A thesis by - COnnecting REpositories and pharmacological investigation of a benzimidazole...

Clinical and pharmacological investigation

of a benzimidazole anthelmintic against

donkeys’ worm infestation.

A thesis by

Sawsan Mohmmed Ahmed Imam

B. V. Sc. 2005 University of Nyala

Supervisor

Prof. Tigani Hassan Al amin

Department of Medicine, pharmacology and toxicology. Faculty of

Veterinary Medicine, University of Khartoum.

CoSupervisor

Dr. Hisham Ismail Seri

Department of Clinical Studies. Faculty of Veterinary Science

University of Nyala

A thesis submitted to the graduate College University of Khartoum in

partial fulfillment of the requirements of master degree in veterinary

Medicine (Pharmacology)

March 2009

Dedication

This work is dedicated to

Soul of my Father, my great Mother

my kind brothers, my lovely aunties

Asmaa and Mariam

TABLE OF CONTENTS

Table of contents………………………………..….………………………. I

List of tables…………….…………………………..…………..………….. IV

List of figures………………………………………..…….……….…...... VI

Acknowledgements……………….………………….…………………… VIII

English Abstract………………………………………………………......... IX

Arabic abstract………………………………………….…………….….. XI

Chapter one: Introduction and literature review

Introduction ………….…….………………………………...….……….. 1

1.1 Albendazole ……………………………………………………. 4

1.1.1 Identity…………………………………..……….…..………... 4

1.1.2 Chemical name……………………….……….…….…...……… 5

1.1.3 Molecular formulae…………………………………..…………. 5

1.1.4 Molecular weight………………………………………………. 5

1.1.5 Appearance……………………………………………………. 5

1.1.6 Structural formula………………………………..….…...…...... 5

1.1.7 Mode of action…………………..…………………...………… 5

1.1.8 Toxicity of Albendazole…………………………………..….... 6

1.1.9 Pharmacokinetics………………………….………………….. 9

1.1.10 Use of Albendazole in animals……………………....……...... 13

1.2 Ivermectin ……………………..…………...……………….... 16

1.2.1 Efficacy of Ivermectin against equine nematodes………….. 17

1.3 Prevalence of gastro- intestinal nematodes in donkeys and

horses…………………………………………………………..

19

Chapter two: Materials and Methods

2.1 Survey of gastrointestinal nematodes in donkeys and horses… 21

2.1.1 Study area.……………………………………...…………….. 21

2.1.2 Samples collection and examination….…………..…….……… 21

2.1.3 Intensity of infection.…………………………….…................ 21

2.1.4 Parasitological techniques …….……………………………… 23

2.1.4.1 The modified McMaster technique……………………………. 23

2.1.4.2 Faecal culture and identification of larvae……………………... 23

2.2 Therapeutic efficacy of Albendazole against donkey’s worm

infestation ……………………………………………………..

23

2.2.1 Experimental animals……………………………………......... 24

2.2.2 Experimental drugs…………………………………………… 24

2.2.3 Design of the study……………………………………………. 24

2.2.4 Sampling and Time schedule…………………………………. 24

2.2.5 Necropsy of animals and sample preparation………………… 26

2.2.6 Data analysis…………………………………………………. 26

2.3 An assay of some biochemical parameters in donkeys

medicated with benzimidazoles………….……………………

26

2.3.1 Collection of samples ….…………………………………….. 26

2.3.2 Biochemical methods…………………………………...……… 32

2.3.2.1 Total serum protein…………………………………………… 32

2.3.2.2 Serum albumin………………………………………………… 32

2.3.2.3 Serum urea…………………………………………….………. 32

2.3.2.4 Serum creatinine………………………………………………. 33

2.3.2.5 Serum calcium ………………………..…………………........ 33

2.3.3.6 Serum inorganic phosphorus ………………………………… 34

2.4 Statistical methods…………………………………………….. 34

Chapter three: Results

3.1 Survey of gastrointestinal nematodes in donkeys (Equus asinus)

and horses in South Darfur State……………………………….

35

3.2 Therapeutic efficacy of Albendazole against gastrointestinal

nematodes in donkeys (Equus asinus)…………………....……..

48

3.3 An assay of some biochemical parameters in donkeys (Equus

asinus) medicated with Albendazole….. ……………………….

61

CHAPTER FOUR: DISCUSSION

4 Discussion …………………………………………………….. 68

CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion………………………………………………………. 74

5.2 Recommendations……………………………………………….. 74

References …………………………………………..……….………….. 76

LIST OF TABLES

No. Table Page

3.1 Overall prevalence of gastrointestinal nematodes in donkeys and horses in South Darfur State……………........................................

36

3.2 Mean± SD and range of egg per gram of faeces (epg) in donkeys and

horses infested with gastro-intestinal nematodes……………….…..

39

3.3 Severity of infection with gastro-intestinal nematodes in donkeys and

horses per month…………………………………………………….

40

3.4 Prevalence of gastro-intestinal nematodes in donkeys and horses per

month…………………………………………………………………

45

3.5 Mean± SD and range of egg per gram of faeces (epg) count in

donkeys and horses infested with gastro-intestinal nematodes per

month………………………………………………………………..

46

3.6 Severity of infection with gastro-intestinal nematodes in donkeys and

horses per month…………………………………………………….

47

3.7 Mean faecal egg counts (±SD) and reduction percentage for

Albendazole-treated donkeys……………………………………….

49

3.8 Mean faecal egg counts (±SD) and reduction percentage for Albendazole twice-treated donkeys……………………………….

50

3.9 Mean faecal egg counts (±SD) and reduction percentage for

Ivermectin-treated donkeys…………………………………………

51

3.10 Summary of harvested worms from control and animals treated with

Albendazole (ABZT1) drench at necropsy…………………………

52

3.11 Summary of harvested worms from control and animals treated with Albendazole Twice (ABZT2) drench at necropsy……………………………………………………………

53

3.12 Summary of harvested worms from control and animals treated with

Ivermectin (IVMT) drench at necropsy……………………………..

54

3.13 Changes in total protein concentration (g/dL) following oral

administration of experimental anthelmintics compounds……….

62

3.14 Changes in albumin concentration (g/dL) following oral

administration of experimental anthelmintics compounds…..……

63

3.15 Changes in urea concentration (mg/dL) following oral administration

of experimental anthelmintics compounds……………………..….

64

3.16 Changes in creatinine concentration (mg/dL) following oral

administration of experimental anthelmintics compounds…..……

65

3.17 Changes in calcium concentration due (mg/dL) following oral

administration of experimental anthelmintic compounds…………

66

3.18 Changes in inorganic-phosphorus concentration (mg/dl) following

oral administration of experimental anthelmintics compounds……

67

LIST OF FIGURES

No. Figure Page

2.1 Map of South Darfur state, Sudan………………………………. 22

2.2 Experimental animals allocated and penned in the clinic of Faculty

Veterinary Sciences, Nyala University.........................................

25

2.3 Preparing a donkey for Euthanasia.............................................. 27

2.4 Bleeding of a donkey from the group treated with Albendazole... 28

2.5 Abdominal cavity of Euthanized donkey treated with Albendazole. 29

2.6 Cranial mesenteric artery removed from euthanized donkey treated

with Albendazole…………………………………………………..

30

2.7 Stomach removed from euthanized donkey treated with

Albendazole.................................................................................

31

3.1 Percentage of donkeys to horses in the population sampled……. 37

3.2 Prevalence of gastro-intestinal nematodes in donkeys and horses

per month………………………………………………………….

38

3.3 Severity of infection with gastro-intestinal nematodes in donkeys

and horses per month……………………………………………..

41

3.4 The percentage efficacy of Albendazole (Different dose regimen) and Ivermectin at 14 days post treatment…………………………..

55

3.5 larvae of Strongylus vulgaris in cranial mesntric artry removed form Albendazole treated donkey………………………………..

56

3.6 larvae of gasterophilus in a stomach removed from Albendazole treated donkey…………………………………………………….

57

3.7 Parascaris equorum removed from small intestine of donkey in the control group………………………………………………….

58

3.8 large and small strongyles in a Caecum removed from donkey in the control group…………………………………………………..

59

3.9 Encysted larvae of small strongyles in colon of donkey in the 60

control group…………………………………………………….

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude and sincere appreciation to

my supervisor Prof. Tigani Hassan Department of Medicine, Pharmacology and

Toxicology. Faculty of Veterinary Medicine University of Khartoum and my

co-supervisor Dr. Hisham Ismail Seri Department of Clinical studies, Faculty of

Veterinary Science, University of Nyala, for their unfailing guidance,

constructive criticism, provision of facilities, encouragement and for their

considerable assistance throughout this work.

Special thanks are due to staff members of the faculty of Veterinary

Science, University of Nyala. Special thanks are due to Dr. Hidaia B. Zolain.

Tech. Ahmed Alam Eldin and Mr. A/Allah Younis for the help with

exsanguinations and necropsy of donkeys and to Mr. El Tahir Shueib for the

help with statistical analysis.

My thanks are also due to all members of the Nyala Veterinary Research

Laboratory specially Dr. M. B. A/Wahab and Mr. S. S. Suleiman A. Noga.

The thanks extend to Dr. Awad El kareem A/Allah the head of animal

production development, Federal Ministry of Animal Resource and Fishery; Dr.

M. Tahir (CHF development organization), Mr. Mustafa Arabi for the help in

collection of faecal samples. And to my colleagues in the Ministry of Animal

Resource and Fishery, South Darfur State for all kinds of help they provided.

The author also would like to appreciate the kind help he received from

the staff members of the Department of Radioisotopes, Central Veterinary

Research Laboratory (CVRL) Soba, in this regard special thanks are due to Dr.

Yousif H. A. Elmansoury.

Above all, praise is to Allah, the Compassionate, and the most Merciful

for giving me the health and strength to carry out this work.

English abstract

In this study we decided to throw light on some aspects of donkey’s

helminthes infestation and control and the impact of treatment on liver and

kidney function.

In this study, 1256 animals were examined, 446 horses (Equus cabalus)

and 810 donkeys (Equus asinus) during the period October 2006 to September

2007, in South Darfur State, to investigate infection with gastro-intestinal

nematodes. The overall prevalence of gastro-intestinal nematodes was 29.20%.

November showed the highest incidence of infection (41.50%) while June the

lowest incidence of infection (13.92%). The average arithmetic mean of egg per

gram of faeces (epg) count was 589.97 ± 986.02 and the highest range reported

was on April of 50-13450. The animals harbouring mild infection reported the

highest incidence of 81.35%, while moderate infection reported 8.11% and

10.54 for severe infection. The most prevalent genera of gastro-intestinal

nematodes were Strongylus spp, Cyathostomes spp, Trichostrongylus spp, and

Strongyloides westeri.

Horses showed prevalence of 15.37%. In August showed we observed the

highest incidence (100%), while in March was the lowest incidence (5.26%).

Severity of infection reported were 82.35% for mild, 8.82% for moderate and

8.82% for severe infection. Arithmetic mean of egg per gram of faeces (epg)

count was 975.37±1099 and the highest range reported on November of 50-

13450.

Donkeys showed 37.48% prevalence of gastro-intestinal nematodes. In

January we observed the highest incidence of 55.79%, while in May we

reported the lowest incidence 14.89%. Severity of infection showed 81.25% for

mild, 7.89% for moderate and 10.86% for severe infection. Arithmetic mean of

egg per gram of faeces (epg) count was 750.14±1071.95 and the highest range

reported was in April 50-11800.

The therapeutic efficacy of Albendazole and Ivermectin drench

formulation at the manufacturers recommended dose were evaluated in

controlled trial at Nyala town, South Darfur State, Sudan. The study involved

24 donkeys naturally infected with gastrointestinal nematodes; they were

divided into four groups of equal size. Albendazole was administered orally

once at dose rate of 10 mg/kg body weight for group one (ALB1) and twice

with interval of 14 days post treatment at the same dose rate of 10 mg/kg body

weight for group two (ALB2). Ivermectin was administered orally as single

dose at 200 µg/kg body weight for group three (IVMT), group four was left

without treatment as a control. Treatment efficacy was based on the mean faecal

egg count reduction 14 days post treatment. A faecal egg count reduction of

100% was found after treatment with Albendazole, Albendazole twice and

Ivermectin. At necropsy efficacy percentages of Albendazole, Albendazole

twice and Ivermectin against adult nematode were as follows: Trichostrongylus

axei 67.09%, 100% and 100%; Parascaris equorum 100%, 100% and 100%;

Oxyuris equi 100%, 100% and 100%, Strongylus sp. 98.4%, 100% and 98.81%;

and small strongyles 91.7%, 99.5% and 96.5%. Albendazole, Albendazole twice

and Ivermectin with the single dose showed low to moderate efficacy (33%,

48.84% and 62.71%, respectively) against larvae found the cranial mesenteric

artery aneurisms. No adverse reactions were observed in treated donkeys

during the experiment period.

Blood samples were also collected from the above treated animals to

examine the effect of treatment on different biochemical parameters. Blood

samples were collected at day 0, 1, 3, 7, 14 and 21 days post treatment, then

serum biochemical analysis was conducted using commercial kits. Although,

results obtained showed significant changes during some sampling times but all

the values were within the normal range suggested by other researchers. All

parameters tested were within the normal range by the end of the study.

بسم اهللا الرمحن الرحيم

اخلالصة

بالديـدان الطفيليـة اجلوانب املتعلقـة باإلصـابة مت تسليط الضوء على بعض يف هذه الدراسة

.حيوية يف احليوانات املعاجلةوم بعض القيم الكيمي عالجها و تقي واالسطوانية املعدية املعوية

شـهر سـبتمرب و إىل 2006 مت إجراء مسح و بائي خالل سنة كاملة امتدت من شهر أكتوبر

حيوان من الفصيلة اخليلية و قد كـان عـدد 1256مشل املسح ، الية جنوب دارفور ينة نياال و مبد 2007

خضعت مجيعها للفحص عن اإلصابة الطبيعية بالديدان الطفيلية االسطوانية 810 بينما احلمري 446اخليول

شهر نوفمرب النـسبة سجل، %29.20نسبة اإلصابة : كم يلي" و قد كانت النتيجة إمجاال. املعدية املعوية

متوسـط تعـداد ،%13.92"بينما سجل يونيو النسبة األكثر اخنفاضا % 41.50األعلى بني كل الشهور

-50 يف شهر ابريـل هليسجقد مت ت لتعداد البيض و كان املدى األعلى986.02 ±589.97البيض كان

بينما سجلت اإلصابة % 81.35 النسبة االعلي إلصابة اخلفيفة شكلت ا من ناحية حدة اإلصابة، . 13450

االسـطوانية األجناس التالية من الديدان %. 8.11" توسطة كانت اقل حظا و اإلصابة امل % 10.45احلادة

,Strongylus spp, Cyathostomes spp: التالية سجلت الغالبيـة يف العينـات الـيت مت فحـصها

Trichostrongylus spp, and Strongyloides westeri .

بينمـا % 100سجل شهر أغسطس النسبة األعلى ، %15.37 نسبة اإلصابة يف اخليول كانت

% 8.82لإلصابة اخلفيفة و % 82.35و توزعت حدة اإلصابة ما بني %. 5.26شهر مارس كان األدىن

و 1099 ±975.37لكل من اإلصابة املتوسطة و احلادة و كان متوسط تعداد البيض لزنة جرام من الروث

.نوفمرب سجل يف شهر 13450-50املدى األعلى لتعداد البيض

كانـت يف % 55.79لكل العام و لكن النسبة األعلى % 37.48 نسبة اإلصابة يف احلمري كانت

املتوسـطة ، % 10.86اإلصابة احلادة شـكلت . كانت يف شهر مايو % 14.89شهر يناير بينما األدىن

±750.14متوسط تعداد البيض لزنة جـرام مـن الـروث كـان %. 81.25بينما الطفيفة % 7.89

.11800-50على لتعداد البيض سجل يف شهر ابريل و كان م املدى اال1071.95

و ) املعلـق ( البنـدازول ي من ذكور احلمري لتقدير جناعة كل من عقار 24 أجريت جتربة مشلت

ملجرام للكيلو جـرام لعقـار 10( صى ا من املصنع للعقارين حسب اجلرعات املو ) الشراب( االيفرمكتني

يف عـالج اإلصـابة الطبيعيـة بالديـدان ) مايكروجرام للكيلو جرام لعقار االيفرمكتني 200البندازول و

تلقت اموعـة األوىل وانات بالتساوي إيل أربع جمموعات، قسمت احلي . االسطوانية الطفيلية املعدية املعوية

14 بالفم بفاصل زمين قدره وية واحدة من عقار البندازول بينما تلقت اموعة الثانية جرعتني أيضاً جرعة فم

اموعة الثالثة تلقت جرعة فموية واحدة من عقار االيفرمكتني بينما مت االحتفـاظ يوم من عقار البندازول،

التجربة يف تقدير فعالية الـدوائني علـي اعتمدت . باموعة الرابعة دون عالج للتحكم و املقارنة فيما بعد

القتل الرحيم يف اليـوم مت إجراء يوما من جتريع الدواء و من مثة 14االخنفاض يف تعداد البيض الروث خالل

احلادي و العشرين من جتريع الدواء و ذلك لفحص حمتوي القناة اهلضمية من الديدان االسـطوانية املعديـة

.املعوية

و . للثالثة جمموعات املعاجلـة % 100اض تعداد البيض يف زنة جرام من الروث كان بنسبة اخنف

% 100و % 100و % 67.09بلغــت نــسبة الفعاليــة يف معاجلــة أنــواع الديــدان االســطوانية

Trichostrongylus axei .100% ،100 % ــنس % 100و . Parascaris equorumجلـ

جلــنس %98.81 و Oxyuris equi .98.4%، 100%جلــنس % 100و% 100، 100%

Strongylus spp. . 91.7%، 99.5%، كانت ألجناس % 96.5 وsmall Strongyles لكل من

العقاران أبديا درجة فعالية . عقار البندازول باجلرعة العادية مث اجلرعة املضاعفة و عقار االيفرمكتني على التوايل

% 62.71للبندازول باجلرعـة املـضاعفة و % 48.84للبندازول باجلرعة العادية % 33متوسطة بلغت

املتغذيـة علـى الـدماء بالـشريان Strongylus sppلاليفرمكتني يف عالج يرقات الطور الثالث لديدان

.املساريقي القحفي و فروعه

18 االستطبايب بعقاري البندازول املعلق و االيفرمكتني الشراب يف أجريت دراسة ثانية لتقيم األثر

قسمت احليوانات بالتـساوي ايل . من ذكور احلمري ذات اإلصابة الطبيعية بالديدان االسطوانية املعدية املعوية

تلقت اموعة االوىل جرعة فموية واحدة من عقار البندازول بينما تلقت اموعة الثانيـة ، ثالث جمموعات

اموعة الثالثة تلقت جرعة فموية واحدة ، ندازول يوم من عقار الب 14 بالفم بفاصل زمين قدره جرعتني أيضاً

من العالج و من بعد 21يوم " و أخريا 14 ،7، 3 ،1 يوم صفر، مجعت العينات يف . من عقار االيفرمكتني

اختالفات النتائجسجلت . اليت مت مجعها خالل التجربة ذلك اجري التحليل الكيموحيوي على عينات املصل

و لكن غالب االختالفات كان ضمن املـستوى الطبيعـي و الزمنية خالل التجربة الفترات معنوية يف بعض

. كل القراءات كانت ضمن املستوى الطبيعي يف اية التجربة" عموما

CHAPTER ONE

Introduction

In Sudan, donkeys are becoming increasingly important in view of their

increased use instead of horses in labour as drought animals as well as carrying

water, and in transportation. This present situation can be observed in many

urban and suburban areas. Moreover, donkeys are used as pack beasts and in

ploughing. As in many other developing countries donkeys in Sudan play and

constitute an important source of cheap energy for agricultural production by

way of traction for cultivation and transport of produce, goods and labour.

With regard to livestock population, Sudan constitutes one of the richest

African and Arab countries. Most of these animals are owned by nomadic or

semi-nomadic tribes living in the semi arid regions. Livestock population was

reported to be about 121 million head, composed of 35.825, 44.802, 37.346,

3.031, 0.65 and 6.35 million head of cattle, sheep, goats, camels, horses and

donkeys respectively (SBAR, 2000). However, donkeys’ population is

relatively large compared to camels and horses. About 30.9% of donkeys’ are

found in Darfur States.

Horses, ponies and donkeys are hosts to a large population of parasites

(Duncan, 1983). Lichtenfels (1975) stated that; equids are hosts for helminths

belonging to 28 genera and 75 species of nematodes, 2 genera and 5 species of

trematodes as well as 3 genera and 24 species of cestodes.

Helminth parasite infection was the main problem reported in donkeys

admitted to veterinary clinic (Ali et al., 2001). Seri and his colleagues (2004)

showed that the prevalence of nematodes infection in donkeys in Khartoum

state was 70.1%. Kheir and Kheir (1981) conducted field survey in South

Darfur State, Sudan, and stated that the prevalence was 56.2% and overall

prevalence of infection with nematode parasites was (58%) in town animals and

(22%) in nomadic areas. In Sennar; of the 218 donkeys examined for parasitic

infestation, 193 donkeys were positive and the prevalence was 88.53% (El

Dirdiri et al., 1986).

Parasites may cause many effects to their hosts (equines): suck blood,

often which cause anaemia and even death; penetrate and destroy the mucosal

cell, severely impairing the host ability to digest and absorb nutrients;

physically obstruct the gut lumen; the damage of other tissues which caused by

migrating larvae (Brander et al., 1982).

The health and welfare of the donkey is of crucial importance to those

who depend on it for their livelihood. Should a donkey fall out of work or die,

the family depending on it may suffer great hardships and even non-recoverable

adversities.

Before 1960 the two most widely used compounds for the control of adult

strongyle worms and Parascaris equorum, and some of the less important horse

worms, were Phenothiazines and Piperazines (Gibson, 1975). And since the

introduction in the early 1960s of the first broad spectrum anthelmintic

Thiabendazole, followed shortly afterward by Tetramisole, there have been

virtual processing of new highly effective broad spectrum anthelmintic and

considerable confusion reigns as to the merits of the different drugs (Armour

and Bogan, 1982).

Details such as the number and timing of treatments recommended in

different areas of the world vary according to local conditions, including

management practices, environmental factors and availability of drugs.

Evaluation of anthelmintics is determined consequent upon knowledge of the

epidemiology of the parasites to be treated and their importance in the

economics of the production.

The prominent importance of donkeys in the Sudan coupled with rarity

and scantiness of data and information concerning helminth parasites infestation

and treatment in this species of domestic animals seems to justify the current

study.

Objectives

The main objectives of this study are:

1. To determine the prevalence of the gastrointestinal helminths of equines in

South Darfur State.

2. To investigate the therapeutic efficacy of the drugs under study against

gastrointestinal nematodes of donkeys.

3. To report on the post treatment biochemical changes, if any.

Literature review

1.1 Albendazole

Benzimidazole anthelmintics have a broad spectrum of activity against

gastrointestinal helminths, including migrating strongyle larvae and lungworm

infections, and are well tolerated by mammals (McKellar and Scott, 1990).

All benzimidazoles have been developed as a result of the extensive

studies that were carried out in a number of laboratories to modify the structure

of thiabendazole. A feature of the newer structures is that they are metabolised

and excreted much more slowly than thiabendazole. This is achieved by

blocking the 5-position. The substitutions of another group in the 5-position,

and the replacement of the thiazole ring by methyl carbamate, have a very

significant effect on the rate of elimination (Brander et al., 1982). The

differences in efficiency between members of this class of drugs against groups

of parasites probably reflect differences in bioavailability of the drugs within

the host animal (McKellar and Scott, 1990).

Benzimidazoles have a low mammalian toxicity and it have proved

impossible to find an LD50 for some of these compounds. Oxenfendazole and

albendazole are teratogenics, and depending on the dose rate, should not be used

in early pregnancy (Armour and Bogan, 1982). In vitro turbidimetric techniques

and competitive colchicine-binding studies demonstrated the inhibitory power

of benzimidazoles on the polymerization of tubulin into microtubules. The

difference in the sensitivity of host and parasites to the effects of

benzimidazoles may be due to difference in the structure of microtubules in

their cells (Davis and Gull, 1983).

1.1.1 Identity

Albendazole is a member of the benzimidazole group. It is orally

administered broad-spectrum anthelmintic, soluble in dimethylsulphoxid, strong

acids and strong bases. It is slightly soluble in methanol, chloroform, ethyl

acetate and acetonitrile. Albendazole is practically insoluble in water.

1.1.2 Chemical name

Methyl [5-(propylthio)-1H benzimedazole-2 yl] carbamate.

1.1.3 Molecular formula

C12 H15 N3 O2 S.

1.1.4 Molecular weight

265.34

1.1.5 Appearance

It is pale green powder. The recent commercial products are white to off-

white (Brander et al., 1982).

1.1.6 Structural formula

1.1.7 Mode of action:

The inhibition of the fumerate reductase system appears to be the mode

of action of the drug, which appears to act on the parasite by absorption

through the cuticle (Brander et al., 1982). Albendazole is thought to act by

binding to parasite -tubulin, inhibiting its polymerization and impairing glucose

uptake. Albendazole is initially oxidized to albendazole sulphoxide, an active

drug, and then further oxidized to albendazole sulphone, which is inactive

(Moskopp and Lotterer, 1993).

1.1.8Toxicity of Albendazole

Acute toxicological effects

World health organization (WHO), the food additive series 25 reported

findings in dead rats included, urinary staining of abdomen, bloody discharge

around nose, and intestinal haemorrhage. Also in another study, necropsy of

dead rabbits showed intestines containing fluid dilated with gas.

Short term toxicological effects

Mice

Two groups of both males and females treated with albendazole in

different doses for three months. All the females and 50% of the males of the

highest dose 1600 mg/kg bw (160 times the recommended dose) died

spontaneously or killed in a moribund condition. From week 9, ear lesions

involving thickening and/or encrustation of the lip were observed in 20% of the

males and 22.2% of the females at 800 mg/kg bw (80 times the recommended

dose), and 100% of the males at 1600 mg/kg bw. Food consumption was

generally decreased in males given 400 mg/kg bw (40 times the recommended

dose) or more but body weight gain was depressed only at1600 mg/kg bw. At

the end of the study haemoglobin (Hb), haematocrit and erythrocyte levels were

reduced at dose of 800 mg/kg bw or more also decreased leucocyte counts

observed in males at (800 mg/kg bw) (Daly and Rinehart, 1980).

Post mortem examination revealed increase in absolute and relative liver

weight at 40 mg/kg bw (4 times the recommended dose) in males and 80 mg/kg

bw (8 times the recommended dose) in females (Daly and Rinehart 1980).

Rats

The toxic signs following administration of higher doses more than 48

mg/kg bw, (4.8 times the recommended dose) were diarrhoea, piloerection,

nasal swelling with blood stained, nasal discharge, death and body weight

depressed. Haemoglobin, haematocrit, erythrocyte and leucocyte counts were

reduced (Simon, 1979a).

At high doses 48 and 168 mg/kg bw, (4.8 and 16.8 times the

recommended dose respectively), adrenal size was increased particularly in

females. Testes were reduced in size at 48 mg/kg bw (Simon, 1979a).

Histopathology revealed hypoplasia in testes, bone marrow, spleen and lymph

nodes at 48 and 168 mg/kg bw (Simon, 1979).

At dose of 45 mg/kg bw (4.5 times the recommended dose) plasma

cholesterol was increased in males and females, potassium was increased in

males and albumin, plasma and erythrocyte cholinesterase were decreased in

females, and urinary protein was increased at 30 and 45 mg/kg bw (3 and 4.5

times of recommended dose, respectively) (Daly and Hogan, 1982).

Numerous gross alternations were noted with high doses in post mortem

including discoloration and/or nodules in the lungs, heart, lymph nodes, spleen,

pancreas, liver, adrenal and kidneys. Some of these organs were also enlarged

or showed adhesions. And thymic tissue was often absent and testes were small

and flaccid (Daly and Hogan, 1982).

Histopathology carried out identified a number of instances, where

bacterial colonization in lung, spleen, kidney, and heart was associated with

necrosis without the usual acute inflammatory response (Daly and Hogan,

1982).

Long term toxicological effects

Mice

Long-term toxicological studies in mice revealed decrease in erythrocyte

and leucocyte counts without toxic signs or effects on food intake and body

weight (Selwyn, 1987). Histopathological tests carried out indicated flaccid or

small testes, testicular tubular degeneration and oligospermia (Selwyn, 1987).

Rats

Studies carried in rats, showed decrease of total leucocytes neutrophil

counts, serum cholesterol was increased in some sampling times (Sauer 1985).

Post mortem findings and histopathology revealed in rats treated at 20mg/kg bw

(2 times of recommended dose) were increased incidence of flaccid testes (Daly

and Knezevich, 1987). Degeneration, atrophy of germinal and relative liver

weight in males and hepatic fatty metamorphosis in both males and females

(Daly and Knezevich, 1987).

Reproductive toxicological effects

In a group of rats fed diet containing albendazole at different doses for

three successive generations. There were no toxic sings, body weight, food

consumption, mating, fertility, pregnancy rate and gestation length, were

normal. During lactation weight gain depressed but only in F1 and F2

(Schroeder and Rinehart, 1980). In female rats uterine examinations in gestation

day 13 showed fewer implantation (not significant) at dose of 30 mg/kg bw (3

times the recommended dose) with no effect on resorption (Boutemy, 1980).

Genotoxic effects

Studies on Chinese hamster ovary cells indicated negative result for

genotoxicosis by albendazole (Galloway, 1981).

Teratogenic effects

Mice

There was no overt maternal toxicity or effects on resorption incidence,

foetal weight and external-visceral and skeletal development of foetus following

administration of albendazole (Killeen and Rapp, 1975).

Rats

At dose of 6 - 62 mg/kg bw (0.6 and 6.2 times of recommended dose)

skeletal abnormalities were increased and greater with increases in resorption

and external malformations, decreased foetal weight. The major malformations

were cranio-facial and bone defect (Martin, 1980). The dose of 30 mg/kg bw (3

times the recommended dose) showed foetal limbs abnormalities (Christian,

1984).

At 40 mg/kg bw (4 times the recommended dose) no toxicity or effect on

gestation and parturition. Small lungs and anasarca possibly related to treatment

in foetus (Johnson, 1981).

Rabbits

In rabbits, the maternal mortality was increased in 30 mg/kg bw (3 times

the recommended dose) and reduction in implantation in gestation day 7 – 19

(Killeen and Rapp, 1975).

Sheep

The pregnant ewes given albendazole in gestation day 17 were allowed to

deliver naturally, but premature delivery in more ewes was noted. All premature

lambs were stillborn; consequently the number of live lambs was reduced. Post-

mortem revealed increased incidence of prognathia, scoliosis, spinal bifid,

reduced tail and poorly developed or absent kidneys (Tash and Harper, 1977).

1.1.9 Pharmacokinetics

Mice

The single gavage dose of albendazole C14-ring labelled albendazole

given to mice revealed, over 72 hours period, that 20.5% of the administered

dose was recovered in the urine. Albendazole sulphoxide and two other

metabolites accounted for 81% of label. Low level of parent drug, albendazole

sulphoxide and three other metabolites were detected (Parish et al., 1979a). The

sulphone metabolite has higher plasma level than sulphoxide (Souhaili El Amri

et al., 1988). Both the sulphoxide and the sulphone metabolites decreased to

very low levels at 18 hours (Delatour et al., 1984).

Albendazole induces certain hepatic drug-metabolising enzymes, which

may be responsible for the enhancing degradation of sulphoxide to sulphone

following repeated administration (Souhaili El Amri et al., 1988).

Rats

In rats 31% of administered radioactive dose was recovered in the urine

over 72 hours period. Sulphoxide, 2-aminosulphone and three other metabolites

accounted for 89% of the label. Albendazole sulphone and two other

metabolites were also found (Parish and Gyurik, 1979). The sulphoxide and the

sulphone derivatives resulted in the urinary excretion. The urinary metabolites

were qualitatively similar to those observed after albendazole administration

(Parish and Gyurik, 1979).

Sheep

Albendazole was absorbed unchanged from the rumen and passed

through the stomach while the metabolites were secreted or diffused in this

organ (Marianer and Bogan, 1980).

After single oral dose of labelled albendazole on day 1, in the liver the

label was mainly on form of sulphoxide and sulphone metabolites, which were

progressively converted to 2-amino sulphone which was the primary metabolite

on day 8. Over 72 hours the sulphoxide and 2-amino sulphone accounted 60 –

70% of urinary excretion of the drug. Low levels of parent drug and the

sulphone with 6 other metabolites were detected (Colman et al., 1977).

Cattle

The metabolism to the sulphoxide and the sulphone was rapid and the

compounds were present in plasma for up to 40 hours (Prichard et al, 1985).

In calves at dose of 20 mg/kg bw (4 times the recommended dose), over 7 days

period 47% of the administered dose was recovered in urine; the sulphoxide,

sulphone and 2-amino sulphone accounted 70% of the excreted dose (Parish et

al.,1977 )

Although albendazole excreted in the animal’s milk, biliary elimination

presumably accounts for a portion of the elimination as evidenced by biliary

concentrations of albendazole similar to those achieved in the plasma.

Limited examinations of kidney revealed a similar metabolic profile (Kaeer et

al., 1977)

Human

Oral bioavailability of albendazole appears to be enhanced when it is co-

administered with a fatty meal as evidenced by higher plasma concentrations of

albendazole sulphoxide as compared to the fast state. Albendazole not detected

in plasma, but its main metabolites could be found and the half-life of

albendazole sulphoxide is 10 and 15 hours (Jung et al., 1992).

Albendazole sulphoxide is 70% bound to plasma protein and is widely

distributed through the body; it has been detected in urine, bile, liver, cyst wall,

cyst fluid and cerebral spinal fluid (CSF). Analysis of the urine showed the

presence of the sulphoxide, the sulphone and their amino derivatives with three

other metabolites (Rossignol and Maisonneuve, 1984).

Equine

Albendazole when given to donkeys at dose of 10 mg/kg bw; the parent

molecule of the drug was not detected in the plasma, but its sulphoxide and

sulphone metabolites were detected, demonstrating that albendazole was

completely metabolised by first-pass mechanism in donkeys. The sulphoxide

metabolite of albendazole was significantly higher than that of the sulphone

metabolite (Gokbulut et al., 2005).

Faecal concentration of parent molecule of the drug after albendazole

administration was significantly metabolised, probably by gastro-intestinal

microflora, to its sulphoxide metabolites that showed a similar profile to the

parent molecule in the faecal sample (Gokbulut et al., 2005). Plasma

concentration of albendazole was significantly lower when compared with

oxifendazole (Gokbulut et al, 2005).

Benzimidazoles are extensively metabolized in all animal species.

Generally, the plasma elimination half-lives of the parent drugs are short and

the metabolic moieties predominate in plasma and tissues and in the excretion

of the host as well as in parasites recovered from Benzimidazole-treated animals

(Lanusse and Prichard, 1993; Delatour and Parish, 1986).

The less soluble Benzimidazole compounds have a longer residence in

the fore stomachs of ruminants from which they are absorbed over prolonged

periods and thus remain in the plasma for a relatively long time. Since an

equilibrium exists between the plasma and gastrointestinal tract, the duration of

exposure of gut parasites to an effective concentration of the drug is extended

(Mackellar and Scott, 1990). Extremely insoluble anthelmintics may be less

effective, since they may not be absorbed and are excreted unchanged in the

faeces. This may explain the difference between the plasma concentrations of

oxfendazole (fenbendazole sulphoxide, FBZSO) following oral administration

and its inter-convertible metabolite fenbendazole (FBZ) (Ngomuo et al., 1984).

A large proportion of the less soluble FBZ is known to be excreted in the faeces

of ruminants (Duwel, 1977).

Oxfendazole, fenbendazole and albendazole (ABZ) are commercially

available sulphur-containing benzimidazoles that commonly undergo

microsomal oxidation in liver. Sulphide benzimidazoles (FBZ and ABZ) are

reversibly metabolized to their sulphoxide derivatives (Marriner and Bogan,

1980, 1981; Gyurick et al., 1981; Mohammed Ali et al., 1987). Irreversible

sulphonation follows sulphoxidation and is a slower oxidative step resulting in a

sulphone metabolite (Averkin et al., 1975).

The pharmacokinetics of FBZ and FBZSO have been studied in horses in

which the metabolic inter-conversion of those compounds appears to differ

substantially from ruminants. In the horse, the bioavailability and residence time

of the tested benzimidazoles (and metabolites) were lower and shorter,

respectively, than in ruminants (Mckellar et al., 2002; Gokbulut, 2000; Marriner

and Bogan, 1985, 1981).

Sulphide and sulphoxide benzimidazoles are known to bind to nematode

tubulin (Lacey et al., 1987) and therefore have activity against nematode

although sulphides exert an inhibitory activity on tubulin at lower

concentrations than sulphoxides. In most species examined, the sulphoxide

moiety predominates in plasma and is thought to confer activity against gut

dwelling nematodes following secretion across the gastrointestinal wall into the

gut lumen where it may undergo sulpho-reduction (Mckellar and Scott, 1990).

Maximum plasma concentration (Cmax) of albendazole sulphoxide

(ALBSO) (0.08µg/mL) and albendazole sulphone (ALBSO2) (0.04 µg/mL)

were obtained at (tmax) 5.71 and 8.00 h respectively, following administration of

albendazole. The area under the curve (AUC) of the sulphoxide metabolite

(0.84 µg.h/mL) of ABZ was significantly higher than that of the sulphone

metabolite (0.50 µg.h/mL).

Although (ABZ) is not licensed for use in Equidae. Its metabolites

presented a greater plasma kinetic profile than FBZ which is licensed for use in

horses. A higher metabolic capacity, first-pass effects and lower absorption of

benzimidazoles in donkeys decrease bioavailability and efficacy compared to

ruminants (Gokbulut et al., 2005).

1.1.10 Use of Albendazole in animals

Cattle

Albendazole had been shown to be highly effective against lungworms;

also it is very active against adult liver flukes in cattle when given at twice the

recommended dosage rate for roundworm therapy (Armour and Bogan, 1982).

Sheep

Albendazole is effective in chronic fasciolaisis treatment, also

albendazole is certainly effective against the adult and developing larval stage

of D. filarial and naturally occurring infection with the other small lungworms

in sheep and goat (Armour and Bogan, 1982).

Equine

There is a paucity of data available in the literature on the toxic or side

effects in equine and albendazole is not used in these species (Gokbulut et al.,

2005). A higher metabolic capacity, first-pass effects and lower absorption of

benzimidazoles in donkeys decrease bioavailability and efficacy compared to

ruminants (Gokbulut et al., 2005).

High intestinal concentrations could be effective against gastrointestinal

nematodes that inhabit the gut lumen, but very low plasma concentrations of

albendazole may not be effective against migrating fourth larval stages of large

strongyles or lung worms. Repeated dosage regimes of albendazole or co-

administration with metabolic inhibitors could be used to treat migrating larval

or tissue stages of strongyles and lungworms in donkeys (Gokbulut et al, 2005).

10 mg/kg BW of albendazole may be used for treatment of

strongyloidosis of horses and donkeys (Kassai, 1999). In Kentucky agricultural

experiment station, tests were conducted between February 1977 and June 1981

using 10 adolescent horses which were proved to be naturally infected with

population B small strongyles. Effective reductions (97% to 100%) were

recorded for treatments with Oxibendazole, Albendazole, and Thiabendazole.

The larval count indicated that all of the compounds were 100% effective in

removing larvae of S. vulgaris and S. edentates. Albendazole removed 94% to

100% of 11 species and 84% of the 12th species (Cyathostomum coronatum).

Removal of immature small strongyles by oxibendazole and albendazole in the

tests (15% and 38%) was somewhat less than the 71% for Oxibendazole in an

earlier study (Drudge et al., 1979). Population B small strongyles have been

reported to be resistant to thiabendazole, fenbendazole, and oxfenbendazole but

not to oxibendazole (Drudge et al., 1979). Five species of small strongyles were

resistant, whereas 19 species of small strongyles and 2 species of large

strongyles (Strongylus vulgaris and Strongylus edentates) were removed

efficaciously by all six benzimidazoles. Albendazole is closely related analogue

of oxibendazole and is active against the 5 species of Benzimidazole resistant

small strongyles.

Worm counts indicated that the efficacy of albendazole against larval stages

of small strongyles, measured as the percentage of worms removed by

albendazole treatment was limited, particularly for L3. The efficacy of

albendazole against these helminth adults was somewhat higher, but

considerable differences between species also were observed (Eysker et al.,

1988).

The effect of albendazole against larval stages was lower than that against

adult worms; this low effect mainly is the result of a limited efficacy of

albendazole against larval stages (Drudge et al., 1984). Low efficacy of

albendazole treatment can be a result of anthelmintic resistance (Eysker et al.,

1988)

Benzimidazole resistance is a common phenomenon in Cyathostomes. It

has been recorded in 5 species of Cyathostomes by Drudge et al., (1979).

In a series of experiments on the epidemiology and control of

cyathostome infections in Shetland ponies between 1984 and 1986, the effect of

repeated early-season albendazole (Eysker et al., 1986, 1988) and oxfendazole

(Eysker et al., 1989) treatments was evaluated. The results of these studies

invariably showed reduction in faecal egg output after the first treatment but

poor effect of later treatments.

Apparently the repeated use of one Benzimidazole within one grazing

season is of little prophylactic value (Eysker et al., 1989a). An important aspect

of the epidemiology of cyathostome infections in equids is the occurrence of

inhibited development. Gibson (1953) observed that some weeks after each of a

series of phenothiazine treatments, strongyle-type eggs reappeared in the faeces

of horses, even after housing period under helminth-free conditions of 3 years.

His explanation of this phenomenon was the existence of an equilibrium

between inhibited larvae in, and adult worm on, the gut wall leading to

resumption of development of inhibited larvae after removal of adult worms.

Later, Ogburne (1975) found evidence for an emergence of fourth-stage

larvae (L4) from the intestinal wall at the end of the winter, indicating a

seasonal pattern of inhibited development similar to many Trichostrongylidae of

ruminants.

Anthelmintics resistance in cyathostomin nematodes (‘small strongyles’) of

horses is a well-known phenomenon worldwide. Benzimidazole (BZ) resistant

cyathostomins have been reported from North and South America, South

Africa, Australia. New Zealand and many European countries (reviewed by

Kaplan, 2002) including Greece (Papadopoulos et al., 2000).

Benzimidazole resistant populations of cyathostomin nematodes were

detected on seven of ten Turkish horse farms (Cirak et al., 2004). According to

FECRT data, Benzimidazole resistance in stronglyids was observed only at

Dubrovsky (Ukraine) horse farm (FECRT=68%). No resistance to macrocyclic

lactones in strongylids or in Parascaris equorum was observed (Kuzmina and

Kharchenko, 2008). BZ resistance in horse strongylid nematodes in Ukraine

was found only in cyathostomins, or small strongyles (Borgsteede et al., 1997,

Kuzmina et al., 2002).

Horse strongylids are known to be resistant to Benzimidazoles (BZ) and

tetrahydropyrimidines (Kaplan, 2002, 2004), while the only case of resistance in

cyathostomins to microcyclic lactones was reported in donkey in the UK

(Trawford et al., 2005).

1.2 Ivermectin

Ivermectin was the first macrocyclic lactone anthelmintic, introduced as

a veterinary anti-parasitic agent in France in 1981 and marketed as a mixture of

22, 23 dihydro- B1a (>80%) and 22, 23 dihydro- B1b (<20%) (Fisher and

Morzik, 1989).

Ivermectin is today elixir in the world of parasites chemotherapy, it is a

potent agent, active against many internal and external parasites in domestic

animals, and it is vogue and highly effective with a wide margin of activity and

safety. In equines, a variety of adverse reactions have been reported in horses

after parenteral administration of Ivermectin at the recommended dosage of

200µg/kg body weight (Reed, 1983). These reactions have occurred in a small

percentage of treated horses and the drug is now sold as a paste for oral

administration. Ivermectin have an excellent efficacy for an important range of

gastro-intestinal nematodes of equine (Campbell et al., 1989).

1.2.1 Efficacy of Ivermectin against equine endoparasites:

Draschia spp and Habronema spp

Three species, H. musca, H. microstoma and D. megastoma, occur in the

stomach of equine. The largest one is the H. microstom. The first 2 are free in

the host’s stomach; D. megastoma lives in nodules in the stomach wall.

Diagnosis of Habronemiasis is often impossible because the larvae can seldom

be found in the faeces (Hall, 1985).

In horse, Herd and Donham (1984), showed 1-2 intramuscular doses of

ivermectin at 200 µg/kg were highly effective against Draschia spp and

Habronema spp., while DiPietro (1982) reported 100% efficacy.

Gasterophilus spp

The larvae of several species of this genus are parasites of equines and are

known as bots fly, the third- stage larvae causes inflammation and ulceration of

the mucous membrane of the stomach and duodenum in addition to blood

sucking (Soulsby, 1982; Hall, 1985).

At dose rate of 200-300 µg/kg when Ivermectin given intramuscularly to

horses, the efficacy was 99% against larvae of Gasterophilus spp, the few larvae

in the treated horses were located in the large intestine contents and were not

attached to the intestinal epithelium (DiPietro, 1982).

Parascaris equorum

This is found in the small intestine of horse kind. The infection and

migration is through the blood stream via the right ventricle of the heart to the

lungs (Hall, 1985).

When given orally as paste formulation, Ivermectin at 200 µg/kg totally

eliminated the passage of P. equorum in the natural infected horses (Cobra et

al., 1986). Ivermectin is also active against immature stages of P. equorum, in

ponies, larval burdens were determined at necropsy following treatment with

the paste at 200 µg/kg. When necropsy was performed 2 weeks after treatment,

the reduction in lung larvae burden was 100% (French et al., 1988) and the

reduction in intestinal larvae burden was also 100 % (DiPietro et al., 1987).

Both the paste and the liquid Ivermectin formulations have been shown to

be highly active against both the early (L3) tissue phase of P. equorum and

against the later intestinal (L4) phase of P. equorum. The efficacy has been

discussed by Boraski (1987).

Strongloides westeri

This is occurs in the small intestine, mainly in the duodenum and jejunum

which causes irritation, resulting in diarrhea, especially in young foals. 200

µg/kg of Ivermectin was effective against S. westeri in foals in a paste

formulation (Ryan and Best 1985).

Trichostrongylus axei

A small worm (3-8mm) found in the stomach and duodenum of horse

kind. In horses T. axie causes chronic catarrhal gastritis (Hall, 1985).

Stongylus spp

There are three species, Strongylus equinus, S. vulgaris and S. edentatus

occur in the large intestine including the caecum of equines (Soulsby, 1982).

Generally strongylidae worms are not severe pathogens, unless they occur in

large numbers when their mouth parts cause extensive damage by sucking in the

mucous membrane, thus causing ulcers. Larvae, however, in the characteristic

nodules, produced damage, which result in bacterial invasion and consequently

ulcers. When larvae leave the nodules there is extensive bleeding (Hall, 1985).

Fourth-stage larvae S. vulgaris penetrate the intima of the sub-mucosal

arterioles and migrate in the vessels towards the cranial mesenteric artery they

are to be found here from 14 days after infection onwards associated with

thrombi and later aneurysms (Soulsby, 1982). The oral paste and injectable

formulations of Ivermectin at 200 µg/kg was 100% effective against 8-week-old

Stongylus vulgaris, as revealed by necropsy 5 weeks after treatment (Klei et al.,

1984). Also as intramuscular injectable formulation at dose rate 200 µg/kg body

weight Seri and his colleagues (2005) showed that efficacy of Ivermectin

against arterial stages of Strongylus vulgaris was only 69, 23%.

Small Strongyles

Known as Cyathostomes worldwide, they occur in the large intestine of

the equides all over the world (Soulsby 1982). Burrows and his colleagues

(1985) used faecal examination to demonstrate the efficacy of Ivermectin paste

formulation at 200 µg/kg against benzimidazole-resistant small Strongylus, the

egg counts remained zero at least 3 weeks in horses. Also 200-300 µg/kg were

effective against Cyathostomes in percentage of 99% and the efficacy was 86%-

97% against immature stages of Cyathostomes according to 200-300 µg/kg dose

rate respectively (DiPietro 1982).

Oxyuris equi

A worm lives in the large intestine of horses; males are about 9 – 12 mm

and females up to 150 mm. According to Seri et al., (2005) the efficacy of

Ivermectin injectable formulation at dose of 200 µg/kg was 100%.

1.3 Prevalence of gastro- intestinal nematodes in donkeys and horses

Horses, ponies and donkeys are hosts to a large population of parasites

(Duncan, 1983). Helminths parasite infection was the main problem reported in

donkeys admitted to veterinary clinic (Ali et al., 2001).

Swedish nationwide study showed that strongyle infections were highly

prevalent in Swedish horse herds: 78% of 1183 examined horses on 110 farms

were found to shed nematode eggs (Lind, 2005).

In Ethiopia, All of 338 samples examined were found positive for

helminth eggs. Strongyle spp. (100%), Parascaris equorum (50%) and Oxuris

equi (3%). 81.7% of donkeys sampled were severely infected, 8.3% heavily,

3.8% moderately and 6.2% mildly (Ayele et al., 2006).

In Chad, Garber (1970) reported prevalence of 89% for Strongyle spp.

and Cyathostomes spp. 72% for Parascaris equorum, and 6% for Strongloides

westeri. In Kenya, Strongyle spp. prevalence was 57.6%, Cyathostomes spp.

was 15.4% and 20.7% reported for Parascaris equorum (Mukhwana, 1994). In

Morocco, the prevalence of different gastro-intestinal nematodes species

reported as follow: Cyathostomes spp. 52%, Parascaris equorum 37% and

93.5% was for Trichuris spp. (Abdelkarim, 1991).

In Sudan, Seri and his colleagues (2004) reported that, the prevalence of

nematodes infection was 70.1% in Khartoum state, Strongylus spp. 35.8%,

Cyathostomes spp 36.7% Parascais equrum 10.7%, Trichostrongylus axei 12%

Strongloides westeri 3.4%.

Kheir and Kheir (1981) conducted a field survey in South Darfur State,

and stated that the overall prevalence was 56.2% and overall incidence of

infection with nematode parasites was found (58%) in town animals and (22%)

in nomadic areas. Also five nematode genera were encountered in donkeys in

the same area: Strongylus spp 43.6%, Oxyuris spp 10.5%, Strongyloides spp.

3.9%, Parascaris eqourum 4.4% and Trichuris spp. 0.9%. In addition to

donkeys in south Darfur state, prevalence of gastrointestinal nematodes in

horses in Bahr Al Arab area was 18.5%.

Another study in Sennar state revealed that out of the 218 donkeys

examined for the parasitic infestation, 193 donkeys were positive and the

prevalence was 88.53%, the prevalence of Strongylus spp was 100% Oxyuris

spp. was 0.5%, both highly and moderately infested animals noted 36.27% and

27.46% for slightly infection (El Dirdiri et al., 1986).

Recently by postmortem examination, Ahmed (2008) reported that 97.8%

of examined donkeys at Nyala town were infected with one ore more of gastro-

intestinal parasite. He found that the distribution recovered parasites from

different parts of GIT were as follows: Stomach (92%) Small intestine (19.6%)

Caecum (88%) Colon (80%) and Rectum (73.9%). The author reported the

occurrence of Habronema SPP (40%), Trichostrongylus axei (30%), G.

Intestinales (92%), G. Nasalis (77%), Parascaris equorum (18%),

Anaplocephala perfoliata (4.4%), Gastrodiscus aegyptacus (8.7), Large

Strongyles (84%) and Small Strongyles (65%).

CHAPTER TWO

Chapter two

Materials and Methods

2.1. Survey of gastro-intestinal helminths in donkeys and horses

A field survey was conducted to study the prevalence of helminth

parasites in donkeys (Equus asinus) and horses (Equus cabalus). The current

study was conducted to spot light on the prevalence and intensity of infection

with gastro-intestinal helminth parasites in South Darfur State during the period

October 2006 to September 2007.

2.1.1 Study area

The study was conducted in South Darfur State which is located in

western Sudan between latitudes 9º- 30º N and longitudes 13º-15º E (Figure

2.1). Faecal samples were collected from donkeys and horses from different

locations in South Darfur State.

2.1.2 Sample collection and examination

A total number of 1256 animals (446 horses and 810 donkeys) were

sampled for fresh faecal samples, fresh faecal samples were collected directly

from the recta of individual donkeys and horses in long plastic bags, after

labeling of the bags, the samples were submitted as soon as possible to the

diagnostic laboratory of the Veterinary Research laboratory, Ministry of

Sciences and Technology in Nyala. Egg count was done using modified

McMaster technique (Anonymous, 1986) and the eggs were identified

according to Soulsby (1982).

2.1.3 Intensity of infection

The severity of the infection was obtained from the number of egg per

gram of faeces and was classified according to Soulsby (1982) as follows:

500 egg per gram of faeces Mild infection

Figure 2.1 Map of South Darfur state, Sudan.

800-1000 egg per gram of faeces Moderate infection

1500-2000 egg per gram of faeces severe infection

2.1.4 Parasitological techniques

2.1.4.1 The modified McMaster technique

A modified McMaster technique (Anonymous, 1986) was used to count

the egg per gram (epg) of faeces as following:

1. Three grams of faeces were mixed with 42 ml of tap water and the faecal

suspension was passed through the 80 µm square sieve to remove debris.

2. The filtrate was collected in a clean dry container.

3. 15 ml of this filtrate was taken into a centrifuge tube and centrifuged at

1500 rpm for 2 minutes and the supernatant was then discarded.

4. The sediment was emulsified by gentle agitation and saturated Na Cl was

added until the volume became equal to the initial aliquot of the filtrate.

5. The centrifuged tube was inverted several times to obtain an even

suspension of the contents.

6. The two chambers of the McMaster slide were filled using a clean Pasteur

pipette.

7. The average number of eggs present in the chambers was multiplied by

100 to obtain the number of egg per gram of faeces (epg).

2.1.4.2 Faecal culture and identification of larvae

A pooled culture of positive faecal samples was examined for larval

identification, and where possible 100 third stage larvae were identified as

described by Anonymous (1986).

2.2 Therapeutic efficacy of Albendazole against donkey’s worm infestation

The aim of this study was to investigate the therapeutic efficacy of

Albendazole at two different dose regimens as an anthelmintic in donkeys

harbouring natural worm infestation and to compare the results obtained with

that of Ivermectin.

2.2.1 Experimental Animals

In this study we utilized 24 male donkeys (3-10 years). Before starting the

study, animals were examined to prove infestation with gastrointestinal

helminth parasites. Animals were kept in the premises of the Department of

Clinical Studies, Faculty of Veterinary Science, and University of Nyala (Figure

2.2). They were provided with tap water and allowed to graze in pasture freely.

2.2.2 Experimental drugs

Albendazole suspension: Albendex 25mg/ml (Avico®. Jordon).

Ivermectin drench: Avimec liquid (Avico®. Jordon)

2.2.3 Design of the study

Experimental animals were allocated into four groups and penned

according to treatment groups. The first three groups were treated and the last

group was remained untreated as control group.

The animals in the three treatment groups received treatment as follows:

- Albendazole – treated group 1 (ABZT1) received a single oral dose of

Albendazole at the manufacturer recommended dose i-e 10 mg/kg body weight.

- Albendazole – treated group 2 (ABZT2) received two oral doses of

Albendazole 14 days apart, at the manufacturer recommended dose i-e 10

mg/kg body weight.

- Ivermectin treated group (IVMT) received a single oral dose of Ivermectin

drench at the manufacturer recommended dose i-e 200 µg/kg body weight.

Then donkeys were monitored for possible adverse reactions for 2 hours

after administration of each drug.

2.2.4. Sampling and Time schedule

The experiment extended for 21 days. Faecal samples were collected at 0

(before treatment), 1, 3, 7, 14, and 21 days post treatment. Necropsy of the

animals was done at day 21 post treatment for all donkeys.

Figure 2.2 Experimental animals allocated and penned in the clinic of

Faculty Veterinary Sciences, Nyala University.

2.2.5 Necropsy of animals and samples preparation

Animals were euthanized for worm recovery as described by Reinecke

and Le Roux (1972). After donkeys were euthanized as shown in figures (2.3-

2.7). The thoracic and abdominal cavities were opened by making an incision

along the ventral line of the animal and the left half of the thorax and the

abdominal wall was removed. The organs from the thoracic and the abdominal

cavities were removed from the carcass. The different organs from the gastro-

intestinal tract were then isolated by tying double ligatures around the gut to

separate it in the stomach, small intestine, caecum, colon and rectum. The

contents of the different organs were removed and then sieved through 150 mm

sieve to obtain residue samples. The residues preserved in 10% formalin.

Residue samples of ingesta were examined macroscopically. Nematodes

present were placed in a specimen bottle containing 10% formalin. Helminths

were identified at a later stage by placing them on glass slide, examining them

microscopically and classifying them according to Lichtenfels (1975).

2.2.6 Data analysis

The anthelmintic efficacy of Albendazole was estimated using faecal egg

count reduction test (FECR) for helminths burden. Arithmetic mean of the egg

counts and helminths burden were calculated to determine the mean percentage

reduction within each group, according to the following formula:

FECR % = Pre-treatment EPG - Post-treatment EPG X 100

Pre-treatment EPG

2.3 An assay of some biochemical parameters in donkeys medicated with

benzimidazole:

2.3.1 Collection of samples:

Blood samples were collected from the jugular vein of each animal and

were allowed to clot for 24 hours then the sera harvested and freezed for

serological tests.

Figure 2.3 Preparation and control of a donkey for Euthanasia.

Figure2.4 bleeding of a donkey from the group treated with Albendazole.

Figure 2.5 Abdominal cavity of Euthanized donkey treated with

Albendazole.

Figure 2.6 Cranial mesenteric artery removed from euthanized donkey

treated with albendazole.

Figure 2.7 Stomach removed from euthanized donkey treated with

Albendazole

2.3.2 Biochemical methods

2.3.2.1 Total Serum Protein (TSP)

The Biuret method as described by King and Wooton (1956) was utilized

to assess TSP using commercial kits (Spectrum, Egypt).

Principle of the test

In alkaline medium the copper reacts with the peptide bonds of protein to

form the characteristic pink to purple biuret complex. Sodium potassium

tartarate prevent copper hydroxide precipitation and potassium iodide prevents

the auto-reduction of copper. The colour intensity is directly proportional to the

protein concentration. The change in colour was measured using

spectrophotometer (Jenway 6105 U.V./vis. Spectrophotometer, U.K.).

2.3.2.2 Serum Albumin

Concentration of serum albumin was measured by Bromocresol green

method according to Bartholomew and Delany (1966).

Principle of the test

Measurement of serum Albumin based on its binding to the indicator dye

bromochresol green (BCG) in pH 4.3 to form a blue-green coloured complex.

The intensity of the blue-green colour is directly proportional to the

concentration of albumin in the sample. The assay was conducted using

commercial kits (Spectrum, Egypt).

2.3.2.3 Serum Urea

Urea is the major end product of protein nitrogen metabolism. It is

synthesized by the urea cycle in the liver and excreted through the kidneys. The

circulating levels of the urea depend upon protein intake, protein catabolism and

kidneys function. Elevated urea level can occur due to renal impairment or in

some diseases such as diabetes, infection, congestive heart failure, and during

different liver diseases. Determination of blood urea nitrogen is most widely

used screening test for renal function together with serum creatinine.

The principle of the test

Urea is hydrolyzed in the presence of water and urease to produce:

Urea + H2O urease › 2NH3 + CO2

The free ammonia in an alkaline pH and in the presence of the indicator

forms coloured complex proportional to the urea concentration (mg/dl) in the

specimen.

Serum urea concentration was measured by an enzymatic colorimetric

method using a commercial kit (Randox laboratories Ltd., United Kingdom)

according to Fawcett and Scott (1960). The intensity of the developing colour

was measured at 600 nm using Jenway spectrophotometer (Jenway 6105 U. V.

/vis. Spectrophotometer, U. K.).

2.3.2.4 Serum creatinine

Creatinine is derived from creatine and the creative phosphate in the

muscle tissue, and may be defined as a nitrogenous waste product. Creatinine is

not reutilized but it is excreted from the body through the urine via the kidneys.

Principle

Creatinine reacts with picric acid under alkaline condition to form a

yellow-red complex. The intensity of the developing colour was measured at

wavelength 492nm, using Jenway spectrophotometer (Jenway 6105 U. V. /vis.

Spectrophotometer, U. K) according to Bartels et al., (1972).The commercial

kits used were also from the same manufacturer (Spectrum, Egypt).

2.3.2.5 Serum Calcium

Calcium is the fifth most common element in the body, most of which

(98%) is present in the skeleton. One half of the remaining calcium is found in

extracellular fluid and the rest in tissue. Calcium has a crucial role in bone

mineralization and is also vital for basic physiological processes such as blood

coagulation, neuromuscular conduction, and normal muscle tone.

The principle of the test

Calcium ions react with O-cresolphthalein complexone (O-CPC) under

alkaline condition to form violet coloured complex. The colour intensity of the

complex formed is directly proportional to the calcium concentration (mg/dl). It

is determined by measuring the increase in absorbance at 578nm using

commercial kit (Spectrum, Egypt). The calcium values were calculated in

mmol/l of serum according to Sarkar and Chauhan (1967) and Barnett et al.,

(1973).

2.3.2.6 Serum inorganic phosphorus

The body contains phosphorus entirely in the form of phosphates

distributed fairly equally between extracellular and intracellular compartments.

About 85% of extracellular phosphate occurs in inorganic forms as

hydroxyapatite. In plasma or serum, most phosphate exists in the inorganic

form; this fraction is present as the mono- and dihydrogen forms. The relative

proportion is varying with the pH. Intracellular phosphate occurs mainly in as

phospholipids and phosphoproteins; this fraction is termed organic phosphate.

Assay principle

Inorganic phosphate reacts with ammonium molybdate in sulphuric acid to

form no reduced phosphomolbdate. The concentration of phosphomolbdate

formed is directly proportional to the inorganic phosphate concentration. It is

determined by measuring the increase in absorbance at 340 nm. The serum

inorganic phosphorus was determined according to Taussky and Shorr (1953)

and Goldenburg and Fernandez, (1966).

2.4. Statistical Methods

SPSS 11.5 for windows computer package was utilized to assess

significant differences, if any. Paired T-test was used to compare between

means.

CHAPTER THREE

Chapter three

Results

3.1 Survey of gastro-intestinal nematodes in donkeys and horses:

In the current study a total of 1256 donkeys and horses were examined

for helminth gastrointestinal nematodes, during the period October 2006 to

September 2007. The overall prevalence of infection with gastro-intestinal

nematodes in equines was 29.20% (Table 3.1). During the survey period,

November showed the highest incidence of infection (41.50%), while June

showed the lowest percentage 13.92% (Figure 3.2).

Considering severity of infection, animals with mild infection were

dominant (81.35%), while animals with severe infection were 10.54%, and

animals with moderate infection were the lowest one 8.11% (Table 3.3). The

highest incidence of mild infection was reported in August (93.75%) and the

lowest incidence was on April 64.29%. In animals harbouring moderate

infection, the lowest infection level was 0% and reported in August and the

highest moderate infection level was reported in March 20.69%. Severe

infection reported 0% in the lowest incidence level in June while 28.57% in

April as the highest (Table 3.3).

The results of faecal culture and identification of larvae revealed the

dominance of following helminth genera: Strongylus spp, Cyathostomum spp,

Trichostrongylus spp, and Strongyloides westeri. No efforts to larvae count had

done.

In this study, a total of 443 horses were involved in the current survey.

The prevalence of infection was 15.35% (Table 3.5). March reported the lowest

prevalence level of 5.26% and in November was the highest one 50% (Table

3.5). In February we failed to examine any horse. A total number of 810

donkeys were involved in this study. The prevalence of infection was 37.48%.

The highest prevalence infection was reported in January 55.79% and the lowest

one was 14.89% reported in May (Table 3.5).

Table 3.1 overall prevalence of gastrointestinal nematodes in donkeys and horses in South Darfur State

Type Total number Infected animals Prevalence%

Horses 446 68 15.73

Donkeys 810 302 37.48

Total 1256 370 29.78

Mean per month 104.42 30.83 29.20

Figure 3.1Percentage of donkeys to horses in the population sampled.

Figure 3.2 Prevalence of gastro-intestinal nematodes in donkeys and horses

per month

Table 3.2 Mean± SD and range of egg per gram of faeces (epg) in donkeys

and horses infested with gastro-intestinal nematodes

Month Mean ± SD Range

January 400.86 ± 640.41 50-3800

February 890.91±1,231.63 50-4500

March 655.17 ±859.65 50-3600

April 1390.03 ±2401.26 50-11800

May 457.89 ±526.32 50-1700

June 290.00± 242.44 50-900

July 283.75 ±290.53 50-1600

August 340.00± 364.10 50-1500

September 565.71 ±1366.55 50-8000

October 889.29 ±1,641.45 50-10400

November 608.49 ±1,725.63 50-13450

December 307.58 ±542.29 50-2400

Total 589.97 ±986.02 50-13450

Table 3.3 Severity of infection with gastro-intestinal nematodes in

donkeys and horses per month

Month Mild% Moderate% Severe%

January 87.93 3.45 8.62

February 72.73 18.18 9.09

March 68.97 20.69 10.34

April 64.29 7.14 28.57

May 84.21 5.26 10.53

June 81.82 18.18 0

July 92.68 4.88 2.44

August 93.75 0 6.25

September 77.14 5.71 17.14

October 67.86 10.71 21.43

November 88.52 4.92 6.56

December 78.79 15.15 6.06

Total 81.35 8.11 10.54

Figure 3.3 Severity of infection with gastro-intestinal nematodes in donkeys

and horses per month

The Mean ± SD for egg per gram of faeces (epg) was 975.37 ±

1099 for the whole year with a range of 50-13450 (epg). December reported the

highest mean epg count 2300 ± 0 with a range of 0-2300(epg). July showed

Mean ± SD of 300±0 of egg per gram of faeces with range of 0-300 (epg) as the

lowest epg count (Table 3.6). Also in table (3.6) we could observe that the

Mean ± SD of egg per gram of faeces for the 12 months in donkeys was

750.14±1071.95 with a range of 50-11800 (epg). The Highest range was

reported in April 100-11800 (epg) with the highest Mean ± SD in the whole

year 1888.89±2855.51 (epg). August reported the lowest level 261.54±185.02

(epg) with a range of 100-700 (Table 3.6).

The range of egg per gram of faeces showed several variations in the

minimum level during the whole year. In February was too high that reported

300 epg while 200 epg as a minimum epg level was reported twice in both May

and June respectively.100 epg was reported several times also 50 epg (Table

3.6).

Considering severity of infection, in mild infection the highest incidence

of mild infection was reported in June and July with 100% of the animals

showing mild level of infection, while no animal was infected on December

exhibited mild level of infection (Table 3.7). Moderate level infection showed

8.82% as the highest level was reported in March (33.33%) and 0% was the

lowest level reported in several months. The highest severe level of infection of

100% was reported in November and 0% was the lowest level reported several

months (Table 3.7).

In donkeys, concerning severity of infection, mild infection was the

prevalent with a 81.25%, while moderate infection showed 7.89% and 10.86%

for the severe infection (Table 3.7). The highest mild infection percentage

reported was 100% in August. While 55.56% was reported twice as the lowest

mild level of infection. The moderate level infection generally showed small

percentage 7.89% when compared to mild and severe infection. The highest

percentage of moderate infection was reported in June 20%, and the lowest

moderate infection 0% was reported in August and September respectively.

Severe infection reported the highest percentage in April 38.89%. Both June

and August reported 0% as the lowest percentage (Table 3.7).

Table 3.4 Prevalence of gastro-intestinal nematodes in donkeys and horses

per month

Prevalence% Month

Horses Donkeys

January 10.64 55.79

February ND 34.38

March 5.26 46.43

April 17.86 30.51

May 20.34 14.89

June 10.64 15.63

July 9.09 37.25

August 10.00 26.00

September 18.33 24.00

October 20.83 50.00

November 50.00 42.11

December 7.69 48.53

Total 15.73 37.48

Table 3.5 Mean± SD and range of egg per gram of faeces (epg) count in

donkeys and horses infested with gastro-intestinal nematodes per month

Mean ± SD Range Month

Horses Donkeys Horses Donkeys

January 340±357.77 402.83± 664.01 50-900 100-3800

February ND 890.91± 1,231.63 ND 300-4500

March 3600±1417.75 603.85±791.19 50-2800 100-3600

April 405.56±766.56 1888.89± 2855.51 100-3300 100-11800

May 441.67±529.94 485.71± 561.04 50-1700 200-1700

June 160±54.77 420.00± 294.96 50-200 200-900

July 300±0 282.89±298.26 300 50-1600

August 850±919.24 261.54±185.02 50-1500 100-700

September 1485±1768.91 1485.00±1,768.91 50-8000 100-8000

October 1185±1768.91 1335.29± 1822.82 50-10400 50-6250

November 637.23±1822.82 637.23±1,822.82 50-13450 50-2650

December 2300±0 307.58±542.29 2300 50-2400

Total 975.37±1099 750.14±1071.95 50-13450 50-11800

Table 3.6 Severity of infection with gastro-intestinal nematodes in donkeys

and horses per month

Mild% Moderate% Severe%

Month Horses Donkeys Horses Donkeys Horses Donkeys

January 80.00 88.68 20.00 1.89 0 9.43

February ND 75.00 ND 16.67 ND 8.33

March 33.33 73.08 33.33 19.23 33.33 7.69

April 90.00 55.56 0 5.56 10.00 38.89

May 83.33 85.71 8.33 - 8.33 14.29

June 100.00 80.00 0 20.00 0 -

July 100.00 94.74 0 2.63 0 2.63

August 50.00 100.00 0 - 50.00 -

September 81.82 75.00 18.18 - 0 25.00

October 90.00 55.56 0 16.67 10.00 27.78

November 85.71 85.71 14.29 7.14 0 7.14

December 0 78.79 0 18.18 100.00 3.03

Total 82.35 81.25 8.82 7.89 8.82 10.86

3.2 Therapeutic efficacy of Albendazole and Ivermectin drench in donkeys

3.2.1 Egg per gram reduction

The result of mean egg per gram of faeces and the range in addition to the

reduction percentage of egg per gram of faeces for the three treated groups from

day zero to day 21 are presented in tables (3.8, 3.9 and 3.10).

On day 3, Albendazole showed reduction of 97.13% egg per gram count

(EPGC), while Albendazole with the two doses showed 98.90%. In Ivermectin

treated group 98.94% of egg per gram count (EPGC) was reported.

All three groups reported 100% reduction of egg per gram of faeces on

day 7 and till day 21 when animals were euthanized.

3.2.2 Post-mortem findings

The results of post-mortem findings are presented in tables (3.11, 3.12

and 3.13) for the three groups. Albendazole reported efficacy of 97.13% at the

single dose while albendazole at the two doses reported 98.90%, but the

efficacy against L4 Strongylus vulgaris found in cranial mesenteric arteries was

33% for Albendazole and 59.08 %for Albendazole twice. On the other side,

Ivermectin reported 62.71% efficacy against L4 Strongylus vulgaris.

Table 3.7 Mean faecal egg counts (±SD) and reduction percentage for

Albendazole-treated donkeys

Days Arithmetic mean

(epg) Range Reduction%

0 3621.43±3583.05 500-10400 -

1 1221.43±1526.94 200-4500 66.27%

3 21.43±39.34 50-100 98.25%

7 0 0 100%

14 0 0 100%

21 0 0 100%

Table3.8 Mean faecal egg counts (±SD) and reduction percentage for Albendazole twice-treated donkeys

Days Arithmetic mean (epg) Range Reduction %

0 2462.50 ± 2137.50 1300-4500 -

1 1250.00 ± 574.46 900-2100 49.24%

3 100.00 ±141.42 0-300 92%

7 0 0 100%

14 0 0 100%

21 0 0 100%

Table 3.9 Mean faecal egg counts (±SD) and reduction percentage for

Ivermectin-treated donkeys

Days Arithmetic mean (epg) Range Reduction %

0 783.33 ± 625.03 50-1900 0

1 1683.33 ± 1586.72 0-3500 -114.89%

3 16.67 ± 40.82 0-100 99.01%

7 0 0 100%

14 0 0 100%

21 0 0 100%

Table 3.10 Summary of harvested worms from control and animals treated with Albendazole (ABZT1) drench at necropsy.

Albendazole Organs examined Control

No. Reduction %

Cranial mesenteric artery

Strongylus vulgaris 303 203 33.00

Stomach

Gasterophilus spp. 349 273 21.78

Habronema spp. 291 147 49.48

Trichostrongylus axei 79 26 67.09

Small Intestine

Parascaris equorum 25 0 100.00

Strongloid westeri 31 10 67.74

Caecum


Strongylus spp. 841 157 81.33

Cyathostomes spp. 6012 0 100.00

Colon and Rectum



Oxyuris equi 3151 0 100.00

Table 3.11 Summary of harvested worms from control and animals treated with Albendazole Twice (ABZT2) drench at necropsy.

Albendazole Twice Organs examined Control

No. Reduction %



Stomach

Gasterophilus spp. 349 393 (12.61)



Small Intestine



Caecum




Colon and Rectum




Table 3.12 Summary of harvested worms from control and animals treated with Ivermectin (IVMT) drench at necropsy.

Ivermectin Organs examined Control

No. Reduction %



Stomach




Small Intestine



Caecum




Colon and Rectum




Figure 3.4 The percentage efficacy of Albendazole (Different dose regimen) and Ivermectin at 14 days post treatment.

Figure 3.5 larvae of Strongylus vulgaris in cranial mesntric artry removed form Albendazole treated donkey.

Figure 3.6 larvae of gasterophilus in a stomach removed from Albendazole

treated donkey.

Figure 3.7 Parascaris equorum removed from small intestine of donkey in the control group

Figure 3.8 Parascaris equorum expeled from small intestine of donkey in the

Ivermectin Drench treated group on day 3 post treatment.

Figure 3.9 lareg and small strongyles in a Caecum removed from donkey in

the control group

Figure 3.10 Encysted larvae of small strongyles in colon of donkey in the

control group.

3.3 An assay of some biochemical parameters in donkeys (Equus asinus)

medicated with Albendazole:

As shown in table (3.13) there was no significant (P>0.05) difference in

the three treated groups in total protein concentration following administration

of the experimental anthelmintics through out the whole experiment period (21

days).

In table (3.14) we could observe that there is significant (P<0.05)

increase in albumin concentration 24 hours following treatment with Ivermectin

when compared with day zero. While animals in the other two treatment groups

exhibited only fluctuation in albumin concentration.

As we could observe in table (3.15) there is no significant difference in

the treated groups during the whole experiment period in urea concentration.

As shown in table (3.20) animals treated with albendazole at two doses

14 days apart showed significant decrease in creatinine concentration in days 3

and 7 following administration of albendazole and the decrease although non-

significant remained to the end of the experiment.

Serum Calcium and serum inorganic-phosphorus results obtained were

presented in tables (3.17) and (3.18), respectively. Serum Calcium, in

Albendazole treatment groups showed significant increase in days 1, 3 and 14 in

animals treated with one dose, and in days 7 and 14 following treatment with

albendazole at two doses 14 days apart.

In animals treated with two doses of albendazole, inorganic phosphorus

showed significant increase 14 days following treatment.

Table 3.13 Changes in total protein concentration (g/dL) following oral

administration of experimental anthelmintics compounds

ABZT1 ABZT2 IVMT DAY

Mean ± SD Mean ± SD Mean ± SD

0 09.94±2.24 08.47±0.88 8.02±1.22

1 09.06±1.50 09.64±1.24 8.76±0.73

3 10.96±2.56 12.52±5.55 8.49±0.70

7 08.53±0.73 10.30±0.95 8.56±0.84

14 08.21±0.33 08.97±0.58 8.02±0.51

21 09.53±0.39 10.74±1.87 8.86±0.72

Table 3.14 Changes in albumin concentration (g/dL) following oral




0 3.14±0.36 2.88±0.44 2.88±0.44

1 3.15±0.33 3.25±0.37 3.88±0.43*

3 3.06±0.36 3.18±0.42 2.91±0.41

7 2.95±0.59 3.84±0.66 3.74±0.81

14 2.95±0.50 3.28±0.28 2.73±0.33

21 3.16±0.21 3.66±0.86 3.19±0.57

(*) Means on the same column have asterisk are significantly different with day

zero

Table 3.15 Changes in urea concentration (mg/dL) following oral




0 8.57±1.61 09.26±1.94 7.47±1.37

1 7.01±0.35 09.16±3.14 7.10±0.73

3 7.60±1.18 09.19±3.28 8.14±0.82

7 7.68±1.13 11.57±3.86 8.41±1.01

14 8.12±3.80 06.89±2.14 7.57±1.42

21 9.71±2.82 09.55±2.72 8.35±2.12

Table 3.16 Changes in creatinine concentration (mg/dL) following oral




0 0.89±0.18 1.10±0.16 0.86±0.13

1 0.95±0.24 0.89±0.25 0.83±0.14

3 0.73±0.12 0.55±0.35* 0.88±0.02

7 0.85±0.05 0.35±0.28* 0.96±0.07

14 0.93±0.12 0.74±0.37 0.81±0.18

21 1.26±0.80 0.55±0.65 0.82±0.33


zero

Table 3.17 Changes in calcium concentration due (mg/dL) following oral administration of experimental anthelmintic compounds



0 08.21±0.75 08.19±1.23 08.54±0.96

1 10.34±1.01* 09.22±1.01 09.75±1.68

3 11.43±1.35* 09.94±1.59 10.16±1.83

7 09.18±1.58 11.21±1.09* 10.25±1.89

14 09.78±0.89* 11.36±1.14* 09.81±1.64

21 09.81±2.85 09.82±1.00 08.82±0.95


zero

Table 3.18 Changes in inorganic-phosphorus concentration (mg/dl)

following oral administration of experimental anthelmintics compounds



0 4.63±2.57 3.81±1.79 3.90±0.84

1 5.18±1.58 5.75±2.53 4.11±1.26

3 7.26±2.97 7.58±3.99 5.79±1.56

7 6.59±2.10 9.34±5.47 5.74±3.38

14 4.79±0.71 6.46±0.74* 5.30±2.35

21 5.39±1.87 9.66±5.24 5.60±1.18


zero .

CHAPTER FOUR

Chapter four

Discussion

From the results obtained in this study, it is interesting to note that, the

overall prevalence for both donkeys and horses with CIT parasited was found to

be 29.785%, this is in harmony with the results obtained by Kheir and Kheir

(1981) in Bahr El Arab (22%), while it is less than that reported in Nyala town

(58%), this may be referred to the large number of animal examined in this

study (1256 animal) when compared to Kheir and Kheir (390 animals), and also

to the availability of anthelmintics compounds. In Sennar-Sudan also El Dirdiri

et al., (1986) reported a similar percentage of infection with gastro intestinal

diseases (27%).

Prevalence of gastrointestinal nematodes in horses noticed in this study

was 15.73%, which is in close agreement with that reported by Kheir and Kheir

(1981) in Bahr El Arab (18.5%). Results obtained in this study concerning the

prevalence of helminth nematodes in donkeys was 37.84% this result is very

low when compared with that reported by Seri et al., (2004) in Khartoum state-

Sudan (70.1%), while Kheir and Kheir (1981) reported that the overall

incidence of infection with nematode parasites was found higher in town

animals (58%) than in animals kept in nomadic areas (22%).

The overall Mean ± SD for egg per gram of faeces (epg) was 589.97 ±

986.02 and the range was 50-11800 (epg), this is low when compared with Seri

et al., (2004) (1016.6 ± 363.6) (epg), this may be attributed to the large

percentage of mild infection obtained in this study (81.35%) when compared to

that of Seri et al., (2004) who reported 58.6% in donkeys examined in

Khartoum state.

The highest Mean ± SD was observed in April (hot season) 1390.03 ±

2401.26 with a range of 50-11800 (epg), and the lowest was in June (rainy

season) 290.00 ± 242.44 with a range of 50-900 (epg).

Concerning severity of infection reported in this study, 81.35% for mild

infection, both moderate and severe burdens shared the lower incidence (8.11%

and10.54% respectively) these findings are in agreement with Seri et al., (2004)

in donkeys (58.6%, 21.9% 19.5%) for mild, moderate and severe infections

respectively.

However, all the findings obtained in the survey during the whole year

were affected with the extensive miss-use of anthelmintics by animal’s owners

in absence of veterinary authorities where sometimes sub doses, over dosage of

anthelmintics, wrong timing of de-worming and a lot of misconceptions of de-

worming were applied, what shows apparent differences in the results in this

study.

Therapeutic efficacy of Albendazole in donkeys in this study, in both

groups of animals treated with either single dose or two doses of Albendazole at

10 mg/kg were resulted in 100% reduction in (epg) count on day 7 post

treatment. Kuzmina and Kharchenko (2008) obtained a similar result at day10

post treatment in horses treated with a dose rate of 5mg/kg body weight in

Ukraine to control Strongylus spp.

Following post-mortem, Albendazole administered at single dose resulted

in low efficacy of 49.48% against Habronema sp., and Trichostrongylus axei

67.09%, Strongyloid westeri 67.74%; but expressed high efficacy against

Parascaris equorum100%; and 81.33%, 98.40% for Strongylus spp. Present in

caecum, colon and rectum respectively; Cyathostomes spp 100% 98.14% for

caecum, colon and rectum respectively. This result explained by the

justification of Gokbulut et al., (2005) who stated that, a higher metabolic

capacity, first-pass effect and lower absorption of benzimidazoles in donkeys

decrease bioavailability and efficacy compared to ruminants, this explanation

also justify the increase in efficacy when we used the double dose for the

second group in this study

Albendazole at single and multiple dose regimen showed efficacy of

21.78% and (12.61)% against Gasterophilus spp. Albendazole at two doses 14

days apart showed the following efficacy percentages against: Habronema spp.

91.75%; Trichostrongylus axei100.%; Parascaris equorum 100% , Strongloid

westeri 100% ; Strongylus spp .in the caecum, colon and rectum 100%, 99.21%

respectively. Cyathostomes spp in the caecum, colon and rectum 99.17% and

100% respectively; and 100% for Oxyuris equi. These results are in agreement

with that obtained by Kuzmina and Kharchenko (2008) in Ukraine. Besides the

strongylid nematodes, eggs of Parascaris equorum, Oxuyris equi, Strongyloides

westeri and Habronema spp. were found in faecal samples a day before

treatment. No eggs of these nematodes were found in horse faeces on the 10th

and 14th days after treatment with the both drugs.

In this study, Ivermectin showed 100% faecal egg count reduction on day

7 this is in agreement with Seri et al., (2005) who reported the same result when

used ivermectin injectable formulation intramusclarlly at dose rate of 200µg/kg

body weight in donkeys in sudan, the efficacy against Habronema spp was

99.31% this result is in close agreement with that reported in horse by Herd and

Donham (1984), who showed 1-2 intramuscular doses of Ivermectin at 200

µg/kg were highly effective against Draschia spp and Habronema spp., while

DiPietro (1982) revealed 100% efficacy. When Ivermectin given

intramuscularly to donkeys at dose rate of 200 µg/kg, the efficacy was 100%

against larvae of Gasterophilus spp. (Seri et al., 2005), this result is in

agreement with the results (100%) obtained in this study. Ivermectin

successfully (100%) removed P. equorum from the small intestine when given

orally as paste formulation at 200 µg/kg. Ivermectin totally eliminated the

passage of P. equorum in the natural infected horses (Cobra et al., 1986). In

case of T. axie Ivermectin (100%) eliminated T. axie from the small intestine of

donkeys this also the same condition when used at dose rate of 200 µg/kg in

intramuscular formulation (Seri et al., 2005). In this study, Strongylus spp were

98.81% eliminated from caecum and 92.65% from the colon and rectum, which

is in close agreement when compared with that reported by Seri et al.,

(2005)100% when they used Ivermectin injectable formulation intramuscularly

for donkeys at dose rat of 200 µg/kg, a result which may be attributed to the

route of administration that affects the bioavailability of the drug in this study.

Cyathostomes spp were 100% eliminated from both caecum and colon in

this study, this result also is in agreement with that obtained by Seri et al.,

(2005). Costa et al., (1998) when utilized Ivermectin at same dose rate of 200

µg/kg for equine in Brazil as a paste formulation reported an efficacy of 100%

against Oxiuris equi, the same result was also obtained in this study. In this

study, Ivermectin expressed moderate efficacy against Strongylus vulgaris

larvae which found in the cranial mesenteric arteries 62.71% this result is to be

considered in the same range with that reported by Costa et al., (1998) and Seri

et al., (2005) and the results were 67.8%, 69,23% respectively.

In this study, the values obtained for the total protein values in the three

treatment groups were higher than reference values obtained by other

researchers worldwide and not in harmony with the values reported by Zinkl et

al., (1990) 7.2±0.7 in American donkeys and that 6.82 ±0.40 reported by Mori

et al., (2003) in Brazilian donkeys, we attribute this difference to the breed

and/or the technical method used. Daly and Hogan (1982) reported increasing of

urinary protein in rats treated with albendazole at 30 and 45 mg/kg bw (3 and

4.5 times of recommended dose, respectively).

In the current study, Albumin concentration fluctuated within the normal

level for albendazole treated groups which ranged between 2.88±0.44 g/L on

day 0 for Albendazole treated group 1 to 3.66±0.86 g/L on day 21 for the

animals treated with two doses with non significant differences, and this is on

line with reference range 3.48 ± 0.148 reported in India by Gupta (1994) and

not in harmony with Daly and Hogan (1982), who reported increased level of

albumin in male rats when treated with a dose of 45 mg/kg. We referred that to

the difference between the two doses. Ivermectin treated animals showed

significant increase in day one, this finding is in agreement with Seri et al

(2006), who reported significant increase in day 2 post treatment with

Doramectin in donkeys and also in horses Herd et al (1985) reported similar

results.

Urea concentration showed no significant difference in the groups treated

with Albendazole with the tow doses and Ivermectin. Herd and Kociba (1985)

reported that in horses treated with Ivermectin and also Seri et al., (2006) in

donkeys treated with Doramectin had different result which revealed significant

increase and decrease of Urea some sampling times. We may refer this to the

tow different routes of administration which affected the bioavailability of the

drugs.

Serum creatine concentration was decreased in two occasions

respectively on days 3 and 7 for the animals treated with Albendazole at two

doses.

In this study serum calcium concentration showed significant increase in

two occasions on days 1and 3, respectively, then a significant decrease once in

day 14 for Albendazole recommended dose treated group and also significant

increase in days 7 and 14 for Albendazole treated twice. Martin (1980) observed

that at dose of 6 - 62 mg/kg bw albendazole a skeletal abnormalities were

increased and external malformations and major malformations were cranio-

facial and bone defects in rats. No significant difference in calcium level in

Ivermectin treated group and this is in harmony with Seri et al., (2006).

Serum inorganic phosphorus concentration in the animals treated with

double dose of Albendazole showed significant increase in day 14. No

significant differences in inorganic phosphorus level in Ivermectin treated

group, this is not in harmony with Seri et al., (2006) and Herd and Kociba

(1985). We attributed this to the different route of administration for the drug

between the studies.

At the end we may conclude that administration of albendazole and

Ivermectin did not show any toxic or adverse effects on liver and kidney

functions.

CHAPTER FIVE

Chapter five Conclusion and Recommendation

5.1 Conclusion

In this study we throw light on some equine gastrointestinal proplems

of helminth nematodes in South Darfur State, the study is composed of three

aspects.

The first one, was the survey conducted for a whole yaer, a total number

of 1256 animals ( 446 horses and 810 donkeys) were examined to determine the

overall prevalnce of gastro-intestinal nematodes, which was 29.20%, the

prevalence in horses was 15.37% and in donkeys reported to be 37.48% . The

second aspect was to evaluate the therapeutic efficacy of Albendazole drench

formulation and to compare the obtained results with a commercial formulation

of Ivermectin drench formulation against gastro-intestinal nematodes;

Albendazole when used in the single dose expressed mean efficacy of 97.46%

and 98.55% with the two doses administered at 14 days apart, while Ivermectin

showed mean efficacy of 99.06% against gastro- intestinal nematodes. The third

aspect was to evaluate the effect of medication with Albendazole and

Ivermectin in donkeys naturally infected with gastro-intestinal nematode.

Fortunately the three dose protocols showed no adverse or untoward effects in

blood biochemical constituents.

5.2 Recommendations

1- Donkeys need more attention to apply stratigic programmes for

controling gastro-intestinal nematodes what makes donkeys more healthy

and productive.

2- Scheduled deworming programmes should be designed and appllied on

three occasions every year viz: january, july and october including both

donkeys and horses.

3- Deworming portocols should start with Ivermectine drench followed by

Albendazole suspention and finish with Ivermectin drench.

4- The new anthelmintic drugs (Doramectin, Abmectin and Moxidectin)

should be tested and used in equines.

5‐ Research institues should encourage studies in both sudanese donkeys and horses in the coming future.

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