

Spring 50 10 20.00

Summer 100 13 13.00

Autumn 50 26 52.00

Sex Female 205 36 17.56

Male 95 24 25.26

Age Below 2 Yrs 110 18 16.36

Over 2 Yrs 190 42 22.11

Overall 300 60 20.00

23

Table 1.2: Prevalence of fascioliasis in crossbred cows at farms of Lahore district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 1 4.00

Jan 2013 25 2 8.00

Feb 2013 25 3 12.00

Mar 2013 25 5 20.00

Apr 2013 25 5 20.00

May 2013 25 2 8.00

Jun 2013 25 1 4.00

Jul 2013 25 2 8.00

Aug 2013 25 3 12.00

Sep 2013 25 11 44.00

Oct 2013 25 10 40.00

Seasons

Winter 100 9 9.00

Spring 50 10 20.00

Summer 100 8 8.00

Autumn 50 21 42.00

Sex Female 190 17 8.95

Male 110 31 28.18

Age Below 2 Yrs 170 18 10.59

Over 2 Yrs 130 30 23.08

Overall 300 48 16.00

24

Table 1.3: Prevalence of fascioliasis in crossbred cows at veterinary hospitals of

Lahore district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 1 4.00

Jan 2013 25 2 8.00

Feb 2013 25 3 12.00

Mar 2013 25 9 36.00

Apr 2013 25 11 44.00

May 2013 25 2 8.00

Jun 2013 25 2 8.00

Jul 2013 25 3 12.00

Aug 2013 25 2 8.00

Sep 2013 25 12 48.00

Oct 2013 25 13 52.00

Seasons

Winter 100 9 9.00

Spring 50 20 40.00

Summer 100 9 9.00

Autumn 50 25 50.00

Sex Female 130 18 13.85

Male 170 45 26.47

Age Below 2 Yrs 190 29 15.26

Over 2 Yrs 110 34 30.91

Overall 300 63 21.00

25

Table 1.4: Prevalence of fascioliasis in household crossbred cows of Lahore district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 4 16.00

Dec 2012 25 1 4.00

Jan 2013 25 2 8.00

Feb 2013 25 3 12.00

Mar 2013 25 5 20.00

Apr 2013 25 7 28.00

May 2013 25 2 8.00

Jun 2013 25 1 4.00

Jul 2013 25 1 4.00

Aug 2013 25 2 8.00

Sep 2013 25 4 16.00

Oct 2013 25 9 36.00

Seasons

Winter 100 10 10.00

Spring 50 12 24.00

Summer 100 6 6.00

Autumn 50 13 26.00


Male 120 25 20.83

Age Below 2 Yrs 208 20 9.62

Over 2 Yrs 92 21 22.83

Overall

300 41 13.67

26

Table 1.5: Prevalence of fascioliasis in slaughtered crossbred cows of Sialkot district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 4 16.00

Dec 2012 25 3 12.00

Jan 2013 25 2 8.00

Feb 2013 25 5 20.00

Mar 2013 25 11 44.00

Apr 2013 25 4 16.00

May 2013 25 1 4.00

Jun 2013 25 2 8.00

Jul 2013 25 3 12.00

Aug 2013 25 5 20.00

Sep 2013 25 16 64.00

Oct 2013 25 11 44.00

Seasons

Winter 100 14 14.00

Spring 50 15 30.00

Summer 100 11 11.00

Autumn 50 27 54.00

Sex Female 160 25 15.63

Male 140 42 30.00

Age Below 2 Yrs 170 25 14.71

Over 2 Yrs 130 42 32.31

Overall 300 67 22.33

27

Table 1.6: Prevalence of fascioliasis in crossbred cows at farms of Sialkot district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 1 4.00

Jan 2013 25 2 8.00

Feb 2013 25 2 8.00

Mar 2013 25 10 40.00

Apr 2013 25 5 20.00

May 2013 25 2 8.00

Jun 2013 25 1 4.00

Jul 2013 25 1 4.00

Aug 2013 25 3 12.00

Sep 2013 25 16 64.00

Oct 2013 25 5 20.00

Seasons

Winter 100 8 8.00

Spring 50 15 30.00

Summer 100 7 7.00

Autumn 50 21 42.00


Male 140 36 25.71

Age Below 2 Yrs 185 22 11.89

Over 2 Yrs 115 29 25.22

Overall 300 51 17.00

28

Table 1.7: Prevalence of fascioliasis in crossbred cows at veterinary hospital

of Sialkot district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 2 8.00

Jan 2013 25 3 12.00

Feb 2013 25 2 8.00

Mar 2013 25 6 24.00

Apr 2013 25 6 24.00

May 2013 25 1 4.00

Jun 2013 25 2 8.00

Jul 2013 25 2 8.00

Aug 2013 25 4 16.00

Sep 2013 25 10 40.00

Oct 2013 25 8 32.00

Seasons

Winter 100 10 10.00

Spring 50 12 24.00

Summer 100 9 9.00

Autumn 50 18 36.00

Sex Female 224 30 13.39

Male 76 19 25.00

Age Below 2 Yrs 145 20 13.79

Over 2 Yrs 155 29 18.71

Overall 300 49 16.33

29

Table 1.8: Prevalence of fascioliasis in household crossbred cows of Sialkot district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 2 8.00

Dec 2012 25 1 4.00

Jan 2013 25 1 4.00

Feb 2013 25 4 16.00

Mar 2013 25 7 28.00

Apr 2013 25 6 24.00

May 2013 25 2 8.00

Jun 2013 25 1 4.00

Jul 2013 25 1 4.00

Aug 2013 25 3 12.00

Sep 2013 25 9 36.00

Oct 2013 25 11 44.00

Seasons

Winter 100 8 8.00

Spring 50 13 26.00

Summer 100 7 7.00

Autumn 50 20 40.00

Sex Female 252 33 13.10

Male 48 15 31.25

Age Below 2 Yrs 195 21 10.77

Over 2 Yrs 105 27 25.71

Overall 300 48 16.00

30

Table 1.9: Prevalence of fascioliasis in slaughtered crossbred cows of Attock district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 4 16.00

Dec 2012 25 2 8.00

Jan 2013 25 3 12.00

Feb 2013 25 5 20.00

Mar 2013 25 12 48.00

Apr 2013 25 3 12.00

May 2013 25 1 4.00

Jun 2013 25 2 8.00

Jul 2013 25 5 20.00

Aug 2013 25 4 16.00

Sep 2013 25 10 40.00

Oct 2013 25 12 48.00

Seasons

Winter 100 14 14.00

Spring 50 15 30.00

Summer 100 12 12.00

Autumn 50 22 44.00

Sex Female 198 26 13.13

Male 102 37 36.27

Age Below 2 Yrs 155 26 16.77

Over 2 Yrs 145 37 25.52

Overall 300 63 21.00

31

Table 1.10:Prevalence of fascioliasis in crossbred cows at farms of Attock district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 2 8.00

Jan 2013 25 2 8.00

Feb 2013 25 4 16.00

Mar 2013 25 6 24.00

Apr 2013 25 9 36.00

May 2013 25 1 4.00

Jun 2013 25 1 4.00

Jul 2013 25 2 8.00

Aug 2013 25 4 16.00

Sep 2013 25 9 36.00

Oct 2013 25 11 44.00

Seasons

Winter 100 11 11.00

Spring 50 15 30.00

Summer 100 8 8.00

Autumn 50 20 40.00

Sex Female 190 19 10.00

Male 110 35 31.82

Age Below 2 Yrs 185 23 12.43

Over 2 Yrs 115 31 26.96

Overall 300 54 18.00

32

Table 1.11: Prevalence of fascioliasis in crossbred cows at veterinary hospital

of Attock district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 1 4.00

Jan 2013 25 1 4.00

Feb 2013 25 4 16.00

Mar 2013 25 4 16.00

Apr 2013 25 8 32.00

May 2013 25 1 4.00

Jun 2013 25 1 4.00

Jul 2013 25 2 8.00

Aug 2013 25 4 16.00

Sep 2013 25 10 40.00

Oct 2013 25 7 28.00

Seasons

Winter 100 9 9.00

Spring 50 12 24.00

Summer 100 8 8.00

Autumn 50 17 34.00

Sex Female 160 17 10.63

Male 140 29 20.71

Age Below 2 Yrs 190 19 10.00

Over 2 Yrs 110 27 24.55

Overall 300 46 15.33

33

Table 1.12: Prevalence of fascioliasis in household crossbred cows of Attock

district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 1 4.00

Jan 2013 25 1 4.00

Feb 2013 25 3 12.00

Mar 2013 25 4 16.00

Apr 2013 25 7 28.00

May 2013 25 1 4.00

Jun 2013 25 0 0.00

Jul 2013 25 1 4.00

Aug 2013 25 4 16.00

Sep 2013 25 10 40.00

Oct 2013 25 9 36.00

Seasons

Winter 100 8 8.00

Spring 50 11 22.00

Summer 100 6 6.00

Autumn 50 19 38.00


Male 155 30 19.35

Age Below 2 Yrs 135 16 11.85

Over 2 Yrs 165 28 16.97

Overall 300 44 14.67

34

Table 1.13: Prevalence of fascioliasis in slaughtered crossbred cows of Multan

district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 2 8.00

Jan 2013 25 1 4.00

Feb 2013 25 1 4.00

Mar 2013 25 6 24.00

Apr 2013 25 3 12.00

May 2013 25 1 4.00

Jun 2013 25 1 4.00

Jul 2013 25 2 8.00

Aug 2013 25 2 8.00

Sep 2013 25 8 32.00

Oct 2013 25 5 20.00

Seasons

Winter 100 7 7.00

Spring 50 9 18.00

Summer 100 6 6.00

Autumn 50 13 26.00


Male 120 22 18.33


Over 2 Yrs 160 22 13.75

Overall 300 35 11.67

35

Table 1.14:Prevalence of fascioliasis in crossbred cows at farms of Multan district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 3 12.00

Dec 2012 25 1 4.00

Jan 2013 25 1 4.00

Feb 2013 25 1 4.00

Mar 2013 25 3 12.00

Apr 2013 25 5 20.00

May 2013 25 1 4.00

Jun 2013 25 1 4.00

Jul 2013 25 1 4.00

Aug 2013 25 2 8.00

Sep 2013 25 3 12.00

Oct 2013 25 7 28.00

Seasons

Winter 100 6 6.00

Spring 50 8 16.00

Summer 100 5 5.00

Autumn 50 10 20.00


Male 140 16 11.43


Over 2 Yrs 95 17 17.89

Overall 300 29 9.67

36

Table 1.15:Prevalence of fascioliasis in crossbred cows at veterinary hospital of

Multan district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 2 8.00

Dec 2012 25 1 4.00

Jan 2013 25 1 4.00

Feb 2013 25 3 12.00

Mar 2013 25 3 12.00

Apr 2013 25 4 16.00

May 2013 25 1 4.00

Jun 2013 25 0 0.00

Jul 2013 25 1 4.00

Aug 2013 25 4 16.00

Sep 2013 25 4 16.00

Oct 2013 25 6 24.00

Seasons

Winter 100 7 7.00

Spring 50 7 14.00

Summer 100 6 6.00

Autumn 50 10 20.00


Male 85 17 20.00


Over 2 Yrs 130 18 13.85

Overall 300 30 10.00

37

Table 1.16: Prevalence of fascioliasis in household crossbred cows of Multan district

Parameter No.

Examined

No.

Infected

%age

of Infection

Months

Nov 2012 25 1 4.00

Dec 2012 25 2 8.00

Jan 2013 25 1 4.00

Feb 2013 25 1 4.00

Mar 2013 25 3 12.00

Apr 2013 25 6 24.00

May 2013 25 1 4.00

Jun 2013 25 1 4.00

Jul 2013 25 2 8.00

Aug 2013 25 1 4.00

Sep 2013 25 4 16.00

Oct 2013 25 6 24.00

Seasons

Winter 100 5 5.00

Spring 50 9 18.00

Summer 100 5 5.00

Autumn 50 10 20.00


Male 135 19 14.07


Over 2 Yrs 130 16 12.31

Overall 300 29 9.67

38

Table 1.17: Prevalence of fascioliasis in slaughtered crossbred cows in four district

Parameter Examined Infected Infection

percentage

Months

Nov 2012 100 15 15.00

Dec 2012 100 9 9.00

Jan 2013 100 8 8.00

Feb 2013 100 14 14.00

Mar 2013 100 36 36.00

Apr 2013 100 13 13.00

May 2013 100 4 4.00

Jun 2013 100 7 7.00

Jul 2013 100 14 14.00

Aug 2013 100 17 17.00

Sep 2013 100 48 48.00

Oct 2013 100 40 40.00

Seasons

Winter 400 46 11.50

Spring 200 49 24.50

Summer 400 42 10.50

Autumn 200 88 44.00

Sex Female 743 137 18.44

Male 457 88 19.26

Age Below 2 Yrs 575 82 14.26

Over 2 Yrs 625 143 22.88

Overall 1200 225 18.75

39

Table 1.18: Prevalence of fascioliasis in crossbred cows at farms in four districts


percentage

Months

Nov 2012 100 12 12.00

Dec 2012 100 5 5.00

Jan 2013 100 7 7.00

Feb 2013 100 10 10.00

Mar 2013 100 24 24.00

Apr 2013 100 24 24.00

May 2013 100 6 6.00

Jun 2013 100 4 4.00

Jul 2013 100 6 6.00

Aug 2013 100 12 12.00

Sep 2013 100 39 39.00

Oct 2013 100 33 33.00

Seasons

Winter 400 34 8.50

Spring 200 48 24.00

Summer 400 28 7.00

Autumn 200 72 36.00


Male 500 120 24.00

Age

Below 2

Yrs 745 75 10.07

Over 2 Yrs 455 107 23.52

Overall 1200 182 15.2

40

Table 1.19: Prevalence of fascioliasis in crossbred cows at veterinary hospital in

four districts


percentage

Months

Nov 2012 100 11 11.00

Dec 2012 100 5 5.00

Jan 2013 100 7 7.00

Feb 2013 100 12 12.00

Mar 2013 100 22 22.00

Apr 2013 100 29 29.00

May 2013 100 5 5.00

Jun 2013 100 5 5.00

Jul 2013 100 8 8.00

Aug 2013 100 14 14.00

Sep 2013 100 36 36.00

Oct 2013 100 34 34.00

Seasons

Winter 400 35 8.75

Spring 200 51 25.50

Summer 400 32 8.00

Autumn 200 70 35.00


Male 471 121 25.69

Age Below 2 Yrs 695 80 11.51

Over 2 Yrs 505 108 21.39

Overall 1200 188 15.67

41

Table 1.20:Prevalence of fascioliasis in household crossbred cows in four districts

Parameter Examined Infected Infection percentage

Months

Nov 2012 100 10 10.00

Dec 2012 100 5 5.00

Jan 2013 100 5 5.00

Feb 2013 100 11 11.00

Mar 2013 100 19 19.00

Apr 2013 100 26 26.00

May 2013 100 6 6.00

Jun 2013 100 3 3.00

Jul 2013 100 5 5.00

Aug 2013 100 10 10.00

Sep 2013 100 27 27.00

Oct 2013 100 35 35.00

Seasons

Winter 400 31 7.75

Spring 200 45 22.50

Summer 400 24 6.00

Autumn 200 62 31.00


Male 458 107 23.36


Over 2 Yrs 492 92 18.70

Overall 1200 162 13.50

42

Table 1.21: Season wise prevalence of fascioliasis in crossbred cows in four

districts

Crossbred Cows Winter Spring Summer Autumn Overall

Slaughter

house

Total No 400 200 400 200 1200.00

No. infected 46 49 42 88 225.00

%age 11.5 24.5 10.5 44.0 18.75

Farms

Total No 400 200 400 200 1200.00

No. infected 34 48 28 72 182.00

%age 8.5 24.0 7.0 36.0 15.17

Veterinary

Hospitals

Total No 400 200 400 200 1200.00

No. infected 35 51 32 70 188.00

%age 8.8 25.5 8.0 35.0 15.67

Household

Total No 400 200 400 200 1200.00

No. infected 31 45 24 62 162.00

%age 7.8 22.5 6.0 31.0 13.50

Overall

Total No 1600 800 1600 800 4800.00

No. infected 146 193 126 292 757.00

%age 9.13 24.13 7.88 36.50 15.77

43

Table 1.22: Sex wise prevalence of fascioliasis in crossbred cows in four districts

Crossbred Cows Slaughter

houses Farms

Veterinary

Hospital

House

hold Overall

Female

Total No 743 700 729 742 2914.00

No. infected 137 62 67 55 321.00

%age 18.44 8.86 9.19 7.41 11.02

Male

Total No 457 500 471 458 1886.00

No. infected 88 120 121 107 436.00

%age 19.26 24.00 25.69 23.36 23.12

Overall

Total No 1200 1200 1200 1200 4800.00

No. infected 225 182 188 162 757.00

%age 18.75 15.17 15.67 13.50 15.77

44

Table 1.23: Age wise prevalence of fascioliasis in crossbred cows in four

districts

Crossbred Cows Slaughter

houses Farms

Veterinary

Hospital

House

hold Overall

Below 2

years age

Total No 575 745 695 708 2723.00

No. infected 82 75 80 70 307.00

%age 14.26 10.07 11.51 9.89 11.27

Above 2

years age

Total No 625 455 505 492 2077.00

No. infected 143 107 108 92 450.00

%age 22.88 23.52 21.39 18.70 21.67

Overall

Total No 1200 1200 1200 1200 4800.00

No. infected 225 182 188 162 757.00

%age 18.75 15.17 15.67 13.50 15.77

45

Table 1.24: Prevalence of fascioliasis in crossbred cows in four district

Districts Slaughter

houses Farms

Veterinary

Hospital

House

hold Overall

Lahore

Total No 300 300 300 300 1200.00

No. infected 60 48 63 41 212.00

%age 20.00 16.00 21.00 13.67 17.67

Sialkot

Total No 300 300 300 300 1200.00

No. infected 67 51 49 48 215.00

%age 22.33 17.00 16.33 16.00 17.92

Attock

Total No 300 300 300 300 1200.00

No. infected 63 54 46 44 207.00

%age 21.00 18.00 15.33 14.67 17.25

Multan

Total No 300 300 300 300 1200.00

No. infected 35 29 30 29 123.00

%age 11.67 9.67 10.00 9.67 10.25

Overall

Total No 1200 1200 1200 1200 4800.00

No. infected 225 182 188 162 757.00

%age 18.75 15.17 15.67 13.50 15.77

46

Table 1.25: Prevalence of fascioliasis in lactating and non-lactating crossbred

cows at farms in four districts

District Parameter No. Examined No. Infected %age of Infection

Lahore

Lactating 50 7 14

Non Lactating 50 9 18

Sialkot

Lactating 50 8 16


Attock

Lactating 50 5 10


Multan

Lactating 50 6 12


47

Fig 1.3: Overall Season wise prevalence of fascioliasis in crossbred cows in four

districts from November 2012 to October 2013

Fig. 1.4: Overall District wise prevalence of fascioliasis in crossbred cows in four


48

Fig. 1.5: Overall Sex wise prevalence of fascioliasis in crossbred cows in four


Fig 1.6: Overall Age-wise prevalence of fascioliasis in crossbred cows in four


49

Fig 1.7: Overall Month wise prevalence of fascioliasis in crossbred cows in four


50

1.5 DISCUSSION

Fascioliasis is a disease of public health and economic importance [64]. Several

workers studied in Pakistan that F. hepatica is endemic in nature in sheep, goats, cattle,

buffaloes and humans [65, 5, 66&67]. Incidence of fascioliasis in an area is mainly

influenced by a multi factorial system which is composed of parasitic agents, hosts,

environmental effects as temperature, humidity, rainfall and pan evaporation including

transmission process. In helminthiasis the parasites, their transitional host and final host

make a link to cause a potential epidemiological danger. It is worth mentioning that the

presences and localization of such type of link may be documented earlier as to bring it

under control.

In this study, epidemiological statistics on fascioliasis due to F. gigantica and F.

hepatica was collected from clinically sick crossbred cows at slaughter houses, livestock

farms, veterinary hospitals and household animals from Lahore, Sialkot, Attock and

Multan districts of the Punjab province. Similar fascioliasis species were also detected in

a survey conducted by Khan [24] in five districts of Punjab, occurrence of disease was

found to be 25.46%, due to F. gigantica (22.40%) and by F. hepatica (3.06%).

Fascioliasis causes acute as well as chronic diseases [7]. The pathology and epidemiology

of both the species is different therefore, It is important to know about the species of

Fasciola, in our region [68&69].

In this study, the overall results obtained from slaughter houses, livestock farms,

veterinary hospitals and household crossbred cows are slightly different from each others.

At slaughter houses of four districts of Punjab province total 1200 animals were

examined in a year out of these 225 (18.8%) found positive for fascioliasis. At farms in

four districts of Punjab total 1200 animals were examined out of which 182 (15.2%)

found positive for fascioliasis. In veterinary hospitals of four districts of Punjab total 1200

animals were examined in a year out of which 188 (15.7%) found positive for fascioliasis.

Where as in household animal the total 1200 animals were examined out of which 162

(13.5%) found positive for fascioliasis. This indicates that the prevalence was highest at

slaughter houses (18.8%) then at veterinary hospitals (15.7%), followed by Farms

(15.2%) and lowest in household animals (13.5%). The observed overall prevalence of

fascioliasis in Crossbred cows in four districts is close to those obtained by various

workers in previous studies in different countries [51,55,56,57,19,58,70,71,20 &72].

51

This study is also in close agreement with the report of [73] from northern

Ethiopia, who reported 24.3% prevalence. However, it is much lower than that of many

other studies from different abattoirs in the country and elsewhere in Africa. [31] reported

90.7% prevalence of fascioliasis in cattle slaughtered at Gondar abattoir, while some

workers [29] recorded a prevalence of 46.2% at Jimma abattoir. In this study it seems that

parasitic infection rate is much less than above stated workers in several countries.

However, Phiri from Zambia [74] Pfukenyi and Mukaratirwa from Zimbabwe [71]

reported 53.9 and 31.7% prevalence, respectively. On the other hand, a lower prevalence

of fascioliasis (14.0%) has been observed in slaughtered cattle at Wolaita Soddo abattoir

[48]. The result is much lower as compare to that of previous report of Biniam Tsegaye

[75] and lower than that of the findings of [76], who recorded 41.41%, 25.2%, 22.4% and

19.3% prevalence of fascioliasis in cattle, respectively. These findings are in partial

conformity to the results of prevalence of bovine fascioliasis reported in Quetta,

Baluchistan [23] and that of Khan and Maqbool [63] from slaughter houses (22.6%)

livestock farms (17.5%), veterinary hospital (10.82%) and on household cattle (10.82%)

under different management conditions.

This difference is due to different environmental areas and diverse ecological

conditions. The difference in the prevalence percentage may also be accredited to manage

mental conditions, parasites and use of fasciolicide drugs.

In the present study the overall district wise prevalence of fascioliasis was

reported the highest in crossbred cows at Sialkot (17.92%) followed by Lahore (17.67%),

Attock (17.25%) while the lowest (10.25%) at Multan. The difference in prevalence

percentage may be due to the marshy and rice-grown areas. The local climatic factors

have influence on the occurrence of fascioliasis and variation in its prevalence i.e.

presence or absence of rivers, water ponds, lakes and incidence of appropriate

intermediate hosts. The fascioliasis is limited to those geographical areas where snails

population is found. Areas with poor drainage, higher annual rainfall and different type of

soil which has humidity are very much suitable for prevalence of disease and its spread

[77]. Due to its great proliferation power Fascioliasis has large colonization capabilities in

its ruminant host and vector species [16].

Seasonal reproduction in fascioliasis indicated high ova production in that season,

having highest activity in monsoon period and after monsoon period. In Punjab province

rains start in July-August and at the end of September. Plenty of snails are found on the

52

pasture during this period. While grazing the animals ingest metacercariae and develop

fascioliasis infection. Therefore, an increased incidence of disease was studied during

autumn. When the data of fascioliasis was analyzed on monthly and seasonal prevalence

basis it was revealed that maximum prevalence occurred during the month of August.

Khan [24] also indicated that favourable temperature with humidity help in the hatching

of ova and encysting cercariae with increase in the population of snails. These are the

most important factors that influence the prevalence of fascioliasis. Moisture and

temperature remain favourable during three seasonal periods for the rapid growth of the

life cycle of Fasciola and infection of snails especially during raining season. [78, 79,

80&81] also reported similar findings.

Age of animals is found to be a main factor in the prevalence of infection. The

P-value was found (P<0.001) in cows having age two years and above (21.67%) and than

that of animals aged below 2 years (11.27%) as shown in Table 1.23. Which are in

conformity to the previous studies of [82&83]. The highest infection was found in

animals above six year of age (62.62%), in four to six years (57.28%) than in two to four

years (42.56%) and up to 2 year (17.87%) [82]. The highest fascioliasis infection was

found in older animals because of long time contact to infected entity and grazing in the

immerse areas. Age association was found in this study is in conformity to the results of

[84]. They found some animals less than two years of age were infected as compared to

that of older animals. [85] also found that the animals more than 2-years were affected

significantly by fascioliasis than the animals less than 2-years of age. Same results were

found by [86&87] who reported higher infection rate in older animals. Whereas, this

study is in contradiction to the prevalence of F. gigantica that is higher in young cattle

(<2 years old) than that of adult cattle (>2 years old) as described by [73]. The resistance

is due to the acquired immunity development in the older animals as opined by earlier

investigators.

In this study it was found that infection was maximum in males 23.12% as

compared to female animals 11.02 %. It was also found that infection was lower in

female animals than males, it relates to the practice of keeping female animals in good

feeding and management as compare to that of male animals that are usually let loose for

grazing on the pastures. Some workers [88] had reported that the prevalence of

fascioliasis was higher in male animals than that of female animals and this is in support

to this study, unlike goats and buffaloes with higher prevalence rate in females. But in the

53

present study the results are not in agreement to the results of [82&83] they observed

(70.289%) in the females and (55.238%) males so prevalence is higher in female

buffaloes than male buffaloes. Phiri [62] also found significantly higher prevalence in

females as compared to males.

1.6 CONCLUDING REMARKS

Study of prevalence of fascioliasis at four districts of Punjab province having

different ecological conditions revealed that the infection rate was the highest in

crossbred cows at Sialkot followed by Lahore then Attock and the lowest at Multan.

Overall the highest season wise prevalence was recorded during autumn at livestock

farms then slaughtered animals followed by veterinary hospitals during summer and the

lowest in household animals in winter. The higher infection rate of fascioliasis was

observed in adults crossbred cows, over two years of age than that of younger, below two

years of age. Male animals were more commonly affected than females.

54

2.1 SUMMARY

Snails have been known to have an important role being intermediate hosts for

helminth parasites of man, animals, birds and fishes. Climatic conditions and utilization

of rivers as a source of irrigation in Punjab Pakistan play vital role in snails distribution.

In the present study almost seven snail species were collected from four districts of

Punjab province which belong to Pulmonata and Prosobranchia. These were Lymnaea,

Bulinus, Indoplanorbis, Bellamya, Gyraulus, Physa and Oncomelania. District wise

prevalence of snails revealed that the highest at Sialkot 16.34% followed by Attock

14.15% then Lahore 11.53% and lowest at Multan 9.33%. Season wise prevalence of

snails was higher in summer 16.15% and autumn 16.07% followed by spring 8.25% and

lowest during winter 8.17%. Factors which affect population of snail have also been

studied under field conditions including temperature, humidity, rainfall and pan

evaporation.

2.2 INTRODUCTION

The most important as intermediate host for fascioliasis is lymnaeid snails [89].

Intermediate host of liver flukes hermaphroditic fresh water snail species that belongs to

the family lymnaeidae (gastropods: Basommatophora) inhabiting water bodies [90].

These fresh water lymnaeid snails transmit fascioliasis, a pathogenic liver parasitosis

caused by trematode specie of the Genus Fasciola which affects human and livestock

species almost everywhere [16]. The distribution of fascioliasis depends upon the

presence of the specific intermediate host, related to the presence of appropriate (suitable)

water bodies and adequate climatic conditions. Temperature has a marked effect on vital

step of life cycles of flukes, such as the production of cercariae in the molluscan host in

both the aquatic life cycles [91, 92, 93, 94, 95, 96, 97, 98, 99&100] and terrestrial life

CHAPTER 2

PREVALENCE AND ECOLOGY OF VARIOUS

GENRA OF SNAILS IN PUNJAB

55

cycles [101&102]. Cercarial output by snails is a key component of the transmission

success of trematodes [103&104].

The life cycle of both F. gigantica and F. hepatica is essentially similar

[105&106]. Other fresh water snails of the genus Lymnaea may also serve as intermediate

hosts for most of the trematode diseases. Indoplanorbis exustus is a planorbid freshwater

snail which is a host for Schistosoma dermatitis which was reported by [107] to be

commonly spread in most parts of Pakistan and India. Bellamya bengalenis is freshwater

gastropods which serves as intermediate hosts for many blood and liver fluke parasites

[108, 109&110].

In Pakistan freshwater snails are of considerable importance and found throughout

the year except dormancy in extreme hot and cold weather [111].

In Pakistan studies on different fresh water snails have been carried out in the

various provinces including Punjab, Sindh and KPK [81,112,113,114,115, 116,117&118].

Punjab province has a wide resources and large well established irrigation system from

Ravi, Chenab and Jhelum rivers, and fresh water snails had the attention of few authors

and detailed studies are still needed.

It is clear from the factors that affect snail population in Punjab, that temperature

has a great effect population of snails. In Punjab the optimum temperature required for

breeding and reproduction of snails exists during summer season and autumn season. It

was generally noted that lymnaeid snails were found to reach their peak in autumn.

Temperature of 20oC brings the optimum constant for snail growth [119&117]. It was

noted that the overproduction process was not affected by the light period of day length.

Most snails collected in winter were young and in summer they were many. The average

pH essential for the breeding and reproduction of snail is (slightly alkaline 7.2-7.6).

Development pattern of the cercariogenous, stages (larval) appear crucial to

produce cercariae. In Fasciola, more or less long finite production of cercarial appears

related to the number of generations of cercariogenous rediae and independent of the

different species of lymnaeid vector are involved [120&121].

In previous studies include the presence of snail habitats on pasture, proportion of

grazed grass in the diet, length of grazing season, stocking rate, type of drinking water

supply and grazing on mowed pastures, whilst the other factors such as herd size affect

risk of fluke infection, risk via an unknown or combination of mechanisms [122&123].

56

These factors have been found to vary between studies, depending upon the local

environment and farming systems [124].

Lymnaeid snail transmitting F. gigantica can be distinguished morphologically

and as habitat requirement from those of transmitting F. hepatica [125]. Several snails

species may contribute to the transmission of fascioliasis in Egypt among these the main

intermediate host, is water bodies of the Nile Delta are L. truncatula was found naturally

infected with F. gigantica, while Radix natalensis is the essential intermediate host for F.

gigantica based on filed and experimental studies. Larval stages of F. gigantica were

detected in planorbid as Biomphalaria Alexandria in Egypt [126,127,128&129]. Broad

information acquired through studies over many decades indicates a clear performance of

F. hepatica for Galba truncatula and of F. gigantica for Radixnatalensis [89&90].

In Pakistan, during wet summer (July, August) they are in a state of flood while

during hot summer (May and June) many ponds get dry. It seems likely that humidity,

temperature, pH, depth of pond, Vegetation and availability of snails as intermediate host

play an important role [130,131,119,132,86,133,134,135,136,137&138] also recorded the

similar results.

Most herdsmen graze their herds along the river banks during the prolonged dry

season when the pasture is hardly available or in poor quality. These animals are exposed

to the high risk of fascioliasis infection [139].

In Pakistan studies on different fresh water snails have been carried out in various

provinces as Punjab, KPK and Sind [81,112,113,114,115,116,117&118].

Punjab province has a wide resources or extensive irrigation system from Ravi,

Chenab and Jhelum rivers where fresh water snails had the attention of few authors and

detailed studies are still required. In province of Punjab fascioliasis has been found when

development and survival of metacercariae takes place in favourable environmental

where fresh water snails act as their intermediate host.

The relationship of snails was also noted to water depth. It was found essential to

inquire on some properties of the distribution and seasonal incidence as well as other

factors affecting the fresh water snails population as represented by those inhabiting

Punjab province as a whole and in its different districts i.e. Lahore, Sialkot, Attock and

Multan.

57

To find out seasonal distribution of various species of snails also invites to

determine the factors affecting the population of fresh water snails. In Pakistan, much

work has been done on snails but no work has been done on the role of snails in the

epidemiology of fascioliasis through snails in the buffaloes and cattle of Pakistan in

relation to the environmental conditions like temperature, humidity, rainfall and pan

evaporation.


Attached as appendix -B

2.4 RESULTS

During this study period from November 2012 to October 2013, 8273 snails were

studied that were relating to 7-genera as Lymnaea, Indoplanorbis, Bulinus, Physa,

Gyraulus, Bellamaya and Oncomelania.

These snails were collected from four districts as Lahore, Sialkot, Attock and

Multan. The presence of cercariae was studied in all the snails. It was also studied that

there was a specific pattern of month wise and season wise prevalence with level of

infection that varied significantly in snails of some genera that have been collected from

four districts of Punjab, having different land slope, pattern and climatic effects.

Lahore district

A total of 1952 snails were collected during this study. Out of these Lymnaea

were 186 (21.34% infected), Indoplanorbis 330 (14.98% infected), Bulinus 119 (8.31%

infected), Physa 759 (13.51% infected). Gyraulus 197 (6.40% infected), Bellamaya 193

(0% infected), Oncomelania 168 (6.43% infected). Month wise and seasonal prevalence

of snails and presence of cercariae in Lahore district are shown in Table 2.1 and 2.5. Out

of 1952 snails molluscs, 225 (11.53%) were having cercariae. Indoplanorbis, Physa and

Bulinus are the three snails were present in abundance which were quite rich in infection.

The maximum snails 825 (14.84% infected) were collected in summer and the

minimum 213 (6.77% infected) in spring (Table 2.5). It was found that the overall

infection was 11.53 %. The highest month-wise prevalence 22.50 % of trematode

infection was found in August where as the lowest prevalence 3.14 % was found during

January. Bellamya was found to be uninfected with larval treamatodes.

58

Sialkot district

A total of 2402 snails were collected from Sialkot district during this study period.

Out of these Lymnaea were 218 (29.26% infected), Indoplanorbis 420 (22.29% infected),

Bulinus 170 (13.73% infected), Physa 861 (18.92% infected). Gyraulus 250 (9.74%

infected), Bellamaya 249 (0% infected), Oncomelania 234 (10.40% infected). Monthly

and seasonal prevalence of snails and the presence of cercariae in Sialkot district are

shown in Table 2.2 and 2.6. Out of 2402 snails molluscs, 392 (16.34%) were harboring

infection. Indoplanorbis, Physa and Bulinus are the three snails were present in

abundance which were quite rich in infection.



infection was 16.34 %. The highest month-wise prevalence 27.30 % of trematode



Attock district

A total of 2163 snails were collected from Attock district during this study period.


Bulinus 145 (11.19% infected), Physa 811 (16.42% infected). Gyraulus 216 (7.92%

infected), Bellamaya 219 (0% infected), Oncomelania 199 (9.49% infected). Month wise

and seasonal prevalence of snails and the presence of cercariae in Attock district are

shown in Table 2.3 and 2.7. Out of 2163 snails molluscs, 306 (14.15%) were harboring

infection. Indoplanorbis, Physa and Bulinus are the three snails were present in

abundance which were quite rich in infection.



infection was 14.15%. The highest month-wise prevalence 24.80 % of trematode



Multan district

A total of 1756 snails were collected from Multan district during this study period.


59

Bulinus 101 (4.46 % infected), Physa 734 (10.66% infected). Gyraulus 172 (3.66%

infected), Bellamaya 162 (0% infected), Oncomelania 142 (5.72% infected). Month wise

and seasonal prevalence of snails and the presence of cercariae in Multan district are

shown in Table 2.4 and 2.8. Out of 1756 snails molluscs, 164 (9.33%) were having

cercariae. Indoplanorbis, Physa, Bulinus and Oncomelania are the three snails were

present in abundance which were quite rich in infection.



infection was 9.33 %. The highest month-wise prevalence 20.36 % of trematode infection

was found in August where as the lowest prevalence 2.21 % was found during January.

Bellamya was found to be uninfected with larval treamatodes.

Overall prevalence of snails in four districts of Punjab province

A total of 8273 snails were collected out of these 13.14 % were found infected.

Out of the 2402 were collected from Sialkot having infection rate 16.34 % followed by

Attock 2163 (14.15%) then Lahore 1952 (11.53%) and lowest at Multan 1756 (9.33%)

(Fig 2.9).

Genus wise prevalence of snails with infection rate at Lahore district indicated

that Lymnaea, Indoplanorbis, Physa and Bulinus are the predominant genra having

infection rate 21.34, 14.98, 13.51 and 8.31 % respectively. However, the highest numbers

of snails (759) were collected of genus Indoplanorbis.

Genus wise prevalence of snails with infection rate at Sialkot district indicated

that Lymnaea, Indoplanorbis, Physa and Bulinus are the predominant genra having

infection rate 29.26, 22.29, 18.92 and 13.73 % respectively. However, the highest

numbers of snails (861) were collected of genus Indoplanorbis.

Genus wise prevalence of snails with infection rate at Attock district indicated that

Lymnaea, Indoplanorbis, Physa and Bulinus are the predominant genra having infection

rate 25.15, 18.79, 16.42 and 11.19 % respectively. However, the highest numbers of

snails (811) were collected of genus Indoplanorbis.

Genus wise prevalence of snails with infection rate at Multan district indicated

that Lymnaea, Indoplanorbis, Physa and Oncomelania are the predominant genra having

60

infection rate 19.59, 12.32, 10.66 and 5.72 % respectively. However, the highest numbers

of snails (734) were collected of genus Indoplanorbis.

From the results it is indicated that at Sialkot district rice is grown in abundance in

the paddy fields. Constant standing of water helps in breeding and reproduction of snails

and also for the cercariae and metacercariae of the Fasciola.

When analysis of variance was applied highly significant difference (p<0.001)

was noted among Lymnaea and Indoplanorbis with rest of the five genre for the presence

of Fasciola cercariae in four districts of Punjab province. Season wise infection rate of

snails was found highly significant (p<0.001) during summer than winter and spring

whereas, not significant with autumn. District wise prevalence was found highly

significant (p<0.001) at Sialkot as compared to other districts (Fig 2.9).

61

Table 2.1: Month-wise prevalence (%) of infection of different snails at Lahore district from

November 2012 - October 2013

Month Parameter Lymn Indo Buli Phy Gyr Bella Onco Overall

Nov

2012

Collected 7 28 8 60 16 14 12 145

Infected 1 4 0 7 1 0 0 13

(%) 12.50 12.86 0.00 12.00 5.63 0.00 0.00 8.70

Dec

2012

Collected 0 28 0 56 16 19 12 131

Infected 0 3 0 6 1 0 0 10

(%) 0 9.64 0 11.25 5.63 0.00 0.00 7.55

Jan

2013

Collected 8 22 6 54 19 18 16 143

Infected 1 1 0 2 1 0 0 5

(%) 11.25 4.02 0.00 3.31 4.69 0.00 0.00 3.14

Feb

2013

Collected 4 20 10 48 8 6 7 103

Infected 0 2 1 4 1 0 1 8

(%) 0.00 9.00 9.38 7.50 11.25 0.00 12.50 7.85

Mar

2013

Collected 3 16 8 52 12 12 6 110

Infected 0 3 1 3 1 0 1 8

(%) 0.00 16.88 11.25 5.19 7.50 0.00 14.06 7.39

Apr

2013

Collected 5 16 6 48 12 8 8 103

Infected 0 2 1 3 1 0 0 6

(%) 0.00 11.25 14.06 5.63 7.50 0.00 0.00 6.10

May

2013

Collected 3 12 10 46 8 6 7 92

Infected 0 1 1 2 1 0 0 5

(%) 0.00 7.50 9.38 3.95 11.25 0.00 0.00 4.89

Jun

2013

Collected 14 36 10 76 20 24 21 201

Infected 1 6 1 3 1 0 1 13

(%) 6.25 17.50 9.38 3.55 4.50 0.00 4.33 6.27

Jul

2013

Collected 48 39 14 80 24 26 25 256

Infected 9 6 2 23 2 0 2 43

(%) 18.75 16.07 12.50 28.13 7.50 0.00 7.26 16.88

Aug

2013

Collected 50 45 23 92 23 22 20 276

Infected 16 11 2 28 2 0 4 62

(%) 32.14 24.11 7.76 30.33 7.76 0.00 18.00 22.50

Sep

2013

Collected 30 36 15 79 20 22 16 218

Infected 9 9 1 14 1 0 2 35

(%) 29.61 25.00 5.92 17.05 4.50 0.00 11.25 16.07

Oct

2013

Collected 12 32 10 68 18 16 18 174

Infected 3 3 1 10 1 0 1 18

(%) 22.50 8.44 9.38 14.56 4.89 0.00 5.11 10.37

Total

Collected 186 330 119 759 197 193 168 1952

Infected 40 50 10 103 13 0 11 225

(%) 21.34 14.98 8.31 13.51 6.40 0.00 6.43 11.53

62

Table 2.2: Month-wise prevalence (%) of infection of different snails at Sialkot district from



Nov

2012

Collected 10 40 16 70 22 18 19 195

Infected 2 8 2 10 2 0 1 24

(%) 17.31 20.25 11.25 14.22 8.04 0.00 4.69 12.45

Dec

2012

Collected 0 36 9 64 20 23 21 173

Infected 0 5 0 21 2 0 0 27

(%) 0 12.50 0.00 32.34 9.00 0.00 0.00 15.63

Jan

2013

Collected 6 28 8 61 24 20 17 163

Infected 0 5 0 3 0 0 0 8

(%) 0.00 19.29 0.00 4.44 0.00 0.00 0.00 4.96

Feb

2013

Collected 6 24 11 57 12 11 15 136

Infected 2 0 3 5 0 0 1 11

(%) 32.14 0.00 24.11 9.51 0.00 0.00 5.92 7.94

Mar

2013

Collected 4 23 10 55 14 16 8 130

Infected 1 6 2 6 3 0 2 20

(%) 22.50 27.16 18.75 11.41 18.75 0.00 22.50 15.18

Apr

2013

Collected 5 20 8 52 13 12 9 118

Infected 1 5 1 3 3 0 1 13

(%) 18.75 22.50 11.25 5.19 21.09 0.00 10.23 10.64

May

2013

Collected 11 16 12 62 10 8 12 132

Infected 0 4 1 2 2 0 1 9

(%) 0.00 22.50 7.50 2.88 17.31 0.00 7.50 6.82

Jun

2013

Collected 16 44 14 88 26 30 28 246

Infected 2 10 3 5 3 0 3 25

(%) 11.25 22.50 18.75 6.14 10.55 0.00 9.64 10.23

Jul

2013

Collected 54 46 20 90 27 31 29 298

Infected 14 12 5 32 3 0 4 69

(%) 26.47 25.22 22.50 36.16 9.93 0.00 12.50 23.29

Aug

2013

Collected 58 52 29 104 30 28 26 326

Infected 26 16 4 34 4 0 5 89

(%) 45.31 31.15 12.50 32.88 12.16 0.00 20.45 27.30

Sep

2013

Collected 33 48 18 83 27 26 26 261

Infected 12 14 2 23 3 0 5 59

(%) 35.67 30.00 10.23 28.13 9.93 0.00 17.58 22.43

Oct

2013

Collected 16 42 16 75 24 25 24 222

Infected 5 9 3 18 2 0 3 39

(%) 28.13 21.23 16.88 23.94 7.50 0.00 11.25 17.40

Total

Collected 218 420 170 861 250 249 234 2402

Infected 64 94 23 163 24 0 24 392

(%) 29.26 22.29 13.73 18.92 9.74 0.00 10.40 16.34

63

Table 2.3:Month-wise prevalence (%) of infection of different snails at Attock district from



Nov

2012

Collected 8 33 14 68 18 16 16 172

Infected 1 5 1 9 1 0 1 18

(%) 11.25 16.46 6.62 13.24 5.11 0.00 5.63 10.47

Dec

2012

Collected 5 32 7 60 17 20 15 156

Infected 1 4 0 17 1 0 0 23

(%) 18.75 11.25 0.00 28.50 5.36 0.00 0.00 14.42

Jan

2013

Collected 4 24 6 56 20 18 14 142

Infected 0 3 0 2 2 0 0 6

(%) 0.00 11.25 0.00 3.21 9.00 0.00 0.00 4.45

Feb

2013

Collected 5 20 9 52 10 9 12 116

Infected 1 1 2 5 0 0 1 9

(%) 18.75 4.50 20.45 8.65 0.00 0.00 7.50 7.76

Mar

2013

Collected 3 20 8 50 13 14 7 116

Infected 1 5 1 4 2 0 1 13

(%) 28.13 22.50 11.25 7.14 14.06 0.00 12.50 10.86

Apr

2013

Collected 6 18 8 51 11 10 7 111

Infected 1 2 1 1 1 0 1 6

(%) 16.07 9.78 11.25 1.76 8.04 0.00 12.50 5.67

May

2013

Collected 10 16 10 60 10 8 10 124

Infected 1 3 0 1 1 0 1 6

(%) 9.38 16.88 0.00 1.50 9.38 0.00 8.65 5.08

Jun

2013

Collected 14 40 12 80 23 26 24 219

Infected 1 8 2 4 2 0 2 18

(%) 6.62 20.25 15.00 4.50 7.76 0.00 7.50 8.21

Jul

2013

Collected 52 42 16 84 26 28 27 274

Infected 12 9 4 27 3 0 3 57

(%) 22.50 21.63 22.50 32.14 10.55 0.00 9.93 20.66

Aug

2013

Collected 52 48 25 96 27 26 24 298

Infected 19 14 3 32 3 0 5 74

(%) 36.35 28.13 10.89 32.81 9.93 0.00 18.75 24.80

Sep

2013

Collected 33 40 16 82 22 24 23 240

Infected 11 11 2 18 2 0 4 47

(%) 32.93 27.00 11.25 22.06 8.04 0.00 15.52 19.50

Oct

2013

Collected 14 36 14 72 20 20 19 195

Infected 4 6 2 15 1 0 2 30

(%) 26.47 17.50 12.50 21.25 4.50 0.00 9.38 15.22

Total

Collected 204 369 145 811 216 219 199 2163

Infected 51 69 16 133 17 0 19 306

(%) 25.15 18.79 11.19 16.42 7.92 0.00 9.49 14.15

64

Table 2.4: Month-wise prevalence (%) of infection of different snails at Multan district from



Nov

2012

Collected 6 24 7 56 14 11 10 128

Infected 0 3 1 5 0 0 0 9

(%) 0.00 11.25 12.50 9.64 0.00 0.00 0.00 7.03

Dec

2012

Collected 0 24 0 54 12 14 16 120

Infected 0 3 0 5 0 0 0 7

(%) 0 11.25 0 8.27 0.00 0.00 0.00 6.00

Jan

2013

Collected 2 20 4 52 18 14 13 122

Infected 0 1 0 1 1 0 0 3

(%) 0.00 4.50 0.00 1.73 5.11 0.00 0.00 2.21

Feb

2013

Collected 3 16 8 48 6 5 5 91

Infected 0 1 0 2 0 0 0 3

(%) 0.00 5.63 0.00 3.75 0.00 0.00 0.00 2.96

Mar

2013

Collected 2 12 6 51 10 8 4 93

Infected 0 2 0 1 0 0 0 3

(%) 0.00 15.00 0.00 1.76 0.00 0.00 0.00 2.91

Apr

2013

Collected 3 14 4 46 10 7 6 91

Infected 0 1 0 2 0 0 1 4

(%) 0.00 6.62 0.00 3.88 0.00 0.00 14.06 3.95

May

2013

Collected 0 10 8 44 8 6 5 82

Infected 0 0 0 1 0 0 1 2

(%) #DIV/0! 0.00 0.00 2.05 0.00 0.00 18.75 2.21

Jun

2013

Collected 15 32 8 72 18 22 20 187

Infected 1 5 0 2 1 0 1 9

(%) 5.92 14.06 0.00 2.50 5.11 0.00 4.50 4.81

Jul

2013

Collected 44 32 11 76 22 21 19 226

Infected 7 5 1 17 1 0 1 32

(%) 16.36 14.06 8.04 22.50 4.02 0.00 4.69 13.96

Aug

2013

Collected 48 40 20 89 20 19 16 252

Infected 14 9 1 23 2 0 3 51

(%) 30.00 22.50 4.50 25.34 9.00 0.00 16.88 20.36

Sep

2013

Collected 27 32 14 76 18 20 12 199

Infected 7 5 1 12 1 0 1 27

(%) 26.47 16.88 6.62 15.39 4.89 0.00 7.50 13.55

Oct

2013

Collected 10 29 10 70 16 14 15 165

Infected 2 2 1 9 1 0 1 15

(%) 17.31 6.25 8.65 12.93 5.63 0.00 5.92 9.28

Total

Collected 161 285 101 734 172 162 142 1756

Infected 32 35 5 78 6 0 8 164

(%) 19.59 12.32 4.46 10.66 3.66 0.00 5.72 9.33

65

Table 2.5: Season-wise prevalence (%) of infection of different snails at Lahore district

from November 2012 - October 2013


Winter

Nov - Feb

Collected 19 98 23 218 59 57 47 522

Infected 2 9 1 19 4 0 1 35

(%) 9.38 9.15 3.88 8.65 6.08 0.00 1.91 6.72

Spring

Mar - Apr

Collected 8 32 14 100 24 20 14 213

Infected 0 5 2 5 2 0 1 14

(%) 0.00 14.06 12.50 5.40 7.50 0.00 6.25 6.77

Summer

May - Aug

Collected 116 132 57 294 75 78 73 825

Infected 26 24 5 55 5 0 6 122

(%) 22.50 18.41 9.51 18.70 7.18 0.00 8.65 14.84

Autumn

Sep - Oct

Collected 42 68 25 147 38 38 34 392

Infected 12 12 2 23 2 0 3 53

(%) 27.59 17.21 7.26 15.90 4.69 0.00 8.04 13.55

Total

Collected 186 330 119 759 197 193 168 1952

Infected 39.6 49.5 9.9 103 12.6 0 10.8 225

(%) 21.34 14.98 8.31 13.51 6.40 0.00 6.43 11.53

66

Table 2.6: Season- wise prevalence (%) of infection of different snails at Sialkot district from



Winter

Nov - Feb

Collected 22 128 44 251 78 72 72 667

Infected 4 18 5 39 4 0 2 70

(%) 16.67 14.06 10.23 15.41 4.59 0.00 2.50 10.52

Spring

Mar - Apr

Collected 9 43 18 107 27 28 17 249

Infected 2 11 3 9 5 0 3 32

(%) 20.45 25.00 15.34 8.40 19.85 0.00 16.07 13.02

Summer

May - Aug

Collected 139 158 75 344 93 98 95 1002

Infected 42 41 12 74 11 0 13 193

(%) 30.39 26.14 15.56 21.45 11.64 0.00 13.24 19.21

Autumn

Sep - Oct

Collected 49 90 34 158 51 51 50 483

Infected 16 23 5 41 5 0 7 97

(%) 33.20 25.88 13.39 26.14 8.79 0.00 14.52 20.12

Total

Collected 218 420 170 861 250 249 234 2402

Infected 64 94 23 163 24 0 24 392

(%) 29.26 22.29 13.73 18.92 9.74 0.00 10.40 16.34

67

Table 2.7: Season- wise prevalence (%) of infection of different snails at Attock district from



Winter

Nov - Feb

Collected 22 109 35 236 64 63 57 586

Infected 3 13 3 32 4 0 2 56

(%) 12.50 11.58 7.67 13.73 5.63 0.00 3.17 9.53

Spring

Mar - Apr

Collected 9 38 16 102 24 24 14 227

Infected 2 6 2 5 3 0 2 19

(%) 20.45 16.41 11.25 4.43 11.25 0.00 12.50 8.32

Summer

May - Aug

Collected 127 146 63 320 86 88 86 915

Infected 32 33 8 63 8 0 10 155

(%) 25.47 22.87 12.82 19.69 9.46 0.00 11.57 16.91

Autumn

Sep - Oct

Collected 46 76 30 154 42 44 42 435

Infected 14 17 4 33 3 0 5 77

(%) 31.03 22.50 11.84 21.68 6.37 0.00 12.74 17.58

Total

Collected 204 369 145 811 216 219 199 2163

Infected 51 69 16 133 17 0 19 306

(%) 25.15 18.79 11.19 16.42 7.92 0.00 9.49 14.15

68

Table 2.8: Season-wise prevalence (%) of infection of different snails at Multan district

from November 2012 - October 2013


Winter

Nov - Feb

Collected 11 84 19 210 50 43 44 462

Infected 0 7 1 13 1 0 0 22

(%) 0.00 8.57 4.69 5.99 1.81 0.00 0.00 4.68

Spring

Mar - Apr

Collected 5 26 10 98 20 15 10 184

Infected 0 3 0 3 0 0 1 6

(%) 0.00 10.55 0.00 2.77 0.00 0.00 8.65 3.42

Summer

May - Aug

Collected 107 114 47 281 68 69 60 746

Infected 23 18 2 42 4 0 5 94

(%) 20.99 15.73 3.81 15.06 5.29 0.00 9.00 12.54

Autumn

Sep - Oct

Collected 38 61 24 146 34 34 27 364

Infected 9 7 2 21 2 0 2 42

(%) 23.94 11.84 7.50 14.22 5.23 0.00 6.62 11.62

Total

Collected 161 285 101 734 172 162 142 1756

Infected 32 35 5 78 6 0 8 164

(%) 19.59 12.32 4.46 10.66 3.66 0.00 5.72 9.33

69

Table 2.9: District-wise prevalence of different snails in four Districts

Districts Parameter Lymn Indo Buli Phy Gyr Bella Onco Overall

Lahore

Total No 186 330 119 759 197 193 168 1952

Infected 40 50 10 103 13 0 11 225

(%) 21.34 14.98 8.31 13.51 6.40 0.00 6.43 11.53

Sialkot

Total No 218 420 170 861 250 249 234 2402

Infected 64 94 23 163 24 0 24 392

(%) 29.26 22.29 13.73 18.92 9.74 0.00 10.40 16.34

Attock

Total No 204 369 145 811 216 219 199 2163

Infected 51 69 16 133 17 0 19 306

(%) 25.15 18.79 11.19 16.42 7.92 0.00 9.49 14.15

Multan

Total No 161 285 101 734 172 162 142 1756

Infected 32 35 5 78 6 0 8 164

(%) 19.59 12.32 4.46 10.66 3.66 0.00 5.72 9.33

Overall

Total No 769 1404 535 3166 834 822 742 8273

Infected 186 248 54 477 60 0 62 1087

(%) 24.23 17.63 10.09 15.07 7.23 0.00 8.36 13.14

70

Table 2.10: Season-wise prevalence of different snails at four Districts

Seasons Parameters Lymn Indo Buli Phy Gyr Bella Onco Overall

Winter

Total No 74 419 122 916 251 235 220 2237

Infected 8 47 9 103 12 0 5 183

(%) 11.01 11.16 7.40 11.20 4.66 0.00 2.05 8.17

Spring

Total No 30 139 58 406 95 87 56 873

Infected 4 24 6 22 10 0 6 72

(%) 11.84 17.46 10.79 5.31 10.40 0.00 11.25 8.25

Summer

Total No 490 550 242 1238 322 333 314 3489

Infected 123 117 27 234 28 0 34 563

(%) 25.18 21.26 11.14 18.90 8.68 0.00 10.91 16.15

Autumn

Total No 175 295 113 605 166 167 153 1674

Infected 51 59 12 119 11 0 17 269

(%) 29.28 20.12 10.37 19.64 6.49 0.00 11.19 16.07

Overall

Total No 769 1404 535 3166 834 822 742 8273

Infected 186 248 54 477 60 0 62 1087

(%) 24.23 17.63 10.09 15.07 7.23 0.00 8.36 13.14

71

Fig.2.1: Showing month-wise prevalence of different snails at Lahore district. Lym:

Lymnaea Indo: Indoplanorbis Buli: Bulinus Phy: Physa Gyr: Gyraulus Bella:

Bellamya and Onc: Oncomelania in Punjab province

Fig.2.2: Showing season-wise prevalence of different snails at Lahore district. Lym:



72

Fig.2.3: Showing month-wise prevalence of different snails at Sialkot district. Lym:

Lymnaea Indo: Indoplanorbis Buli: Bulinus Phy: Physa Gyr: Gyraulus

Bella: Bellamya and Onc: Oncomelania in Punjab province

Fig.2.4: Showing season-wise prevalence of different snails at Sialkot district. Lym:

Lymnaea Indo: Indoplanorbis Buli: Bulinus Phy: Physa Gyr: Gyraulus

Bella: Bellamya and Onc: Oncomelania in Punjab province

73

Fig.2.5: Showing month-wise prevalence of different snails at Attock district. Lym:



Fig.2.6: Showing Season-wise prevalence of different snails at Attock district. Lym:



74

Fig.2.7: Showing month-wise prevalence of different snails at Multan district. Lym:



Fig.2.8: Showing Season-wise prevalence of different snails at Multan district. Lym:


Bellamya and Onco: Oncomelania in Punjab province

75

Fig. 2.9: Showing district-wise overall prevalence of different snails in Punjab province

Fig. 2.10: Showing season-wise overall prevalence of different snails in Punjab province

76

2.5 DISCUSSION

Fresh water snails play intermediate role to transmit fascioliasis. The infected

hosts start releasing eggs that are passed through faeces which develop into miracidia on

hatching in water, between 2 to 4 weeks, depending on temperature [140]. Miracidia

infect snails, of family Lymnaeidae, that further develop from sporocyst to cercariae

within 6 to 7 weeks, depending upon external temperature. For the development of

miracidia the optimal temperature is 24-26 ºC. During extreme summer (43 ºC)

particularly in dry conditions Fasciola eggs are sensitive and may not survive. Pakistan

has ideal environmental conditions for the development of snails.

In the present study 8273 snails were collected from the four districts of Punjab.

Out of these 1087 (13.14 %) were found infected with trematode cercariae. In various

parts of the world different studies were carried out on snails to know the rate of infection

with treamatodes, and the infection rate found in these snails were nearly similar to that of

the present study [141,142,143,144,145,119,134,136&62].

District wise prevalence of snails indicated that the highest prevalence was

recorded at Sialkot (16.34%) followed by Attock (14.15%), Lahore (11.53%) and the

lowest at Multan (9.33%). Pfukeni [146] reported that the prevalence of snails at different

locations of the country is different, because of difference in environmental and

management conditions. [130,147,131&137] also reported similar findings.

Mixed infection may occur in the liver if the same animals if ecological conditions

conducive for replications of both snails species exist and intermingling of cattle from

various grazing areas occur [31]. Whereas, studies in other African countries suggested

that F. gigantica was the predominant species encountered [148,74,149&150], in Europe,

America and Oceania only F. hepatica is concerned [90].

As the highest prevalence at Sialkot (16.34 %) indicates that the environmental

conditions at Sialkot are more suitable for snail breeding. As there are many ponds,

streams and canals in Sialkot district that is why the high occurrence of snail population is

there.

77

Correlates with the meteorological factors associated with the disease pattern.

Pfukeni [136] also reported that humid and water logged areas increase the prevalence of

snails, [131&151] also reported similar result.

During one year study i.e from November 2012 to October 2013 almost 8273

snails were collected from four districts of Punjab province i.e Lahore, Sialkot, Attock

and Multan. Out of which 1087 (13.14%) were found to be infected with larval

trematode. Similar observations were also recorded by [142,152,80,112,136&153] and

[154&136] 29.4% and 30.8% infection respectively.

Heavily infected snails with fascioliasis were Lymnaea 24.23% and

Indoplanorbis 17.63 % same scientists also reported similar results as

[142,152,155,119,68&156]. Lymnaea and Indoplanorbis are found the most heavily

parasitized snails in Lahore, Sialkot, Attock and Multan districts. It was observed that

many snails in the favourable played vital role to form the trematode life cycle and

reasons for this is clearly an indications of the suitability as host for trematode parasites.

In this study some genra of snails i.e Lymnaea and Indoplanorbis were heavily

parasitized in Sialkot and Attock districts than other districts. This difference in the

incidence rate of infection is because of depth of ponds, temperature, humidity, water

flow, rainfall, vegetation assemblage and variety of definitive host species, [131,132,

157,158,134,136,62,138&159] also explained the same results.

In this study it also indicates that the occurrence of trematode cercariae is

seasonal. It was noted that the percentage of infection of snails in different districts varies

throughout the year as the cercariae of Fasciola were not found around the year. During

July to October the incidence of cercarial stage remain high [142,160,161,80,136&162].

Effect of climatic conditions on the prevalence was found to be highly significant [163].

It was also noted that irrigation system of Punjab province plays an important role in

distribution of snails, [131,132&164] also reported similar results. During summer the

snails reach their maximum count 16.15% followed by autumn 16.07% then spring 8.25%

and the lowest in winter 8.17%. Seasons provide optimum requirements for reproduction

and breeding. So the results found in this study are in agreement with [165&166]. High

temp may reduce the survival rate of cercariae as described by [131,161&137] they also

found that the snails growth requires 20ºC temperature. The temp noted in autumn and

summer was also close to this temperature.

78

For survival of snails and flukes, temperate and humidity are very important

factors [131,119,166,81,137&117] also reported that temperature is important for

hatching and development of flukes and there is no growth of parasite in the snails nor

appearance of cercariae even at or below 10 ºC. Similar results were also reported by

[167].

In this study it was observed that animals produce fluke eggs continently

throughout the year while grazing on pastures or having stalled feeding. When the green

fodder is brought from the fields, having incidence of snails hatching remains continue

but varied from June - October and snails infected in the beginning of July produced

cercariae in the late autumn and summer and give rise to disease in animals during late

autumn and summer [119,166&117] also observed the same results.

The overall results of the study showed that Lymnaea was the most important

intermediate host for the larvae of fascioliasis in all the four districts of Punjab province

with 24.23 % infection rate. Whereas, Indoplanorbis with 17.63 % infection stood

second.

Trematode cercariae were found in all the genra of snails and these snails were

found to occur in common ecological behavior like aquatic inhabitant with permanents

water bodies containing still and clear fresh water with abundant vegetation of alfalfa

(Barseem), Nelumbo (Lotus), Ipomcea (Nari), Nymphaea (Lilly) and Typha (Koondar)

besides paddy.

The infection of Fasciola at Lahore, Sialkot, Attock and Multan districts was

found endemic. Climatic conditions and topography of this region provide suitable

ecological conditions for the parasitic infection. Water logged areas having lack of

effective drainage system are suitable for snails breeding and their continuous exposure to

infection. Grazing of infected animals on contaminated pasture acts as a major source of

infection.

Overall prevalence and infection of snails was found to be highest during summer,

autumn, spring and the lowest in winter.

79


Seven genre of snails including Lymnaea, Indoplanorbis, Bulinus, Physa,

Gyraulus Bellamaya and Oncomelania were collected and identified.

Lymnaea snails were found to be shedding cercariae, which are responsible for the

transmission of fascioliasis.

The highest prevalence of these snails was recorded in Sialkot followed by

Attock, Lahore and the lowest in Multan.

Season-wise prevalence of snails was found the highest in summer, autumn,

spring and the lowest in winter. Infection of snails was highly dependent on climatic

conditions and terrain.

80

3.1 SUMMARY

Results of the present study indicate that metrological factors as humidity,

temperature, rain fall and pan evaporation have vital role in the causation of disease.

Temperature and humidity affect the metabolic processes of parasite, snail and host, thus

interfering the parasite reproduction within snail, snail growth and snail survival rate. No

development of various larval stages within and outside of snails of Fasciola takes below

10°C. At 10°C, little development of larval stages of parasite takes place. The ideal

temperature range in this study was recorded 23-28°C and humidity 50 - 70 % where

efficient development of cercariae and snails take place. For completion of life cycle of

Fasciola, rainfall also plays an important role that helps in spreading cercariae from one

place to other. Maximum prevalence was recorded at 80 - 140 mm pan evaporation.

3.2 INTRODUCTION

Fascioliasis is disease cause by liver fluke parasite, mainly in cattle and sheep, it

has cosmopolitan distribution and is zoonotic [168&169]. The climatic factors are having

very crucial role in the transmission of fascioliasis.

The epidemiology of the disease in closely related to environmental factors, such

as temperature, relative humidity and rainfall [77&170].

Epidemiological dynamics of F. hepatica in temperate regions was evaluated by

[77&171]. Knowledge of seasonal distribution of fascioliasis is helpful for prophylaxis

[172].

Fascioliasis has been reported to very over the years due to variation in amount

and paten of rainfall, while difference in prevalence among geographical location is

mainly due to the altitude and the variation in climatic conditions as rainfall and

temperature [173&19].

CHAPTER 3

PREVALENCE OF FASCIOLIASIS IN RELATION

TO METEROLOGICAL PARAMETERS

81

Climatic factors play a pivotal role in Fasciola transmission in many parts f the

world [97&74].

Most important factors pointed out in fascioliasis is temperature [174] beside

other factors such as water velocity, rainfall, habitat stability and geomorphology

[174&175].

The highest prevalence during autumn is due to the presence of optimal conditions

of environment for the transmission progress of parasitic life cycle stages and its growth

[44]. This includes temperature that ranges from 23-26oC for development of ova

[44&176] and maximal growth of snails [177] and humidity level up to 90 % caused by

plenty of water available enhancing embryonation [178], emergence of miracidium from

eggs due to increased activity of cilia [176&179] and liberation of cercariae from snails.

However, fascioliasis was recorded throughout the year [81]. Fresh water Lymnaeid

snails transmit fascioliasis a highly pathogenic liver diseases caused by trematode species

of genus Fasciola that affects human and livestock almost everywhere [39]. The

distribution of fascioliasis both in space (Latitudinal, longitudinal and altitudinal) and

time (seasonal and yearly) depends upon the presence and population of the specific

intermediate host species in its turn linked to the presence of the appropriate mater bodies

and on adequate climate characteristics enabling fluke development.

Temperature of flukes has a pronounced and direct effect on both its life cycles

and cercarial production in intermediate molluscan host [91,92,93,94,95,96,97,98,

99&100] and terrestrial life cycles [101&102]. The transmission success of trematodes is

a key component of cercarial output by snails [103&104]. Development patterns of larval

stages of the cercarial genus found critical to produce cercariae. In Fasciola, more or less

long, finite production of cercariae appears related to the number of generations of

cercarial genus rediae and independent of the different Lymnaeid vector species involved

[120,180&4]. The presence or absence of the intermediate host species in the area can be

attributed to the variations pertaining to the prevalence and seasonal fluctuations of

various fluke species. Ecological factors such as temperature, water quality and speed of

water currents, etc play an important role in the biology of the larval stages of flukes, and

also of the snail hosts [181,182&183]. The incidence of fascioliasis throughout the year is

indicative of the reality that essential requirements for the fluke life cycle completion

high moisture, moderate temperature and incidence of the snail, are continually present in

the region. The epidemiology of fascioliasis has been studied by several authors in

different countries [184,185,186,187,188,97,189,190&191]. All of these authors agreed

82

that climatic factors i.e. rain fall and temperature, play very important role in the

epidemiology of F. hepatica, since the population dynamics of the snail intermediate host

is affected by them.

The variation in rainfall, altitude, temperature, farm management and suitability of

environments may contribute in the dissemination of parasite and intermediate host.

Fascioliasis distribution pattern in the given area is directly proportion to availability of

fresh water snails [192]. Development of Fasciola in snails is dictated by external

temperature. The optimum temperature required for F. hepatica is 10°C and F. gigantica

is 16°C [193]. Optimum temperature 25-27 °C is required for the development of

Fasciola within the snail and low temperature effects the parasite reproduction [194].

Metrological and severity factors play a pivot role in the prevalence of

fascioliasis. Occurrence of fascioliasis is commonly associated to regions having

temperatures ranging between 10-26 °C [195]. Other conditions such as rainfall, slope

and the presence of wetlands, contribute effectively in maintaining the aquatic molluscs

of the genus Lymnaea, which are intermediate host for larval stages of the parasite [77].

Strong of evidence of the links between occurrence of fascioliasis and climatic

factors has led to create predictive models that help prevent disease incidence on a

broader scale that has enabled to develop and implement the improved control strategies

[196].

Several workers developed prediction models for the incidence of fascioliasis

based on meteorological data [197,198,196,199,200,201,39&202].

Recently, climatic changes have been identified as a major driver in increasing

prevalence of disease of intestinal parasites, because their survival mainly depends on a

number of environmental and climatic factors [174,175,198&203].

Climatic fluctuations can produce effects on the dynamics of vector borne tropical

diseases [204]. Shortened winter periods would increase growth potential in many

parasite populations [205].

Due to strong dependence on environment and weather, the distribution and

prevalence of the fascioliasis is higher in places where mean temperature vary from 10-

25°C or a little more [206].

Small increase in air and water temperature promote the proliferation of infective

stages of trematode [143,175,134,135,203,207,164,138,118&208].

Little development of larval stages of parasite takes place at temperature 10°C. No

cercarial transmission occurs at 5°C or 10°C [167]. The best temp range is 22 to 25°C on

83

which development in the snail occurs in an efficient way, likewise humidity range from

55 to 70°C is ideal for the development of cercariae and snail. Rainfall plays an important

role in completion of life cycle of fascioliasis and it helps in the spread of cercariae from

one place to another. The role of pan evaporation has been discussed by various workers

as well.

The present studies were designed to see the effects of meteorological factors

including temperature, humidity, rainfall and pan evaporation on fascioliasis.


Attached as appendix -C

3.4 RESULTS

From the data of Lahore station, the highest (average) temperature from

November 2012 - October 2013 was 30°C and the lowest (average) temperature was

17.9°C. The average relative humidity was 58.7 %. The average rainfall was 103.6 mm

and pan evaporation rate was 93.9 mm in 24 hours.

It was found that an average maximum temperature 32.7°C found during May

2013 and minimum 11.25°C in January 2013 and maximum relative humidity 78% in

August 2013 and minimum 27 % in May 2013. Maximum rainfall 513.20 mm was found

in August 2013 and minimum 0.10 mm was found in December 2012. The highest pan

evaporation rate 260 mm, 230 mm was found in May and June 2013 respectively and

minimum 40.1 mm was found during December 2012.

From the data of Sialkot station, the highest (average) temperature from


17.2°C. The average relative humidity was 62.6 %. The average rainfall was 114.3 mm

and pan evaporation rate was 99.2 mm in 24 hours.

It was found that an average maximum temperature 32.45°C found during Jun



in August 2013 and minimum 1.40 mm was found in December 2012. The highest pan

evaporation rate 239.5 mm in May 2013 and minimum 39 mm was found in December

2012.

84

From the data of Attock station, the highest (average) temperature from November

2012 - October 2013 was 28.5°C and the lowest (average) temperature was 15°C. The

average relative humidity was 59.2 %. The average rainfall was 162.6 mm and pan

evaporation rate was 96.2 mm in 24 hours.

It was found that an average maximum temperature 30.65°C found during June



in August 2013 and minimum 4 mm was found in December 2012. The highest pan

evaporation rate 205.6 mm in May 2013 and minimum 36.3 mm was found in February

2013.

From the data of Multan station, the highest (average) temperature from


19.7°C. The average relative humidity was 53.1 %. The average rainfall was 20.1 mm and

pan evaporation rate was 159.7 mm in 24 hours.

It was found that an average maximum temperature 35.2°C found during June


August 2013 and minimum 28 % in May 2013. Maximum rainfall 64 mm was found in

February 2013 and minimum 0 mm was found in December 2012 and January 2013. The

highest pan evaporation rate 300.9 mm in May 2013 and minimum 45.5 mm was found in

December 2012.

Data obtained from Lahore, Sialkot, and Rawalpindi (Attock) and Multan stations

was statistically examined and an affirmative association of disease occurrence was found

by interpreting the highest and lowest humidity, temperature, rainfall and pan evaporation

(Fig.3.1 - 3.4).

It has been concluded by analyzing the data statistically that there is highly

significant (p<0.0001) relation of humidity, temperature and rainfall to the incidence of

disease whereas this relationship to other metrological factors is non- significant. During

summer and autumn optimal humidity, temperature and rainfall play a vital role in rapid

propagation of the lifecycle of parasite as shown in Fig 3.2.

85

Table 3.1: Average month-wise temp, humidity, rainfall and pan evaporation at Lahore from

November 2012 to October 2013

Month Temperature (°C)

Relative

Humidity %

Rainfall

(mm)

Pan evaporation

(mm) Min Max

Nov-2012 11.3 26.5 56 0.10 67.9

Dec-2012 7.3 20.9 67 11.00 40.1

Jan-2013 5.6 16.9 67 16.20 ***

Feb-2013 9.9 20.5 68 75.00 ***

Mar-2013 14.9 28.1 53 15.10 ***

Apr-2013 19.8 34 37 17.50 ***

May-2013 25.1 40.3 27 6.00 260.3

Jun-2013 26.5 38.7 53 161.40 230.3

Jul-2013 25.2 35.3 72 221.50 204.6

Aug-2013 24.8 32 78 513.20 118.8

Sep-2013 24.4 35.1 65 168.30 124.6

Oct-2013 20.5 32.1 63 37.60 79.8

Average 17.9 30.0 58.7 103.6 93.9

86

Table 3.2: Average month-wise temp, humidity, rainfall and pan evaporation at Sialkot from

November 2012 to October 2013

Month

Temperature (ºC) Relative

Humidity %

Rainfall

(mm)

Pan evaporation

(mm) Min Max

Nov-2012 10.2 24.9 62 3.10 55.2

Dec-2012 6.3 19.9 70 1.40 39

Jan-2013 4.8 15.9 74 45.10 44.8

Feb-2013 8.8 19.5 75 142.10 30.4

Mar-2013 13.5 26.5 62 27.10 95.8

Apr-2013 18.6 32.7 42 2.20 150.3

May-2013 23.6 39.6 28 4.30 239.5

Jun-2013 26.2 38.7 49 69.10 183.4

Jul-2013 25.8 34.2 71 314.40 101.6

Aug-2013 25.1 31.9 80 670.30 63.7

Sep-2013 23.7 33.2 71 76.20 107.4

Oct-2013 20.1 30.9 68 16.30 79.6

Average 17.2 29.0 62.6 114.3 99.2

87

Table 3.3: Average month-wise temp, humidity, rainfall and pan evaporation at Attock

from November 2012 to October 2013

Month


Humidity %

Rainfall

(mm)

Pan evaporation

(mm) Min Max

Nov-2012 13.0 26.5 59 12.10 51.6

Dec-2012 3.7 20.1 60 4.00 39.8

Jan-2013 2.1 18.1 67 13.10 40.1

Feb-2013 6.7 18.6 71 306.10 36.3

Mar-2013 10.9 25.9 58 69.40 90

Apr-2013 14.9 29.7 48 32.40 118.9

May-2013 18.7 36.3 35 13.40 205.6

Jun-2013 23.5 37.8 44 119.30 156

Jul-2013 24.5 34.6 66 317.50 124.1

Aug-2013 23.5 31.9 76 670.30 90.5

Sep-2013 21.6 32.0 68 336.00 100

Oct-2013 17.0 30.2 61 58.10 101.2

Average 15.0 28.5 59.2 162.6 96.2

88

Table 3.4: Average month-wise temp, humidity, rainfall and pan evaporation at Multan

from November 2012 to October 2013

Month


Humidity %

Rainfall

(mm)

Pan evaporation

(mm) Min Max

Nov-2012 13.1 27.3 55 1.10 72.8

Dec-2012 8.1 21.7 65 0.00 45.5

Jan-2013 5.5 19.4 61 0.00 54.9

Feb-2013 11.0 21.6 68 64.50 67.0

Mar-2013 15.0 28.8 54 18.20 144.8

Apr-2013 20.5 34.4 41 5.50 195.2

May-2013 26.1 41.4 28 4.10 300.9

Jun-2013 29.3 41.1 44 47.10 277.8

Jul-2013 30.0 39.5 54 58.00 246.4

Aug-2013 28.1 37.1 63 38.00 182.9

Sep-2013 26.6 37.1 54 0.00 185.0

Oct-2013 22.9 34.5 52 5.00 142.9

Average 19.7 32.0 53.1 20.1 159.7

89

Table 3.5: Prevalence of fascioliasis infection with reference to environmental temperature

at four districts

Month

Lahore

Sialkot Attock Multan

Mean

Temp 0C

Prevalence

%

Mean

Temp 0C

Prevalence

%

Mean

Temp 0C

Prevalence

%

Mean

Temp 0C

Prevalence

%

Nov

2012 18.9 14.0 17.6 12.0 19.8 13.0 20.2 9.0

Dec

2012 14.1 5.0 13.1 7.0 11.9 6.0 14.9 6.0

Jan

2013 11.3 8.0 10.4 8.0 0.0 7.0 12.5 4.0

Feb

2013 15.2 12.0 14.2 13.0 0.0 16.0 16.3 6.0

Mar

2013 21.5 26.0 20.0 34.0 0.0 26.0 21.9 15.0

Apr

2013 26.9 26.0 25.7 21.0 22.3 27.0 27.5 18.0

May

2013 32.7 7.0 31.6 6.0 27.5 4.0 33.8 4.0

Jun

2013 32.6 6.0 32.5 6.0 30.6 4.0 35.2 3.0

Jul

2013 30.3 10.0 30.0 7.0 29.6 10.0 34.8 6.0

Aug

2013 28.4 13.0 28.5 15.0 27.7 16.0 32.6 9.0

Sep

2013 29.8 41.0 28.5 51.0 26.8 39.0 31.9 19.0

Oct

2013 26.3 44.0 25.5 35.0 23.6 39.0 28.7 24.0

Mean 24.0 17.7 23.1 17.9 18.3 17.3 25.8 10.3

± ± ± ± ± ± ± ± ±

STDEV 7.5 13.5 7.7 14.6 12.1 12.7 8.3 7.0

S.E 1.4 2.5 1.4 2.7 2.2 2.3 1.5 1.3

Mean

±

SEM

24.69

±

1.32

42.36

±

3.18

24.8

±

1.38

39.64

±

3.2

24.75

±

1.38

46

±

4.21

24.56

±

1.31

42.18

±

3.49

90

Table 3.6: Maximum and minimum Prevalence of Fasciolosis infestation with reference to

Temperature at four districts of Punjab

Factors Maximum Minimum

Lahore Sialkot Attock Multan Lahore Sialkot Attock Multan

Temperature0C 26.3 28.45 26.79 31.85 32.6 31.6 27.50 35.2

Prevalence % 44 51 39.00 19 6 6 4 3

Fig 3.1: Graph showing maximum and minimum prevalence of fascioliasis in

relation to environmental temp in four districts

91

Table 3.7: Prevalence of fascioliasis in relation to relative humidity in four districts

Month

Lahore Sialkot Attock Multan

Humidity

%

Prevalence

%

Humidity

%

Prevalence

%

Humidity

%

Prevalence

%

Humidity

%

Prevalence

%

Nov

2012 56 14 62 12 59 13 55 9

Dec

2012 67 5 70 7 60 6 65 6

Jan

2013 67 8 74 8 67 7 61 4

Feb

2013 68 12 75 13 71 16 68 6

Mar

2013 53 26 62 34 58 26 54 15

Apr

2013 37 26 42 21 48 27 41 18

May

2013 27 7 28 6 35 4 28 4

Jun

2013 53 6 49 6 44 4 44 3

Jul

2013 72 10 71 7 66 10 54 6

Aug

2013 78 13 80 15 76 16 63 9

Sep

2013 65 41 71 51 68 39 54 19

Oct

2013 63 44 68 35 61 39 52 24

92

Table 3.8: Maximum and minimum prevalence of Fasciolosis infestation with

reference to relative humidity at four districts of Punjab



Humadity % 63 71 61 52 53 28 35 28

Prevalence % 44 51 39 28 6 6 4 4


relation to environmental humidity in four districts

93

Table 3.9: Prevalence of fascioliasis infection in relation to total rain fall in four districts

Month


Rain

Fall

mm

Prevalence

%

Rain

Fall

mm

Prevalence

%

Rain

Fall

mm

Prevalence

%

Rain

Fall

mm

Prevalence

%

Nov

2012 0.1 14.0 3.1 12.0 12.1 13.0 1.1 9.0

Dec

2012 11.0 5.0 1.4 7.0 4.0 6.0 0.0 6.0

Jan

2013 16.2 8.0 45.1 8.0 13.1 7.0 0.0 4.0

Feb

2013 75.0 12.0 142.1 13.0 306.1 16.0 64.5 6.0

Mar

2013 15.1 26.0 27.1 34.0 69.4 26.0 18.2 15.0

Apr

2013 17.5 26.0 2.2 21.0 32.4 27.0 5.5 18.0

May

2013 6.0 7.0 4.3 6.0 13.4 4.0 4.1 4.0

Jun

2013 161.4 6.0 69.1 6.0 119.3 4.0 47.1 3.0

Jul

2013 221.5 10.0 314.4 7.0 317.5 10.0 58.0 6.0

Aug

2013 513.2 13.0 670.3 15.0 670.3 16.0 38.0 9.0

Sep

2013 168.3 41.0 76.2 51.0 336.0 39.0 0.0 19.0

Oct

2013 37.6 44.0 16.3 35.0 58.1 39.0 5.0 24.0

94

Table 3.10: Maximum and minimum prevalence of Fasciolosis infestation with

reference rainfall at four districts of Punjab.



Rain Fall mm 37.6 76.2 58.1 5.0 161.4 4.3 13.4 4.1

Prevalence % 44.0 51.0 39.0 24.0 6.0 6.0 4.0 4.0


relation to rainfall in four districts

95

Table 3.11: Prevalence of fascioliasis infection with reference to pan evaporation mm in four

districts

Month


Pan

Evapora-

tion (mm)

Prev-

alence

%

Pan

Evapor-

ation (mm)

Prev-

alence

%

Pan

Evapor-

ation (mm)

Prev-

alence

%

Pan

Evapor-

ation (mm)

Prev-

alence

%

Nov

2012 67.9 16.0 55.2 11.0 51.6 12.0 72.8 9.0

Dec

2012 40.1 9.0 39.0 7.0 39.8 6.0 45.5 7.0

Jan

2013 *** 11.0 44.8 10.0 40.1 9.0 54.9 4.0

Feb

2013 *** 16.0 30.4 18.0 36.3 16.0 67.0 6.0

Mar

2013 *** 35.0 95.8 40.0 90.0 25.0 144.8 17.0

Apr

2013 *** 31.0 150.3 21.0 118.9 27.0 195.2 18.0

May

2013 260.3 10.0 239.5 6.0 205.6 4.0 300.9 4.0

Jun

2013 230.3 6.0 183.4 6.0 156.0 4.0 277.8 4.0

Jul

2013 204.6 10.0 101.6 13.0 124.1 10.0 246.4 6.0

Aug

2013 118.8 15.0 63.7 16.0 90.5 16.0 182.9 9.0

Sep

2013 124.6 36.0 107.4 51.0 100.0 34.0 185.0 21.0

Oct

2013 79.8 44.0 79.6 35.0 101.2 40.0 142.9 24.0

96

Table 3.12: Maximum and minimum Prevalence of Fasciolosis infestation with

reference to Pan Evaporation mm at four districts of Punjab



Pan Evaporation mm

79.8 107.4 101.2 142.9 230.3 183.4 156.0 54.9

Prevalence % 44.0 51.0 40.0 24.0 6.0 6.0 4.0 4.0

Fig 3.4: Graph showing maximum and minimum prevalence of fascioliasis in relation to

Pan Evaporation (mm) in four districts

97

Table 3.13: Overall maximum / minimum prevalence of fascioliasis infection in relation to

meteorological factors in Punjab

Districts

Prevalence

%

Maximum

Met-factors

Pan

Prevalence

%

Minimum

Met-factors

Pan

Temp 0C

Hum

%

Rain

mm

Temp 0C

Hum

%

Rain

mm

Lahore 44.00 26.30 63 37.6 79.8 6 32.60 53 161.4 230.3

Sialkot 51.00 28.45 71 76.2 107.4 6 31.60 49 69.1 183.4

Attock 39.00 23.61 61 58.1 101.2 4 27.50 44 13.4 205.6

Multan 24.00 28.70 52 142.9 142.9 4 33.75 61 0 54.9

98

3.5 DISCUSSION

The landscape of Punjab province is amongst the most heavily irrigated and canals

can be found throughout the area. Besides that, Punjab receives abundant rainfall.

Temperature and humidity are favourable for the growth and multiplication of Fasciola

and snail, so the incidence order from highest to lowest is as autumn, spring, winter and

the summer. These findings are closely related to those of [46,126,209&210]. Infection

was the highest at Sialkot followed by Lahore, Attock while the lowest was at Multan.

This may be due to the fact that high infection rate was thought to be associated with the

extension of the canal system providing additional areas of swamp and marsh where the

crossbred cows were exposed to infective larvae and metacercariae of helminths as was

also noted by [211,212&213]. They described that moisture and temperature play a vital

role on the incidence of fascioliasis as these factors affect hatching of fluke ova and the

capability of encysting cercariae and snail’s population [214].

In this study, it was observed that temperature, rainfall, humidity and pan

evaporation have intensive effect on the prevalence of fascioliasis due to favourable

environment for intermediate host (Snails). Pan evaporation factor shows maximum

prevalence 51 % at 107.4 mm pan evaporation and lowest 4 5 prevalence at 156.9 mm

pan evaporation that indicates that increase in evaporation deceases incidence as the more

temperature is required for more pan evaporation. The amount of variation explained by

management factors and climate is comparable with two recent studies comparing the

herd level variation [122&69]. Some studies showed that the prevalence of fluke and

other snail borne diseases have strong effect with rainfall, slope, stocking density and

temperature [122,123&215].

Study of the climatic factors indicated that humidity increases after intense rainfall

in July-August (221.5mm and 513.2mm, accordingly), which results that the maximum

prevalence 51 % was recorded at 76.2 mm rainfall and minimum prevalence 4 5% at 4.1

mm rainfall that means prevalence is directly proportional to the rainfall. Risk of infection

in September and October, indicated the positive relation between infection and humidity

as described by [81]. Lowest infection was found in May and June is linked to incidence

of hot and dry weather, as the temperature was high (up to 32°C) and humidity was low in

these months. In January the prevalence was again increased after rainfall when humidity

increases, indicating a strong relation between humidity and the disease as reported by

[81,87,74&31].

99

In this study maximum 51 % prevalence of fascioliasis was recorded at 71 %

humidity and minimum 4 % prevalence at 28 % humidity showing strong effect of the

humidity on causation of disease whereas [172] an enhanced prevalence of fascioliasis in

cattle was found after some weeks when there is an increase in the population of infected

and non-infected snails after rise in humidity.

Fasciola gigantica was the dominant liver fluke in the study animals. This may be

due to the favourable metrological environment under existence of favourable

intermediate host Lymnaea. Small proportion of crossbred cows were found infected with

mixed infection by both the species. Mixed infection may occur in liver of the same

animal if ecological conditions are conducive for replication of both snail species and

intermingling of cattle from various grazing areas occur [31]. Studies in other countries of

Asia and Africa showed that F. gigantica was the predominant species encountered

[148,74,149&150] where as in the Americas, Oceania and Europe only F. hepatica is

concerned [90].

In nature, physical and chemical factors of snails vary significantly. Organisms

live easily in the ambient climatic conditions as they have changed themselves

accordingly. In this study it was found that various climatic conditions as humidity,

temperature, rainfall and pan evaporation play specific role to completion lifecycle of

fascioliasis as described by [141,143,79,81&118].

For breeding and reproduction of snails and parasites an optimum temperature is

required which is found in Punjab during summer season followed by autumn. The

present study shows that maximum 51% prevalence was recorded at 28.45°C temperature

and minimum 3% at 35.2°C. It means there is a strong link between temperature and

prevalence of disease as the temperature increases prevalence decreases after 26°C. in this

study it reveled that during summer and autumn maximum parasites and snails were

found as described by [143,111,122,175,39,134,135,96,203,207, 164&216].

For survival of snails and flukes both temperature and humidity are important

factors [131,119,166,157,137&117].

100


For the development and growth of snails climatic conditions of Pakistan are quite

favourable. Throughout the year snails are found in large quantity in canals and ponds

except they remained dormant for a short period in spring and winter. Development of

flukes outside the final host takes place from autumn to early winter as summer is usually

remain dry.

From the results it is established that humidity and temperature play a vital role to

cause disease. Suitable temperature and humidity are responsible for the severity of

disease. Rainfall plays important role in dissemination of snails and cercariae of Fasciola

from one place to other place.

101

4.1 SUMMARY

One hundred and ten crossbred cows were included in this studies (100 positive

for fascioliasis (infected) and 10 healthy crossbred cows) to compare the efficacy of three

indigenous herbal and one allopathic drugs, including Nigella sativa, Fumaria parviflora

and Flemingia macrophylla with Triclabendazole (ICI) to treat fascioliasis. Effectiveness

was measured by reduction in eggs per gram faeces (EPG) before and after the treatment

of diseased animals. After giving the first dose of Nigella sativa at dose rate of 80, 100

and 120 mg/kg body weight, EPG reduced by 46.34, 51.35 and 66.67 per cent

respectively. After giving the second dose the reduction in EPG was 82.93, 89.19 and

97.44 % respectively. After giving the first dose of Fumaria parviflora at dose rate of 80,

100 and 120 mg/kg body weight, EPG was reduced by 53.66, 56.10 and 50 %


90.48 % respectively. On giving first dose of Flemingia macrophylla at dose rate of 80,

100 and 120 mg/kg body weight, EPG was reduced by 64.10, 73.17 and 74.36 %


94.87% respectively. Efficacy of triclabendazole at first dose level of 10 ml/kg body

weight was 67.50 % and at second dose level it was 97.50%. Order of efficacy from high

to low is triclabendazole, Nigella sativa, Flemingia macrophylla and Fumaria parviflora.

4.2 INTRODUCTION

Parasitic infection is a big threat to the overall industry of livestock [217]. In

Pakistan the highest prevalence of helminthiasis had been found in sheep and goats [218]

and then in cattle and buffaloes [219]. This study was designed because the allopathic

dewormer is very expensive as compared to the indigenous herbal drugs. That is why we

CHAPTER 4

THERAPEUTIC TRAILS WITH HERBAL AND

ALLOPATHIC DRUGS AGAINST FASCIOLIASIS IN

CROSSBRED COWS

102

can save billions of foreign exchange by using effective indigenous herbal drugs. At

present the infection is generally controlled by synthetic drugs and vaccination [220].

The aim of fluke control should be to reduce the infection intensity in snails and in

cattle [197&221]. Factors of the farm management may have an effect on the chances of

cattle getting in touch with infective metacercariae [122]. Fluke prevalence remains high

also in countries where flukicides are used routinely [124]. There are reports of resistance

to commonly used anthelmintics making fluke a threat to animal health, welfare and

agricultural productivity [222,223&224].

Parasitic infection has poor effects on the production of small ruminants causing

heavy financial losses especially where negligence and meager control practices are

common as found in developing countries [225,226,218&227].

The possible reasons of increased rate of fascioliasis can include (a) Due to

offensive use of fasciolicides the resistance developed even including the frequent use of

same dewormer for a longer time and improper doses [228&229], (b) Due to lack of

regular assessment of local existing drugs against parasitic disease or use of improper

drugs against parasitic infections [230] and (c) Socio-economic position of the farmer to

treat the annoyance [230].

The treatment is essential for controlling spread of fascioliasis as infected animals

pass eggs through feces. Ranges of anthelmintics [231] effective in killing of different

developmental stages of parasites are available. Indiscriminate use of anthelmintics has

caused resistance [229] so the disease has not been eradicated. Mixture of some

dewormer found highly efficient to treat the immature and mature flukes [232]. In

developed countries it is supported by analyzing on costs and benefits, but there are

different priorities in developing countries to utilize the limited cash reserves instead for

the treatment of animals. Treating with an anthelmintic four times in a year is successful

against young parasites, but there are few marked places where it will be feasible. The

most frequently used drug in F. hepatica infected dairy herds is Albendazole [59], a broad

spectrum dewormer which is effective against nematodes in the GI tract as well, but have

a negative effect on the production of milk [233]. Oxyclozanide is found to be the only

efficient dewormer against mature flukes over fourteen weeks old [231&234],

triclabendazole is more effective against the entire phases of parasite and better milk

production probably expected by the use of this drugs. Triclabendazole (TCBZ) is a

current dewormer of choice to be used for the treatment of Fasciola hepatica infections,

because of its high activity against both adult flukes and Juvenile [235&236]. The regular

103

use of triclabendazole is creating populations of F. hepatica that are resistant to it

[236&237]. One possible policy to reduce resistance is by manipulating the drug

pharmacokinetics, and increasing its bioavailability and active lifespan, to increase its

effectiveness. Metabolism of benzimidazole kind drugs, as triclabendazole, may be

affected when treated with inhibitors which mark the flavin monooxygenase (FMO) and

Cyto-chrome (CyP450) enzyme pathway. It may direct to an augmentation of the bio-

availability of the active metabolisms [238,239,240,241,242,243,83,244&245]. In turn,

the enhanced bio-availability is considered to improve the efficiency of the medicine

[246,247,248,249,250&251]. Rediae of F. hepatica [247&248] and F. gigantica [251]

can reduce or sterilize the fertility by damaging the gonads of Lymnaeid snails. Fasciola

hepatica in Lymnaea truncatula [252] and Fasciola gigantica in Lymnaea natalensis

[253] harm the snail’s growth in the early phase of infection. But at later stage rate of

growth may be stimulated by Fasciola hepatica [254&251]. When there are intense

infections of Fasciola hepatica it kills the snails [247,255&256]. Rediae of one species of

trematode can consume the sporocyst of the other species [94]. Rediae of Echinostoma

audyi eliminate Fasciola gigantica from snails [257]. Echinostoma revolutum eliminates

existing F. gigantica infections and prevent super infection [258]. In some circumstances

there have been considerable achievements for biological control of schistosomiasis in the

field, mainly with contestant snails and trematode antagonisms, revived by [259&260]

but on the whole there is little impact on the disease. Cattle attain resistance against the

distinct infections with F. hepatica and some or all of the resistance may be a result of

fibrosis of the liver parenchyma, and fibrosis and calcification in the bile duct

[261,262,263&189]. Although dewormer is effective treatment against fascioliasis, which

is an expensive and un-sustainable method due to which drug resistant strains are

developing [264&265]. The substitute to dewormer is the development of a vaccine.

Vaccines are as cost effective as fasciolicide in developed countries. Vaccines in

developing countries would have to be affordable, but would have the benefit that their

efficacy would be independent at all levels of infection in other animals in the

community. Lymnaeid Snails are vulnerable to predators such as, crustaceans, birds,

amphibians, reptiles and rodents as they are having thin shells and without operculum.

The resistance is developing continuously on the part of helminths against

commercial drugs that have increased the interest of researchers in traditionally used

medicinal plants [266,267,268&60]. Traditional use of medicinal plants as anthelmintics

104

(dewormer) has been reported from different parts of the world [269,270,271,272,

273,274&275].

Activities of various botanical anthelmintics have been reported e.g. Artemisia

brevifolia [276&277], Asimina triloba [278], Calotropis procera [279], Nicotiana

tabacum [280], Adhatoda vasica [281], Leonotis ocymifolia [282], Leuceana

leucocephala [283], Jatropha curcas, Chenopodium ambrosioides and Lawsonia inermis

[284]. Ferula costata (Kor.) ver. Naraan (Pashto) belongs to the family Umbelliferae. It

has wide distribution in Pakistan and Afghanistan. It’s distribution in Pakistan is

restricted to Northern Balochistan and Khyber Pukhtoonkhwa [285]. Unpublished reports

claim antiparasitic use of F. costata and other species of Ferula. Extracts of F. costata

[286], F. persica [287], F. hermonis [288] and F. lycia [289] possessed antibacterial

activity. Ferula narthex is locally in use for gastric problems and anti-constipation [2].

The use of medicinal plants for the prevention and treatment of gastrointestinal

parasitism has its origin in ethno veterinary medicine. Some anthelmintic herbal drugs

prepared by the medicinal plants, are effective against helminths parasites and kill them

[290,291,292,293,294,279&224].

However, Pasture drainage is another option but in most cases this is impractical,

prohibitively expensive [274] and is discouraged in the UK for environmental reasons. In

adult cattle most fluke infections are sub-clinical, yet are of economic importance

[295&21]. There is considerable evidence from around the world that fluke infection has

an adverse effect on milk production in dairy cows. Decreased in the milk production is

reported between 8 to 15%, which is 0.7 to 4.2 kg per cattle per day

[233,33,296,2,297,298,299,300&21]. Reduction in butterfat was reported in some studies

[301,233,33,24,2&298].

Some workers reported the effects of fascioliasis on fertility, they have reported

late puberty and an enlarged calving break in young animals [233&249], but some

establish that there is no change [298&302]. The magnitude of the effect depends on the

husbandry system used and the breed of cow, which varies greatly among countries

studied, even within Western Europe [303&298] the habitations, rotation of grazing,

minimum grazing on contaminated pastures and combination of treatment with

fasciolicides [212&274].

In the past Fascioliasis control was limited as there was lack of proper early

diagnostic test. It has been diagnosed / confirmed by the latest studies through molecular

characterizations and adult morphology that the intermediate forms of F. hepatica and F.

105

gigantica is present in the Punjab [304,305,306&307]. However, to ensure it needs to be

considered in a view to the single farm condition, that these methods are effective or not,

and the veterinarian has an important role in advising on holistic fluke control [212].

Currently, control of F. hepatica is generally achieved using anthelmintic

treatment, however more widespread application of herd ELISA status data will allow

greater evidence-based control at farm level [3]. This in turn will contribute to more

appropriate and sustainable use of flukicides treatment in dairy herds [33], which could

result in a reduction in anthelmintic use and reduce selection for anthelmintic resistance

[308].

The high cost of these drugs, its residual concern in food animals and

environmental pollution have awaken interest in medicinal plants as an alternative source

of anthelmintic drugs [309,226,310&311]. There are some reports on plants with

anthelmintic properties from Asia and Africa [312,313,314,315,226,280,316,273&317]

America [311] and Europe [318&310] that have shown that plants may be a good

alternative for the treatment of helminthiasis.

Nigella sativa, Fumaria parviflora and Flemingia macrophylla are mainly found

in the Indo-Pakistan subcontinent. These herbs are used as an anthelmintic in the

customary veterinary rehearses in the Pakistan. Some reports have also been seen on

anthelmintic use of F. parviflora as by the use of this drug there is reduction in faecal egg

count (FEC) [313&310]. Basic aim of this study was to carry out the herbal drug trial to

confirm the anthelmintic activity and efficacy against fascioliasis.


Attached as appendix -D

4.4 RESULTS

Drugs trials

After having gone through the drug trials of three herbal and one allopathic

following results have been found.

Nigella sativa

Reduction in EPG count in the faeces and treatment trials of fascioliasis positive

crossbred cows with this herbal drug at the dose rate of 80mg/kg body is presented in the

Table 4.12 and Fig 4.2. After the treatment with single dose on day 18th

the efficacy of N.

sativa was established 46.34 %. Whereas, on day 28th

when the animal was given the

106

second dose the effectiveness of the drug was elevated to 82.93 percent. It indicates that

on day 18th

and 28th

after the treatment of diseased cows, there is significant increase

(P<0.0001) in the efficacy of the drug.


crossbred cows with this herbal drug at the dose rate of 100 mg/kg body is presented in

the Table 4.13 and Fig 4.3. After the treatment with single dose on day 18th

the efficacy of

Nigella sativa was established 51.35 %. Whereas, on day 28th

when the animal was given

the second dose the effectiveness of the drug was elevated to 89.19 percent. It indicates

that on day 18th

and 28th






the efficacy of

N. sativa was established 66.67 %. Whereas, on day 28th

when the animal was given the

second dose the effectiveness of the drug was elevated to 97.44 percent. It indicates that

on day 18th

and 28th



At dose rate of 120 mg/kg body weight indicated that Nigella sativa remained

efficient against fascioliasis in crossbred cows. But the efficacy of Nigella sativa and

triclabendazole almost remained equal.

Effect on Milk Production

The treatment effect of N. sativa is shown in Table 4.27 below. When the diseased

animals were treated with this herbal drug positive effect was seen on the milk

production. After giving the second dose to the in milk fascioliasis positive animals at the

dose rate of 80, 100 and 120 mg/kg body weight on day 28th

there was an increase in

milk production as 21.38%, 26.85% and 34.90% in groups Al, A2 and A3 respectively.

Effect of drug on pregnancy

There is no evident adverse effect was noticed in the pregnant cows. The study

was carried out on four cows out of which one was in subgroup A1, two were in A2 and

one was in A3 subgroup.

107

Effect of drug on body condition

After the treatment the body condition of the cows improved gradually. The cows

turned into vigorous and healthy.

It has been noticed in Table 4.12 – 4.14 and Fig 4.5 that Nigella sativa remained

very effective at dose rates of 80, 100 and 120 mg/kg body weight. The P-value was

found P<0.001 at all doses levels with that of the control group. Moreover, P-value was

noted P>0.05 while comparing the dose levels of Nigella Sativa to that of triclabendazole.

However, the triclabendazole was found better in terms of percentage as compared to that

of Nigella sativa. Nigella sativa was compared at dose rate of 80, 100 and 120 mg/kg

body weight to triclabendazole and control group as described in Fig 4.5. There was no

side effect of the drug. The body condition of the animals became better and improved

after the treatment.

Fumaria parviflora




the efficacy of

Fumaria parviflora was established 53.66 %. Whereas, on day 28th

when the animal was

given the second dose the effectiveness of the drug was elevated to 85.37 percent. It

indicates that on day 18th

and 28th

after the treatment of diseased cows, there is significant

increase (P<0.001) in the efficacy of the drug.




the efficacy of

Fumaria parviflora was established 56.10 %. Whereas, on day 28th

when the animal was



and 28th






the efficacy of

Fumaria parviflora was established 50 %. Whereas, on day 28th

when the animal was


108


and 28th



At dose rate of 120 mg/kg body weight indicated that Fumaria parviflora

remained efficient against fascioliasis in crossbred cows. But the efficacy of Nigella

sativa and triclabendazole remained enhanced than that of Fumaria parviflora.


The treatment effect of Fumaria parviflora is shown in Table 4.27 below. When

the diseased animals were treated with this herbal drug positive effect was seen on the

milk production. After giving the second dose to the in milk fascioliasis positive animals

at the dose rate of 80, 100 and 120 mg/kg body weight on day 28th

there was an increase

in milk production as 19.49%, 23.59% and 27.84% in groups Bl, B2 and B3 respectively.



was carried out on six cows out of which two were in subgroup B1, one was in B2 and

three were in B3 subgroup.




It has been noticed in Table 4.15 – 4.17 and Fig 4.9 that Fumaria parviflora

remained very effective at dose rates of 80, 100 and 120 mg/kg body weight. The P-value

was found P<0.001 at all doses levels with that of the control group. Moreover, P-value

was noted P>0.05 while comparing the dose levels of Fumaria parviflora to that of

triclabendazole. However, the triclabendazole was found better in terms of percentage as

compared to that of Fumaria parviflora. Fumaria parviflora was compared at dose rate of

80, 100 and 120 mg/kg body weight to triclabendazole and control group as described in

Fig 4.9. There was no side effect of the drug. The body condition of the animals became

better and improved after the treatment.

Flemingia macrophylla




the efficacy of

109

Flemingia macrophylla was established 64.10 %. Whereas, on day 28th

when the animal

was given the second dose the effectiveness of the drug was elevated to 87.18 percent. It


and 28th






the efficacy

of Flemingia macrophylla was established 73.17 %. Whereas, on day 28th

when the

animal was given the second dose the effectiveness of the drug was elevated to 92.68

percent. It indicates that on day 18th

and 28th

after the treatment of diseased cows, there is

significant increase (P<0.001) in the efficacy of the drug.




the efficacy

of Flemingia macrophylla was established 73.36 %. Whereas, on day 28th

when the

animal was given the second dose the effectiveness of the drug was elevated to 94.87

percent. It indicates that on day 18th

and 28th

after the treatment of diseased cows, there is

significant increase (P<0.001) in the efficacy of the drug.

At dose rate of 120 mg/kg body weight indicated that Flemingia macrophylla

remained efficient against fascioliasis in crossbred cows. But the efficacy of Nigella

sativa and triclabendazole remained enhanced than that of Flemingia macrophylla.


The treatment effect of Flemingia macrophylla is shown in Table 4.27 below.

When the diseased animals were treated with this herbal drug positive effect was seen on

the milk production. After giving the second dose to the in milk fascioliasis positive

animals at the dose rate of 80, 100 and 120 mg/kg body weight on day 28th

there was an

increase in milk production as 28.67%, 25.44% and 31.67% in groups Cl, C2 and C3

respectively.



was carried out on six cows out of which two were in subgroup C1, one was in C2 and

three were in C3 subgroup.

110




It has been noticed in Table 4.18 – 4.20 and Fig 4.13 that Flemingia macrophylla

remained very effective at dose rates of 80, 100 and 120 mg/kg body weight. The P-value

was found P<0.05 at all doses levels with that of the control group. Moreover, P-value

was noted P>0.05 while comparing the dose levels of Flemingia macrophylla to that of

triclabendazole. However, the triclabendazole was found better in terms of percentage as

compared to that of Flemingia macrophylla. Flemingia macrophylla was compared at

dose rate of 80, 100 and 120 mg/kg body weight to triclabendazole and control group as

described in Fig 4.13. There was no side effect of the drug. The body condition of the

animals became better and improved after the treatment.

Triclabendazole


crossbred cows with this herbal drug at the dose rate of 10 mg/kg body is presented in the


the efficacy of

triclabendazole was established 67.50 %. Whereas, on day 28th

when the animal was



and 28th


increase (P<0.0001) in the efficacy of the drug. When triclabendazole was compared to

that of control group its efficacy was found significant (P < 0.05) as shown in Fig 4.15.

Efficacy of triclabendazole is maximum than all three herbal drugs. Efficacy of Nigella

sativa was found close to the triclabendazole. There was no side effect of the drug. The

body condition of the animals became better and improved after the treatment.

Effect on milk production

Results shown in the Table 4.27 are described as below. There was a definite

influence of the drug treatment on milk production. The reduction in the milk production

of diseased animals was found but when they were given the second dose there was an

increase in milk production as 35.57% in subgroup D.

111



was carried out on two cows in subgroup D.




It has been noticed in Table 4.21 and Fig 4.14 that triclabendazole remained very

effective at dose rate of 10 mg/kg body weight. The P-value was found P<0.001 at 10

mg/kg body weight with that of the control group. However, the triclabendazole was

found better in terms of percentage as compared to that of other three herbal drugs. There

was no side effect of the drug. The body condition of the animals became better and

improved after the treatment.

112

Table 4.1: Egg per gram (faeces) on different days in crossbred cows at dose rate of

80 mg / kg treated by N. sativa (Gp-A1)

Pre treatment First dose Second dose

Day-zero Day-3rd Day-7th Day-18th Day-21st Day-28th

400 300 200 200 100 100

400 300 200 100 0 0

500 500 400 300 200 100

600 500 400 400 200 0

400 400 300 300 200 100

400 400 300 200 200 100

400 400 300 100 100 0

300 300 200 200 100 100

300 300 200 200 100 100

400 300 300 200 200 100

4100 3700 2800 2200 1400 700

410 370 280 220 140 70

113


100 mg / kg treated by N. sativa (Gp-A2)



400 400 300 200 100 0

300 300 200 100 0 0

400 400 300 300 200 100

300 300 200 100 0 0

500 300 300 300 200 100

400 400 300 200 200 0

300 300 200 100 0 0

400 400 300 200 100 100

300 300 200 100 0 0

400 300 300 200 200 100

3700 3400 2600 1800 1000 400

370 340 260 180 100 40

114

Table 4.3: Egg per gram (faeces) on different days in crossbred cows at dose rate

of 120 mg / kg treated by N. sativa (Gp-A3)



400 300 200 100 0 0

400 300 200 100 0 0

300 200 100 0 0 0

400 400 300 200 100 0

400 300 200 100 0 0

400 400 300 100 0 0

400 300 200 200 100 0

500 500 400 300 200 100

300 300 200 100 0 0

400 300 200 100 0 0

3900 3300 2300 1300 400 100

390 330 230 130 40 10

115


of 80 mg / kg treated by Fumaria parviflora (Gp-B1)



400 300 300 200 100 100

300 300 300 100 100 0

400 400 200 200 100 100

400 400 300 300 100 100

400 400 300 200 100 100

500 400 300 300 0 0

600 300 200 100 100 100

400 400 300 200 100 0

400 300 100 100 0 0

300 200 200 200 100 100

4100 3400 2500 1900 800 600

410 340 250 190 80 60

116

Table 4.5: Egg per gram (faeces) on different days in crossbred cows at dose rate of 100

mg / kg treated by Fumaria parviflora (Gp-B2)



400 300 300 200 100 100

400 200 200 100 100 100

300 300 200 100 0 0

500 400 300 300 100 0

600 500 300 300 100 100

400 300 200 100 100 0

400 300 200 200 100 0

300 300 200 100 0 0

400 400 300 200 100 100

400 300 200 200 100 0

4100 3300 2400 1800 800 400

410 330 240 180 80 40

117


120 mg / kg treated by Fumaria parviflora (Gp-B3)



400 400 300 200 100 0

400 200 200 200 0 0

300 200 200 200 100 100

500 400 300 200 200 0

500 400 400 300 200 0

400 300 200 100 100 0

300 300 300 200 100 0

400 400 300 200 100 100

400 400 300 200 200 100

600 300 300 300 100 100

4200 3300 2800 2100 1200 400

420 330 280 210 120 40

118

Table 4.7: Egg per gram (faeces) on different days in crossbred cows at dose rate of 80

mg / kg treated by Flemingia macrophylla (Gp-C1)



300 300 200 200 100 100

400 300 200 100 100 100

400 200 100 100 0 0

500 300 200 100 100 0

400 400 300 100 100 100

400 200 300 200 100 0

300 300 100 100 100 100

400 300 300 100 100 0

500 500 300 200 100 100

300 300 200 200 0 0

3900 3100 2200 1400 800 500

390 310 220 140 80 50

119


100 mg / kg treated by Flemingia macrophylla (Gp-C2)



400 300 200 200 100 0

300 300 200 100 100 100

400 300 100 0 0 0

400 300 200 200 100 100

300 200 100 0 0 0

400 400 200 100 0 0

400 300 200 100 0 0

400 400 200 100 0 0

500 400 100 0 0 0

600 300 300 300 100 100

4100 3200 1800 1100 400 300

410 320 180 110 40 30

120


of 120 mg / kg treated by Flemingia macrophylla (Gp-C3)



500 300 200 100 0 0

400 400 300 100 100 100

400 400 200 100 0 0

400 300 200 100 100 0

300 200 100 100 0 0

400 300 200 100 100 0

300 300 200 100 0 0

400 300 300 100 100 100

400 400 200 100 0 0

400 300 200 100 0 0

3900 3200 2100 1000 400 200

390 320 210 100 40 20

121


of 10 mg / kg treated by triclabendazole (Gp-D)



300 300 200 100 0 0

300 300 200 100 0 0

500 400 400 200 100 0

500 400 300 200 100 0

400 400 200 200 100 100

400 300 300 200 100 0

500 400 100 100 0 0

400 300 100 0 0 0

400 400 300 100 0 0

300 300 200 100 0 0

4000 3500 2300 1300 400 100

400 350 230 130 40 10

122

Table 4.11: Egg per gram (faeces) at different days in fascioliasis positive untreated

cows control group (Gp-E)

Ser No Treatment

Day-zero Day-3rd

Day-7th

Day-8th

Day-21st Day-28

th

1 500 500 500 400 400 400

2 400 400 400 400 400 350

3 400 400 400 400 350 300

4 600 600 600 600 550 550

5 400 400 400 350 350 300

6 300 300 300 300 300 300

7 400 400 400 400 400 350

8 500 500 500 450 500 500

9 300 300 300 300 250 250

10 400 400 400 400 400 350

Total 4200 4200 4200 4000 3900 3650

Mean 420 420 420 400 390 365

EPG

Reduction% age 0 0 0 4.76 7.14 13.09

Fig 4.1: EPG reduction of control group without any treatment

123

Table 4.12: Efficacy % at dose rate of 80 mg / kg treated by Nigella sativa (Gp-A1)

Animals EPG

pre-treatment

Efficacy %

First dose Second dose

Day3rd Day7th Day18th Day21st Day28th

10 410 9.76 31.71 46.34 65.85 82.93

Fig 4.2: Graphical representation of Nigella sativa 80 mg/kg (Gp A1)

124


Animals EPG

Pre-treatment

Efficacy %



10 370 8.11 29.73 51.35 72.97 89.19

Fig. 4.3 Graphical representation of Nigella sativa 100 mg/kg (Gp A2)

125


Animals EPG

pre-treatment

Efficacy in percentage on different days



10 390 15.38 41.03 66.67 89.74 97.44

Fig. 4.4 Graphical representation of Nigella sativa 120mg/kg body weight(Gp A-3)

Fig. 4.5 Graphical comparison of Nigella sativa 80,100 and 120 mg/kg with

Triclabendazole 10 mg /kg

126

Table 4.15: Efficacy % at dose rate of 80 mg / kg treated by Fumaria parviflora

(Gp-B1)

Animals EPG

pre-

treatment




10 410 17.07 39.02 53.66 80.49 85.37

Fig 4.6; Graphical representation of Fumaria parviflora 80 mg/kg (Gp B1)

127


(Gp-B2)

Animals EPG

pre-treatment




10 410 19.51 41.46 56.10 80.49 90.24

Fig. 4.7: Graphical representation of Fumaria parviflora 100 mg/kg (Gp B2)

128


(Gp-B3)

Animals EPG

pre-treatment




10 420 21.43 33.33 50.00 71.43 90.48

Fig. 4.8 Graphical representation of Fumaria parviflora 120 mg/kg (Gp B3)

Fig. 4.9 Graphical representation of Fumaria parviflora 80,100 and 120 mg/kg with

Triclabendazole 10 mg /kg

129

Table 4.18: Efficacy % at dose rate of 80 mg / kg treated by Flemingia macrophylla

(Gp-C1)

Animals EPG

pre-treatment




10 390 20.51 43.59 64.10 79.49 87.18

Fig. 4.10 Graphical representation of Flemingia macrophylla80 mg/kg (Gp C1)

130


(Gp-C2)

Animals EPG

pre-treatment




10 410 21.95 56.10 73.17 90.24 92.68

Fig. 4.11 Graphical representation of Flemingia macrophylla100 mg/kg (Gp-C2)

131


(Gp-C3)

Animals EPG

pre-treatment




10 390 17.95 46.15 74.36 89.74 94.87

Fig. 4.12 Graphical representation of Flemingia macrophylla 120 mg/kg (Gp-C3)

Fig. 4.13 Graphical representation of Flemingia macrophylla 80, 100 and 120 mg/kg

with triclabendazole 10 mg /kg

132

Table 4.21: Efficacy % at dose rate of 10 mg / kg treated by triclabendazole (Gp-D)

Animals EPG

pre-treatment




10 400 12.50 42.50 67.50 90.00 97.50

Fig. 4.14 Graphical representation of Triclabendazole 10mg/kg (Gp- D)

Fig. 4.15 Graphical representation of Triclabendazole 10 mg/kg with control group

133

Table 4.22: Corporative efficiency of different herbal and allopathic drugs among each other

and with control group against fascioliasis

Drugs used Subgroups and

dose level (mg/kg)

Efficacy percentage on different days



Nigella sativa

A1 (80) 9.76 31.71 46.34 65.85 82.93

A2 (100) 8.11 29.73 51.35 72.97 89.19

A3 (120) 15.38 41.03 66.67 89.74 97.44

Fumaria parviflora

B1 (80) 17.07 39.02 53.66 80.49 85.37

B2 (100) 19.51 41.46 56.10 80.49 90.24

B3 (120) 21.43 33.33 50.00 71.43 90.48

Flemingia macrophylla

C1 (80) 20.51 43.59 64.10 79.49 87.18

C2 (100) 21.95 56.10 73.17 90.24 92.68

C3 (120) 17.95 46.15 74.36 89.74 94.87

Triclabendazole D (10) 12.50 42.50 67.50 90.00 97.50

Control E 0 0 4.76 7.14 13.09

Mean

±

SEM

9.92

±

0.66

29.34 ±

1.95

61.58 ±

4.20

69.41 ±

4.34

73.82

±

4.39

Nigella sativa was found to be the most effective herbal drug at all dose levels as

compare to Flemingia macrophylla and Fumaria parviflora. The efficacy order from

highly effective to least effective is as triclabendazole, Nigella sativa, Flemingia

macrophylla and Fumaria parviflora as shown in table 4.22.

134

Fig. 4.16. Corporative efficiency of different herbal and allopathic drugs among

each other and with control group against Fascioliasis

135

Table 4.23: Milk yield at different dose levels on pre and post treatment with Nigella

sativa

Gp & Dose

Rate

Milk

Yield

(liters) at

Zero day

Milk Yield on different days after treatment

Ist Treatment 2nd Treatment

3rd day 7th day 18th day 21st day 28th day

A1

80 mg/kg

n = 10

6.6 6.06% 11. 39% 15.42% 19. 29% 21.38%

A2

110 mg/kg

n = 10

6.2 5.36% 9.87% 13.03% 18.14% 26.85%

A3

120 mg/kg

n = 10

6.4 6.71% 12.13% 16.54% 22.98% 34.90%

Table 4.24: Milk yield at different dose levels on pre and post treatment with Fumaria

parviflora

Gp & Dose

Rate

Milk

Yield at

Zero day




B1 80 mg/kg

n = 10 6.2 6.16% 7. 25% 12.06% 14. 94% 19.49%

B2 100 mg/kg

n = 10 6.6 4.86% 9.62% 14.38% 20.40% 23.59%

B3 120 mg/kg

n = 10 6.3 6.55% 14.61% 18.81% 24.13% 27.84%

136

Table 4.25: Milk yield at different dose levels on pre and post treatment with Flemingia

macrophylla

Gp & Dose

Rate

Milk

Yield

at

Zero

day




C1 80 mg/kg

n = 10 6.2 3.67% 10. 81% 21. 52% 25. 10% 28.67%

C2 100 mg/kg

n = 10 5.8 6.99% 13.89% 20.78% 23.38% 25.44%

C3 120 mg/kg

n = 10 6.0 8.13% 14.38% 23.31% 28.67% 31.17%

Table 4.26: Milk yield at 10 mg/kg on pre and post treatment with Triclabendazole

(Gp-D)

Gp & Dose

Rate

Milk Yield at

Zero day


1st Treatment 2nd Treatment

3rd 7th 18th 21st 28th

D

10 mg/kg

n=10

6.8 10.30% 16.10% 17.52% 27.88% 35.57%

137

Table 4.27: Milk yield pre and post treatment with three herbal and one allopathic drugs

at different dose levels

Drugs used

Subgroups

and dose

level (mg)

Milk

yield at

zero day


1st Dose 2nd Dose

3rd 7th 18th 21st 28th

Nigella sativa

A1 (80) 6.6 6.06% 11. 39% 15.42% 19. 29% 23.38%

A2 (100) 6.2 5.36% 9.87% 13.03% 18.14% 23.85%

A3 (120) 6.4 6.71% 12.13% 16.54% 22.98% 34.90%

Fumaria

parviflora

B1 (80) 6.2 6.16% 7. 25% 12.06% 14. 94% 19.49%

B2 (100) 6.6 4.86% 9.62% 14.38% 20.40% 23.59%

B3 (120) 6.3 6.55% 14.61% 18.81% 24.13% 27.84%

Flemingia

macrophylla

C1 (80) 6.2 3.67% 10. 81% 21. 52% 25. 10% 28.67%

C2 (100) 5.8 6.99% 13.89% 20.78% 23.38% 25.44%

C3 (120) 6 8.13% 14.38% 23.31% 28.67% 31.17%

Triclabendazole D (10) 6.8 10.30% 16.10% 17.52% 27.88% 35.57%

138

4.5 DISCUSSION

A safe and effective drug for treatment and strategic chemoprophylaxis of

fascioliasis is required having high activity against all stages of Fasciola. Latest allopathic

fasciolicides are effective but many posses adverse effects. Therefore, to discover safe,

newer, economical and effective drug remained open field for research workers. Keeping

in view the efficacy and price of herbal drugs its use is increasing day by day. In this

study the efficacy of different herbal drugs as Nigella sativa, Fumaria parviflora, and

Flemingia macrophylla at dose rate of 80, 100 and 120 mg/kg was evaluated in

comparison to the effective allopathic drug triclabendazole at the dose rate of 10 mg/kg

body weight. The efficacy of the herbal drug was also compared to allopathic drug

triclabendazole, to each other and to control group.

It revealed that the aqueous extract of herbal drug Nigella sativa at various dose

levels as 80,100 and 120 mg/kg body weight when administered on day 18th

, the efficacy

was found as 46.34%, 51.35 % and 66.67 % respectively. Whereas, the efficacy of the

herbal drug Nigella sativa further increased on day 28th

as 82.93 %, 89.19 % and 97.44 %

at dose rate of 80, 100 and 120 mg/kg body weight respectively. Therefore, it was

established that the herbal drug Nigella sativa at given dose levels remained effective

against fascioliasis. It has been observed the efficacy percentage of the herbal drug

Nigella sativa versus the allopathic drug of choice triclabendazole has no significant

difference at all dose levels P<0.05. The clinically sick in milk animals when treated with

Nigella sativa the milk production was increased tremendously by 34.90 % after the

second dose [317].

It revealed that the aqueous extract of herbal drug Fumaria parviflora at different

dose levels i.e. 80,100 and 120 mg/kg body weight when administered on day 18th

, the

efficacy was found as 53.66%, 56.10 % and 50.00 % respectively. Whereas, the efficacy

of the herbal drug Fumaria parviflora further increased on day 28th

as 85.37 %, 90.24 %

and 90.48 % at dose rate of 80, 100 and 120 mg/kg body weight respectively. Therefore,

it was established that the herbal drug Fumaria parviflora at given dose levels remained

effective against fascioliasis. It has been observed the efficacy percentage of the herbal

drug Fumaria parviflora versus the allopathic drug of choice triclabendazole has

significant difference at all dose levels P<0.05. The clinically sick in milk animals when

139

treated with Fumaria parviflora the milk production was increased by 27.84 % after the

second dose. There was no effect found on milk composition and pregnant animals [317].

It revealed that the aqueous extract of herbal drug Flemingia macrophylla at

different dose levels i.e. 80,100 and 120 mg/kg body weight when administered on day

18th

, the efficacy was found as 64.10%, 73.17 % and 74.36 % respectively. Whereas, the

efficacy of the herbal drug Flemingia macrophylla further increased on day 28th

as 87.18

%, 92.68 % and 94.87 % at dose rate of 80, 100 and 120 mg/kg body weight respectively.

Therefore, it was established that the herbal drug Flemingia macrophylla at given dose

levels remained effective against fascioliasis. It has been observed the efficacy percentage

of the herbal drug Flemingia macrophylla versus the allopathic drug of choice

triclabendazole has significant difference at all dose levels P<0.05. The clinically sick in

milk animals when treated with Flemingia macrophylla the milk production was

increased by 31.17 % after the second dose. There was no effect found on milk

composition and pregnant animals [317].

When triclabendazole was given on day 18th

of at the dose rate of 10 mg/kg body

weight the efficacy was found 67.50%. Whereas, on day 28th

the efficacy of the drug was

further enhanced up to 97.50% (in terms of eggs reduction). But in control group the

reduction of eggs was found 13.09% at day 28th

as a consequence of the natural biological

defence. Ultimately it is being revealed that there is no side effect was found by any of

the herbal and allopathic drug used in this study. The milk production was increased by

35.57 % when the sick (Fascioliasis) in milk animals were treated with triclabendazole.

The quality of milk of treated animals was not affected however the colour of milk was

slightly yellowish that remained for 48-72 hours. There was no side effect was noted in

the pregnant animals [319,320,321,317,322, 323&324] also reported similar results.

Reduced flukes burden ultimately increased the milk production which yields

economic benefits to the dairy producers and that leads to the increased fertility. This was

also supported by [321].

Melanthin, Sapginin, Nigella Metbarbain and Melanthiginin are present in Nigella

sativa. Moreover [325] reported that Nigella sativa contains volatile oil 15.1 %, fixed oil

37.5 % and 2 methyl - 4 isopropyl - p-quinine. Nigella sativa showed 84.35 % efficacy

higher than of the control group when Fasciola infected cows were treated at the dose rate

of 120 mg/kg body weight. These results are in agreement to the finding of

[326,327,328,317,329&151], who studied on internal parasitism.

140

Fumaria parviflora (n-hexane chloroform, ethyl acetate and methanol) showed

92.85 % efficacy against paramphistomosis in cattle as reported by [329]. Whereas, in

this study the efficacy of Fumaria parviflora was found 90.48 % against fascioliasis in

cross bred cows. This difference in efficacy percentage may be attributed to the difference

in climatic conditions and host.

Branches and pods of Flemingia macrophylla have been used for ulcers, small

pox, skin diseases and anthelmintic [330&331]. It contains Flemiflavanone A-D,

Narigenin, Genistin, 4-tetrahydroxyisoflavone, Flemichin A, B, C and D, Flemingins ,

Homoflemingin, Chalcone, Lupeol, α-Amyrin, Sitosterol and Procyanidin. [332]. In this

study it found that 94.87 % effectiveness at 120 mg/kg body weight it was 81.78 % higher

than that of control illuminating its use as anthelmintic. In Egypt it was reported by [333]

that efficiency of Pyrantel tartarate and Mebendazole as 79 % and 100 %respectively.

In Iran [334] mentioned that Nigella sativa in being used in traditional drugs. He

also reported that aqueous and ethanolic extracts of Nigella sativa can stimulate milk

production in rats. Administration of effective fascioliasis in June and November is

strongly recommended for appropriate measures to reduce the risk of fascioliasis as

described by [335,175, 298&336].

On reduction in EPG ultimately improves appetite, reduced diarrhea and increase

in plasma protein concentration and albumin globulin ratio which is supported [337,

338,76,339, 340&341]. Submandibular oedema appears in the infected animals when

there is decrease in total serum protein and severe anemia.

In the present study after having gone through the drug trials brought to the

conclusion that allopathic and herbal drugs especially Nigella sativa are equally effective

against fascioliasis. The herbal drug Nigella sativa is economical as compared to

allopathic drugs and easy to administer. Moreover, causative agent (Fasciola) don’t

develop resistance against the herbal drug and it remains effective ever after. Further

studies are required on these herbal drugs to explore or extraction of its active ingredients

with its identification at molecular level and establishing its dosage regimes.


One hundred and ten crossbred cows were used in eleven experiments to evaluate

the efficacy level of three herbal drugs as Nigella sativa, Fumaria parviflora and

Flemingia macrophylla in comparison to one allopathic drug triclabendazole against

fascioliasis. Efficacy of these drugs was established quantitatively by the reduction in

141

eggs per gram faeces (EPG) before and after treatment. The drugs are effective from

highest to lowest as triclabendazole at dose rate of 10 mg/kg body weight, Nigella sativa

120 mg/kg body weight, Flemingia macrophylla and Fumaria parviflora 120 mg/kg body

weight. There were no side effects found by the use of above herbal drugs.

142

5.1 SUMMARY

Thirty crossbred cows ranging from 4-6 years age, having 300-400 kg body

weight suffering from fascioliasis were selected in each group to know the values of

serum enzymes and electrolytes trend. These animals were then treated with three herbal

and one allopathic drug to compare the serum enzyme and electrolytes values before and

after the treatment. When the Efficacy of the drugs was quantified by determining the

difference of eggs per gram faeces (EPG) on pre and post treatment. It was revealed that

during infection there was an increase in mean serum enzymes SGOT (85.37u/L), SGPT

(45.40u/L) and LDH (4157.83u/L) which were decreased to approximately normal values

after treatment as SGOT (78.50u/L), SGPT (43.97u/L) and LDH (4042.47u/L)

respectively. Whereas, some increase in serum electrolyte of infected animals was

observed i.e. sodium (140 mmol/L) and magnesium (0.16 mmol/L) but a little increase in

calcium (0.56 mmol/L) and potassium (5.82 mmol/L). When infected animals were

treated with these herbal and allopathic drugs it played a vital role on various blood

parameters and reduced the elevated values of serum electrolytes as sodium (134.43

mmol/L), magnesium (0.15 mmol/L), calcium (0.54 mmol/L) and potassium (5.07

mmol/L). Which shows that the values of these blood parameters became normal after

treatment. The use of plant origin drugs showed no side effects.

5.2 INTRODUCTION

Fascioliasis is diagnosis on the basis of EPG in faces or by the presence of

specific antibodies in the serum of diseased animals. Recently, the diagnosis is done

through the detection of specific coproantigen of F. hepatica [342]. The specificity and

sensitivity of these diagnostic tests have been found through induced infection [343]

CHAPTER 5

SERUM ENZYMES AND ELECTROLYTE

ANALYSIS IN CROSSBRED COWS INFECTED

WITH FASCIOLIASIS

143

when two different populations negative and positive selected from a fluke free and an

enzootic area respectively [344,342&345]. The latest diagnosis in fascioliasis are

qualitative only, yet the infection level is thought an significant issue to know the

production losses [295&3].

Currently milk samples collected for checking of routine animals productivity and

milk quality are being used for the diagnosis of fascioliasis and other common bovine

diseases [346&201], to reduce the costs involved, disturbance to animals and handling the

sampling. The highly specific and sensitive test for sero-diagnosis of fascioliasis in cows

is MM3-SERO ELISA that may be reliable to use with milk samples. In bulk samples this

method is an excellent in estimating within herd prevalence of infection [347&348]. Due

to the high sensitivity with possibility of more sera samples the immune-enzymatic

techniques as indirect ELISA is more suitable to diagnose the fascioliasis [349]. To detect

early infection, these techniques which are based on the antibodies detection have been

used successfully [350,351,352&353]. During migratory stage of infection, Fasciola

hepatica antigens are found in immune system, and it can be detected by serological

examination as sandwich-enzyme-linked immunosorbent assay S.E.A [354]. When the

parasites move to the bile ducts, minimum antigens are available in the immune system

then the detection of fascioliasis is carried out through fecal or bile samples. There is

huge damage of liver parenchyma takes place during the migratory phase of flukes from

peritoneal cavity to the bile ducts. Therefore, early diagnostic techniques are very

important to reduce losses in cattle. An indirect enzyme-linked immunosorbent assay

I.E.A that allows diagnosis of early fascioliasis. F. hepatica antibodies have been detected

first by indirect-ELISA between three and six weeks after the infection when immature

worms migrating to liver [355]. Long time presence of high levels of immunoglobulin

make the interpretation further difficult even if the animals have been treated successfully

[344]. Langley and Hillyer [354] found antigenemia in infected cows as early as two

weeks after the infection. It is accomplished that indirect and direct enzymatic is useful

and very important to combine 2 assays, ELISA, to achieve knowledge of the infection

status of the host. Results of ELISA using different antigens of F. gigantica for detecting

antibodies against Fasciola in sera may be used in cattle. The diagnostic sensitivity,

specificity and accuracy of the assay can be calculated according to [356&357].

Anemia, eosinophilia and Hypoalbuminemia may be found in fascioliasis and

enzyme activities of liver L.D.H is found in chronic or subacute cases from 12-15 week

144

on metacercariae ingestion [358&359]. Serum enzymes SGOT, SGPT and LDH and

electrolytes such as sodium, magnesium, calcium and potassium increases to some extent

during the infection of liver (fascioliasis).

The cattle are very important animals in our community, so it is necessary to look

into its disease problems and to find the best control methods with best possible drug for

the treatment of fascioliasis.


Attached as appendix -E

5.4 RESULTS

Serum Enzymes

The levels of SGPT, SGOT, LDH values before and after treatment have been

shown in Table 5.1 and Fig 5.1-5.3. The results are analyzed as under.

SGPT (u/L)

The comparison of values before and after treatment showed a non significant

decrease (P>0.05) in SGPT level towards normal on day 28th

. It suggests that there was a

very mild increase in the level of SGPT during the infection.

SGOT (u/L)

The comparison of values before and after treatment showed a highly significant

decrease (P<0.001) in SGOT level towards normal on day 28th

. It suggests that there was

high increase in the level of SGOT during the infection.

LDH (u/L)


decrease (P<0.001) in LDH level towards normal on day 28th


high increase in the level of LDH during the infection.

Serum Electrolytes

The values of Na, K, Ca and Mg before and after treatment have been shown in

Table 5.2 and Figs 5.4-5.7. The results are analyzed as under.

145

Sodium


decrease (P<0.001) in sodium level towards normal on day 28th


high increase in the level of sodium during the infection.

Potassium


decrease (P<0.001) in Potassium level towards normal on day 28th

. It suggests that there

was high increase in the level of Potassium during the infection.

Calcium

The comparison of values before and after treatment showed a significant

decrease (P<0.05) in Calcium level towards normal on day 28th


increase in the level of Calcium during the infection.

Magnesium


decrease (P<0.001) in Magnesium level towards normal on day 28th

. It suggests that there

was high increase in the level of Magnesium during the infection.

146

Table 5.1 Serum enzyme activities in infected cases of fascioliasis on pre and post

treatment with triclabendazole 10 mg/kg body weight

Ser

Pre

treatment

SGPT u/L

Post

treatment

SGPT u/L

Pre

treatment

SGOT u/L

Post

treatment

SGOT u/L

Pre

treatment

LDH u/L

Post

treatment

LDH u/L

1 45 44 82 75 3783 3671

2 46 43 84 76 3893 3779

3 44 39 82 78 4022 3910

4 47 45 88 79 3718 3600

5 45 44 80 75 4290 4180

6 49 47 82 77 4122 4010

7 44 42 89 80 3817 3698

8 43 43 83 76 3785 3672

9 47 44 91 84 3961 3840

10 52 51 79 71 4220 4111

11 41 41 84 77 4423 4309

12 43 41 90 84 3911 3791

13 47 45 84 77 3854 3740

14 44 44 93 87 4432 4309

15 46 47 87 78 4032 3915

16 43 42 82 75 3717 3605

17 44 43 82 75 5135 5023

18 45 44 83 76 4023 3910

19 47 45 90 83 4140 4020

20 48 47 88 81 3968 3850

21 45 44 84 77 4389 4275

22 47 45 89 80 4259 4140

23 46 46 83 77 4403 4290

24 43 41 81 76 4051 3940

25 47 45 85 78 5138 5023

26 42 42 83 76 4023 3910

27 48 47 87 81 3767 3650

28 43 41 89 82 3942 3823

29 45 45 93 87 5133 5010

30 46 42 84 77 4384 4270

Mean 45.40 43.97 85.37 78.50 4157.83 4042.47

Stdev 2.31 2.45 3.84 3.74 397.10 396.87

S.E 0.42 0.45 0.70 0.68 72.49 72.45

147

Fig. 5.1: Graph showing values of SGPT before and after treatment

Fig. 5.2: Graph showing values of SGOT before and after treatment

Fig. 5.3: Graph showing values of LDH before and after treatment

148

Table 5.2 Serum electrolytes activities in infected cases of fascioliasis on pre and post

treatment with triclabendazole10 mg/kg body weight

Ser

Na-Pre

treatment

Na-Post

treatment

K-Pre

treatment

K-Post

treatment

Ca-Pre

treatment

Ca-Post

treatment

Mg-Pre

treatment

Mg-Post

treatment

1 140 134 5.3 5.0 0.54 0.53 0.16 0.14

2 138 131 5.8 5.1 0.58 0.57 0.18 0.17

3 137 132 5.8 5.4 0.57 0.57 0.15 0.14

4 143 133 6.1 5.0 0.54 0.53 0.16 0.15

5 139 132 5.8 5.1 0.56 0.54 0.14 0.13

6 141 139 5.8 5.8 0.53 0.52 0.15 0.14

7 136 132 6.2 5.8 0.57 0.55 0.17 0.16

8 139 136 5.9 5.1 0.55 0.54 0.16 0.15

9 142 134 6.1 5.4 0.58 0.56 0.14 0.13

10 140 133 5.8 5.1 0.55 0.53 0.16 0.15

11 138 134 5.8 5.0 0.54 0.52 0.17 0.16

12 140 132 6.1 4.8 0.56 0.53 0.18 0.16

13 137 132 5.9 5.0 0.57 0.55 0.15 0.14

14 141 139 5.6 5.1 0.55 0.53 0.17 0.17

15 141 139 5.8 5.1 0.58 0.56 0.14 0.12

16 145 133 5.8 5.1 0.54 0.53 0.15 0.14

17 140 134 5.4 5.0 0.56 0.54 0.17 0.17

18 136 132 5.6 4.8 0.53 0.51 0.18 0.15

19 145 136 5.8 5.0 0.58 0.55 0.15 0.15

20 137 133 5.9 5.0 0.55 0.54 0.14 0.13

21 141 139 6.1 5.3 0.57 0.54 0.16 0.15

22 136 134 5.6 4.3 0.54 0.52 0.15 0.12

23 138 136 5.4 4.0 0.58 0.56 0.18 0.17

24 141 137 5.9 5.0 0.53 0.51 0.15 0.16

25 137 134 5.9 5.1 0.55 0.52 0.15 0.13

26 140 136 5.8 5.3 0.57 0.54 0.16 0.14

27 143 132 6.2 5.4 0.54 0.53 0.15 0.15

28 138 134 6.1 5.6 0.58 0.56 0.15 0.14

29 146 133 5.9 5.0 0.55 0.53 0.16 0.15

30 145 138 5.8 4.5 0.53 0.51 0.14 0.12

Mean 140.00 134.43 5.82 5.07 0.56 0.54 0.16 0.15

Stdev 2.88 2.47 0.23 0.37 0.02 0.02 0.01 0.01

S.E 0.53 0.45 0.04 0.07 0.00 0.00 0.00 0.00

149

Fig. 5.4: Graph showing values of sodium before and after treatment

Fig. 5.5: Graph showing values of potassium before and after treatment

150

Fig. 5.6: Graph showing values of calcium before and after treatment

Fig. 5.7: Graph showing values of magnesium before and after treatment

151

5.5 DISCUSSION

Sero-epidemiological studies are the imperative way to diagnose different diseases

so that the early treatment could be rendered. In the serum enzymes and electrolytes study

on 30 crossbred cows having fascioliasis revealed a considerable increase (P<0.001) in

SGOT, In infected animals in comparison with non infected animals. Whereas, there is

almost no change in SGPT in both infected and non infected animals. In this study there

is increase in LDH (4153.87u/L) after infection as described by [358&360] who

demonstrated that the poor production / performance of animals suffering from

fascioliasis is linked with damage of liver that is obvious by increase of enzymatic

activity of lactate dehydrogenase (LDH)

The S.G.O.T level was found increased (85.37 u/L) in infected animals. Since

substantial amount of S.G.O.T activity was noted in almost all of the mammal’s tissues

[337&340]. As the immature flukes cause severe damage to liver etc [14], rise in SGOT

levels were noticed in all infected animals (Fig. 5.2). These results are in accordance with

those of [361&341]. SGOT values significantly decreased with advancing pregnancy and

increased in early lactating cows. [362] reported through haematological survey from

experimental farm and founded significantly higher values of total serum proteins and

SGOT in mature females as compared to immature animals.

The serum glutamic pyruvic transaminase (SGPT) levels (45.40u/L) was found in

infected animals which is slight increase as is shown in Fig. 5.2. The results are in

accordance with those of [341]. In this study it as found that both allopathic and herbal

drugs are equally effective against fascioliasis in cows [363&364].

In the study of [329] it revealed that major elevation in the levels of S.G.O.T,

S.G.P.T and L.D.H was found in animals suffering from endo-parasites. Same

interpretations were found in the cows having paramphistomosis. Damages of tissues by

flukes are the main cause of elevation in the levels of serum enzymes as S.G.O.T.

The results show increasing of S.G.O.T. that agreed with [189] that the elevation

of S.G.O.T. indicates to hepatocyte damage and it important to assessment the degree of

hepatocyte damage or liver fluke infection. [365] indicated that the S.G.O.T. is more

sensitive than S.G.P.T to liver tissue destroying; S.G.P.T is low response to hepatocyte

damage [366]. Increasing of S. T. Bilirubin and S.G.P.T indicates death of liver

152

hepatocyte from fluke infection. In addition to complete or partial bile ducts obstruction

causing returning of Bilirubin to hepatocyte then increased it in serum and elevation of

S.G.P.T [367&368].


In this study it reveled from the data of infected animals that no serum enzyme or

serum electrolyte is directly associated / responsible to the Fasciola burden.

Hypoalbuminemia is associated from medium to high fluke burden. In chronic fascioliasis

S.G.O.T is the only consistent pointer at all stages of infection.

5.7 CONTROL PROGRAMME

The animals should be dewormed in May – June and November – December

(twice a year) with broad spectrum fasciolicides as triclabendazole or with

herbal drug having good efficacy.

To reduce the resistance to drug the fasciolicides should be used alternatively.

The dung should be spread in grazing areas as it generates heat when it

decomposes, that kills parasite eggs and larvae.

Military Farms managed by Pakistan Army and other big farms should use

sizeable land for grazing paddocks and graze the cows alternately with horses

or sheep as horses and sheep carry different parasites.

The feeding and watering should be hygienic to avoid contamination.

Department of livestock should launch a campaign on the prevention of

fascioliasis.

Monthly faecal examination be carried out to evaluate that how fascioliasis

control programme is working.

153

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183

Appendix- A

MATERIALS AND METHODS

To record the epidemiology of fascioliasis in Crossbred cows (Friesian x Sahiwal

and Friesian x Jersey etc) during one year study period i.e. from November 2012 to

October 2013 in different districts of Punjab province i.e. Lahore, Sialkot, Attock and

Multan. Animals having different management conditions and places at slaughterhouses,

livestock farms, veterinary hospitals and household were examined.

Survey of animals at abattoirs, livestock farms, veterinary hospitals and

household

Survey of abattoirs

Monthly visits were carried out at abattoirs of Lahore, Sialkot, Attock and Multan

districts of Punjab Province. The liver and bile ducts of 25 randomly selected animals

were examined for the presence of liver flukes. Flukes were collected, preserved and

identified in positive cases as described by Hendrix [369].

The Fasciola from each liver were gathered with fine forceps and were placed in

plastic beakers having 0.7% saline (NaCl) solution. Afterwards the flukes were killed in

cold water and then fixed and preserved in 80% ethyl alcohol.

Survey of livestock farms

For this study, one crossbred cow farm in Lahore, Sialkot, Attock and Multan

districts of Punjab Province were visited and faecal samples were randomly collected

from 25 animals on monthly basis throughout the year.

Survey of veterinary hospitals

For this study, veterinary hospitals in Lahore, Sialkot, Attock and Multan districts

of Punjab Province were visited and faecal samples were randomly collected from 25

animals on monthly basis throughout the year.

Survey of household

In this study, a total of 25 faecal samples household animal per month were

analyzed. This study was extended for one year. Faecal samples of randomly selected

Crossbred cows were screened out i.e. from November 2012 to October 2013.

184

Seasonal prevalence

Seasonal prevalence was studied by dividing the year into four seasons, i.e. winter

(November - February), spring (March - April), summer (May - August) and autumn

(September - October). Necessary parameters regarding species, breed, age and sex were

recorded. The prevalence of disease in different districts of the Punjab province was

recorded by Thrusfield [370].

Parasitological techniques

Faecal samples were examined for the presence of Fasciola eggs by Direct smear,

Sedimentation technique and Floatation [14]. Eggs were identified on the basis of

morphology and key as described by [371,14&372]. McMaster egg counting technique

[373] was used for fluke egg count (EPG) from positive samples.

Collection of faecal samples

About 5-7 grams of fresh samples were collected directly from the rectum of the

Crossbred cows at the time of defecation. These samples were brought to the Laboratory,

Department of Parasitology, University of Veterinary and Animal Sciences (UVAS)

Lahore. The majority of these collected samples was examined on the same day while a

few were examined on the second day. These were preserved in 10% formalin for

preventing the development and hatching

Examination of faecal samples

Faecal samples were examined by the following methods:-

Direct smear method

The method was adopted as described by Urquhart and Zajac [373&374]. A small

quantity of faecal material was placed on a glass slide, one drop of water was added to it

and then stirred to form a homogenous mixture. After this a cover slip was applied on it

and keep the slide on the stage of microscope, examined under low power objective to

detect the worms and eggs. Three slides were examined from each sample and an average

was taken. The samples found negative were re-examined by using centrifugal floatation

method before declaring as negative.

185

Flotation method

One gram faeces was added to 10ml of Sodium Chloride solution and mixed

thoroughly. Then the suspension was poured into a test tube and more floatation solution

was added to fill the tube to top. A cover slip was placed on the top of the liquid. The tube

and the cover slip were left standing for 10 to 15 minutes then cover slip was removed

vertically and placed on a slide and examined under the microscope [375].

Sedimentation method

Using a spatula, one gram of faeces was mixed with 30ml tap water in a beaker.

The mixture was strained through muslin cloth or strainer. Then was put in centrifuge

tubes as described to centrifuge the samples at 1500-200 rpm. If a centrifuge machine is

not available, allow the mixture to remain undisturbed for 20-30 minutes. Pour off the

liquid from the top of the tube without distributing the sediment at the bottom. Took a

drop from the top layer of the sediment with a Pasteur pipette on a microscope slide. If

the drop seems to be thick, dilute it with a drop of water. A cover slip was applied on the

drop and was examined under the microscope. The procedure was repeated by taking a

drop from the bottom layer of the sediment.

Identification of ovas

F. gigantica and F. hepatica eggs were identified on the basis of morphological

details as described by [14].

Prevalence of Fascioliasis

The prevalence of fascioliasis was recorded as per formula described by

Thrusfield [376].

Prevalence % =

Crossbred cows having a disease at a particular point in time

x100

Crossbred cows in the population at risk at that point in time

186

Appendix- B


This study was designed to record the prevalence of various species of snails over

a period of one year i.e. from November 2012 to October 2013 for the identification of

infected snails with intermediate stages of Fasciola species. The methods used for

conducting this study are as under:-

Collection of snails

During the study period (November 2012 to October 2103) month and season

wise prevalence of snails were recorded. Fortnightly snails were collected from water

bodies of all the four districts of Punjab province i.e. Lahore, Sialkot, Attock and Multan.

A total of 8273 snails were collected. The snails were collected from following areas:

Jinnah Park Lahore

Ravi River side area adjacent to Lahore

Akhrota Syedian Pond Sialkot

Cultivation area Military Farm Sialkot

Kabul River side area adjacent to Attock

Cultivation area Military Farm Attock

Askari Park Multan Cantt

Cultivation area Military Farm Multan

The snails were collected by using a scoop net having metal ring of 36 cm length

and 30 cm diameter with handle of 35cm of long. The snails were brought to laboratory

in a polythene bags along with some of the water vegetation.

In the laboratory they were kept in tanks which were constantly aerated. Aquatic

vegetation (Hydrilla verticillata, Vallisneria spiralis) was placed in the tanks to keep the

water clean for a longer time.

Spinach and lettuce proved to be favorite food of snails. Temperature of the

collection sites was recorded by thermometer which ranged between 0-35.2°C. The pH of

the water was ranged between 7.44-7.56. Most of the snails were found at the depth of 1-

187

3 feet and often found attached on the backside of leaves and sidewalls of the ponds in

shaded area. At the time of visit ecological conditions of the snails were also noted.

The snails brought to the laboratory usually survived for a fairly long time. A

large number of snails were not placed (congested) in one tank and the water was saved to

be polluted too much.

Snail examination

Snails were examined for infection with trematode, they were isolated either in 4

specimen tubes for 24 hours. Water of these tubes was examined with the naked eye

against the light or with a hand lens, after every one hour during the day and on the next

morning. The snails were found infected in this manner, were segregated from the rest.

Some of the cercariae encysted on the walls of the container soon after their emergence. It

was found to be important to note the presence of cysts in such cases. On the other hand

sometimes operculate snails did not protrude their body out of the shell for a long time

and in such cases discharge of the cercariae did not occur. Such snails were re-examined

until the snail protruded its body during the desired time.

Immediately after emergence of cercariae the activity and the resting position of

the cercariae were studied by placing these containers quietly to avoid any disturbance

caused to the cercariae. In living cercariae usually the body and tail are highly contractile.

Identification of snails

Identification was based on snail soft parts and shell morphology as suggested by

[377,378,379&380].

Identification and isolation

The snails collected were placed in a rearing aquarium to keep them alive. Shedding

of cercariae was induced by exposing snails to sunlight for 2 hours on a daily basis [381].

Cercariae were picked and placed on a slide, covered and examined under a stereomicroscope

at a magnification power of 40X and identification was done according to their gross

morphological characteristics, resting position and swimming behavior [382]. Snails were

then dissected by removing the shells to uncover any leftover cercariae, as described by[383].

Genus Lymnaea

Thin shell usually with a prominent and acute spine having a large, often flaring

aperture, tentacles flattened, lip acute and simple, eggs laid in jelly form, radula with a

188

unicuspid central tooth. Jaws composed of three pieces. 1stlarge transversely elongate

piece then 2"d and 3rd

pieces were small with one foot rounded behind.

Genus Physa

Thin sinistral shell, with an acute spire a large lower whorl and a large oval, single

piece foot pointed behind, tentacles were filiform that described species, cosmopolitan.

Mantle reflected over a portion of the shell and fringed with finger like structure i.e.

filaments.

Genus Indoplanorbis

Indoplanorbisbelongs to Planorbidae it is the largest family of aquatic pulmonates

and are widely distributed over the world. The shells of many Planorbidae are right turned

(dextral) but their body is sinistral. The eggs of these hermaphroditic snails are laid in

transparent clutches on plants, stones or other objects. Most members of this genus prefer

to habitats with slow moving or stagnant water like that of ponds, lakes and swamps.

Genus Gyraulus

Gyraulus members possess small shells which are greatly depressed with carinate

or subcarinate peripheries and include three or four rapidly increasing whorls. The

aperture was oblique, the verge has a horny stylet and a separate prostate duct was

present. The radula has bi or tri-cuspid lateral teeth and six cuspid marginal teeth.

Genus Bulinus

Genus Bulinus shells were sinistral, ovate or higher to almost cylindrical and

turreted. The highest range was from 4 to 23 mm. The whorls were usually evenly

rounded. The aperture was high and wide in snails with a low spire and relatively narrow

in specimen with a high spire. The columella margin was either straight, more or less

twisted, or truncate. The sculpture was consisted of spirally arranged rows of small

universe impressions or the shell may possess ribs (costate).

Genus Oncomelania

Members of this genus were having the outer lip of the shell aperture slightly thin

tentacles were slender and long. Operculum was thin and paucispiral. The snout of the

animal was blunt, the tentacles were slender and long and an important characteristic was

the pigmentation at the inner base of the tentacles.

189

Stables and Chappell [140&216] reported that infection period was normally

between May to September. A fairly definite seasonal incidence of cercariae has been

reported. In this study, the total infection with all the genera of cercariae tends to show

three peak months i.e. August, July and September. The classical picture of such peaks

was understood when the life cycle of Fasciola, the habit of various hosts and the

environmental changes are known. All these play important role in such distribution.

These findings are in agreement with [130,152,119,196,134,135,136,146,207,384,

156&216].

190

Appendix- C


Meteorological data including maximum, minimum mean temperature, rainfall,

relative humidity, and pan evaporation rate were collected from meteorological stations of

Lahore, Sialkot, Rawalpindi and Multan districts of Punjab province and the monthly

averages were calculated. The prevalence of fascioliasis was noted in relation to

meteorological factors.

191

Appendix- D


Grouping of animals

One hundred and ten crossbred cows were included in this studies, the 100

(positive for fascioliasis) and 10 healthy crossbred cows (control). These 100 positive

cows were randomly divided into four main groups i.e. A, B, C and D. Animals in group

A, B and C were subdivided further into three groups i.e. A1, A2, A3, B1, B2, B3, C1, C2

and C3 having 10 cows each. Animals in groups A1, A2 and A3 were given herbal drug

Nigella sativa at dosage rate of 80, 100 and 120 mg/kg body weight respectively. Animals

in groups B1, B2 and B3 were given herbal drug Fumaria parviflora at dosage rate of 80,

100 and 120 mg/kg body weight respectively. Animals in groups C1, C2 and C3 were

given herbal drug Flemingia macrophylla at dosage rate of 80, 100 and 120 mg/kg body

weight respectively. Whereas cows in group D were given allopathic drug triclabendazole

at recommended dose rate i.e. 10 mg/kg body weight. The animals in group E infected

with Fasciola were not given any treatment and served as control Details of groups is as

under.

A1-Nigella sativa at dosage rate of 80mg / kg body weight

Group A A2-Nigella sativa at dosage rate of 100 mg/kg body weight

A3-Nigella sativa at dosage rate of 120mg / kg body weight

B1-Fumaria parviflora at dosage rate of 80mg/kg body weight

Group B B2-Fumaria parviflora at dosage rate of 100mg / kg body weight

B3-Fumaria parviflora at dosage rate of 120mg / kg body weight

C1-Flemingia macrophylla at dosage rate of 80mg/kg body weight

Group C C2-Flemingia macrophylla at dosage rate of 100mg/kg body weight

C3-Flemingia macrophylla at dosage rate of 120mg / kg body weight

Group D Triclabendazole at dosage rate of 10 mg/kg body weight

Group E Control (infected untreated cows)

192

The dosage levels of herbal drugs were selected on the basis of preliminary trials in

crossbred cows.

Preparation of aqueous extracts

Herbal drugs Nigella Sativa (seeds), Fumaria parviflora (aerial parts) and

Flemingia macrophylla (aerial parts) were procured from local market and dried under

shade, ground in electric grinder and kept in air tight brown bottles at 4 0C until use. For

preparation of aqueous extracts of Nigella sativa 100 gm of seed powder was added to

one liter of hot water and boiled for 15 minutes and filtered through a cloth. The filtrate

was evaporated under reduced pressure and a viscous residue was obtained. The residue

was then suspended in normal saline. The aqueous solutions of herbal drug Fumaria

parviflora and Flemingia macrophylla were prepared in the same manner except that the

aerial parts of the plant were used instead. During the time of administration the

calculated amount of drug was added in water to make 5% solution and was given by

drench.

The doses for treatment were given on day zero and 18th

. Faecal samples for EPG

were collected and examined on day zero, 3rd

, 7th

, 18th

, 21st and 28

th post treatment.

Efficacy of these herbal drugs were compared to each other and with control and to the

modern allopathic drug i.e. triclabendazole. Side effects of the drugs if any were also

noted.

Egg Counting

Eggs per gram of faeces (EPG) and clinical performance was monitored on day

zero, 3rd

, 7th

and 18th

after treatment. Cows which remained positive after 18th

day were

given a second dose of respective drug on 18th

day and then FEC was made on day 21st

and 28th

. EPG of faeces was made by McMaster egg counting technique (MAFF, 1986).

For this purpose, flotation solution, two beakers, electronic weighing balance, plastic

container, cheese cloth, tea strainer, measuring cylinder, Pasteur pipettes, stirring device,

McMaster counting chamber and compound microscope were used. Flotation solution

having 1.2 specific gravity was prepared by dissolving 400 gm of Sodium Nitrate in one

liter of water. This solution was capable of floating Fasciola eggs from the faecal

samples. Four grams of feces was taken from the sample, weighed and placed in a plastic

container. A quantity of 56 ml flotation fluid was added in the container and stirred.

Faecal suspension was filtered through tea strainer into second container and was stirred

193

with a Pasteur pipette. This pipette was used to withdraw a sub sample as the filtrate was

being stirred. First compartment of McMaster counting chamber was filled with sub

sample. The fluid was stirred again and the second chamber was also filled with another

sub sample. The sub sample of the filtrate was examined under compound microscope at

10X10 magnifications. All eggs within engraved area of both chambers were counted.

Number of eggs within the grid of each chamber was counted and eggs outside the square

were ignored. The number of eggs in both chambers were added and multiplied with 50.

This gave us EPG of faeces.

Efficacy of the drugs

Efficacy of all drugs was calculated on the basis of reduction in faecal egg count

post treatment and by controlled method described by [167].

Efficacy % = a – b x 100

a

a = EPG of faeces before treatment

b = EPG of faeces after treatment

Statistical analysis

In order to analyze and compare the mean ± SEM values for epidemiology

regarding serology and haematology, the statistical analysis was performed by using

analysis of variance (ANOVA). Mean ± SEM values were analyzed within the groups and

compared by Tukeys multiple comparison test and one sample t test where required. In

therapeutic trials to carry out comparison of efficacy on 3rd

, 7th

, 18th

, 21st and 28

th day

among each other the ± SEM values were also analyzed on Bon ferroni post tests in

addition to ANOVA. Level of significance was P<0.05. The data was analyzed by using

Graph Pad Prism programme version 5.00.

Control infected- untreated Eggs per gram of faeces counted in positive cases of fascioliasis of crossbred

cows in control untreated group is shown in Table 4.11 and Fig 4.1. As a result of natural

biological processes the EPG reduction was 4.76 percent 0n 18th

day of observation and

13.09 percent on 28th

day of observation. No significant difference (p value not < 0.05) in

EPG reduction was noted till 28th

day of observation. Signs and symptoms of fascioliasis

did not subside and health condition of all the cows in this group remained poor.

However, in accordance to ethics these animals were given proper medicinal treatment on

the termination of study period i.e. 28 days.

194

Appendix- E


Serum Biochemical Analysis

A total of 30 crossbred cows ranging from 4-6 years age, having 300-400 kg body

weight suffering from fascioliasis were selected in each group on the basis of signs /

symptoms and with 300 EPG of Fasciola in faeces. These cows were separated and kept

under uniform management conditions including feeding, watering and shelter. The cows

were numbered from 1-30 for identification. They were treated with two oral doses of

triclabendazole at dose rate of 10 mg/kg body on day zero and 18th

. The faecal egg count

of all the 30 treated cows was carried out on the 28th

day post treatment and Fasciola eggs

were found nil in all cows. This indicated that all the cows were completely recovered at

that stage.

Collection and processing of blood/serum

Blood samples were collected on day zero before administration of drug and on

day 28th

post treatment. With the help of a sterile disposable syringe under aseptic

conditions, 6-8 ml of blood was collected from jugular vein. For extraction of serum,

samples taken in sterile test tubes without anticoagulant were left for an hour for clotting

to occur. The clot was separated by a fine loop and centrifuged at 3500 rpm for 5 minutes

at least. The supernatant having clear sterile fluid (serum) was aspirated with a Pasteur

pipette and then poured in a screw capped vial. It was stored at 200C under refrigeration

at until processed for analysis. Repeated freezing and thawing was avoided.

Serum Enzymes Analysis

SGPT (Serum Glutamic Oxaloacetic Transaminase , SGOT (Serum Glutamic

Pyruvic Transaminase) and LDH (Lactate Dehydrogenase) enzymes were analyzed from

the serum samples obtained on day zero before treatment and day 28th

post treatment. All

the collected serum samples were analyzed on UVIDEC 430 B double beam

spectrophotometer by JASCO Company.

Serum Electrolytes Analysis

Serum Na+, K

+, Ca

2+ and Mg

2+ electrolytes were analyzed from the serum

samples taken on day zero before treatment and 28th

day post treatment by

spectrophotometer and atomic absorption spectrophotometer

195

Flow diagram of work to conduct serum enzymes and serum

electrolytes

Selection of 30 crossbred cows

suffering from fascioliasis infection

Serum enzymes Serum electrolytes

Pre therapeutic Post therapeutic Pre therapeutic Post therapeutic

SGPT Na+

SGOT K+

LDH Ca2+

Mg2+

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MUHAMMAD AHSAN ALI RANAprr.hec.gov.pk/jspui/bitstream/123456789/2915/1/Muhammad...2016/05/26 · Maria Ahsan who at times could not get my full attention during the study period but