the antiviral effect of acacia nilotica extracts against the mesogenic strain of newcastle disease virus

98 Pages·2008·0.76 MB·English

THE ANTIVIRAL EFFECT OF ACACIA NILOTICA
EXTRACTS AGAINST THE MESOGENIC STRAIN OF
NEWCASTLE DISEASE VIRUS

By

AMEL MAHMOUD MOHAMED SAEED

A Thesis Submitted to the University of Khartoum in Fulfillment for the
Masters Degree in Veterinary Science

Supervisor: Prof. Imadeldin Aradaib

Department of Medicine Pharmacology and Toxicology
Faculty of Veterinary Medicine
University of Khartoum

May 2007
Dedication

To the soul of my father..

To my precious mother….

To my beloved husband…..

To my brothers and sisters..

To my little angel Ruba…

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Acknowledgement

I want to thank a lot of people and colleagues for their great help to finish
this work. First would like to thank my dear husband Osama Abdalla for his
great understanding, patience, encouragement and support. I also like to
thank my supervisor prof. Imadeldin Alamin Aradaib for his support and
good supervision and prof. Abdelrahim Elsayed Karrar for his valuable
advice and encouragement. I appreciate the favor of my dear friend Dr.
Tomador Mohamed Abdallah from the Animal resource research lab for her
great help with her experience in Virology. Thanks are extended to Prof.
Mahasin Elnour the head department of virology at the Central Veterinary
Research Laboratory. I also owe a great favor to Dr. Yahia Hassan Ali from
the same department and would like to thank Dr. Intisar Kamil and the
technicians of the virology department Mona, Omayma and Rihab. Thanks
also extended to prof. M.A.Fadol , Dr. Ihsan Hussein and the technicians
Albager Abdelhaleem, Khidir Abdelgader and Haydar Ahmed.Great thanks
to Dr. Igbal Sayed Ahmed from Poultry department. Thanks also extended to
Amir Mahjoub from food technology department –Faculty of Agriculture U
of K.Iwould like to thank Prof. Mohamed Galal Mohamed Ahmed the
manager of the medicinal and aromatic plant research institute (MAPRI) for
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offering the opportunity of making this study, also I would like to thank
Prof. Aeisha Zuhair Almagboul the head of microbiology department in
MAPRI and I want to thank my colleagues at the lab of molecular biology
Afraa Tajelsir, Samah AbdelRahman, Dr. Khair Allah M.S , Dr. Mohamed
Abdallah and Ahmed Osman.

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Contents

Dedication...........................................................................................................................i
Acknowledgement.............................................................................................................ii
Contents............................................................................................................................iv
List Of Figures.................................................................................................................vii
Arabic Abstract..............................................................................................................viii
CHAPTER ONE...............................................................................................................1
LITREATURE REVIEW................................................................................................1
1. Acacia nilotica................................................................................................................1
1.1. General information of the plant..........................................................................1
1.1.1 Scientific classification..............................................................................1
1.2 Botanical descriptions.............................................................................................2
1. 2.1 Leaves...............................................................................................................2
1.2.2 Flowers..............................................................................................................2
1.2.3 Fruits.................................................................................................................3
1.2.4 Seeds..................................................................................................................3
1.2.5 Maturity age.....................................................................................................3
1.3 Habitat.....................................................................................................................3
1.4. Distribution.............................................................................................................3
1.4.1 Distribution in Sudan......................................................................................4
1.5 Ecology.....................................................................................................................4
1.6. Temperature...........................................................................................................4
1.7 PH range...................................................................................................................4
1.8 Uses...........................................................................................................................5
1.8.1 Firewood-Charcoal..........................................................................................5
1.8.2 Timber-Wood...................................................................................................5
1.8.3 Pulp and paper.................................................................................................5
1.8.4 Forage-Fodder..................................................................................................5
1.8.5 Tannins..............................................................................................................5
1.8.6 Folk medicinal uses..........................................................................................6
1.9 Limitations-Concerns- Additional information...................................................7
1.10Chemical constituents............................................................................................7
1.11 Anti microbial activity for Acacia nilotica..........................................................8
1.11 Hepato protective activity....................................................................................8
2. NEWCASTLE DISEASE.............................................................................................9
2.1 Introduction.............................................................................................................9
2.2 History of the disease..............................................................................................9
2.3 Causative agent.....................................................................................................10
2.3.1 Classification of the virus..............................................................................11
2.3.2 Virion..............................................................................................................12
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2.3.2.1 Size............................................................................................................12
2.3.2.2 Shape........................................................................................................13
2.3.2.3 Structure of the genome.........................................................................13
2.3.2.4 Virus replication......................................................................................14
2.3.2.5 Virus biological and physical properties...............................................16
2.3.2.5.1 Hemagglutination activity...............................................................16
2.3.2.5.2 Hemolysis..........................................................................................18
2.3.2.5.3 Pathogenicity....................................................................................18
2.3.2.5.4 Thermo stability...............................................................................19
2.3.2.5.5 Changes in cell culture.....................................................................19
2.4 Epidemiology.........................................................................................................20
2.4.1 Host range.......................................................................................................21
2.4.2 Reservoirs.......................................................................................................22
2.4.3 Transmission..................................................................................................22
2.4.3.1 bird-to-bird transmission.......................................................................22
2.4.3.1.1 Respiratory route:............................................................................23
2.4.3.1.2 Contamination with faeces..............................................................24
2.4.3.1.3 Vertical transmission.......................................................................24
2.4.3.2 Transmission from birds to humans.....................................................25
2.4.3.3 Transmission from humans to birds.....................................................25
2.5 Pathotypes of the disease......................................................................................26
2.5.1 Pathogenicity..................................................................................................27
2.5.2 Pathogenesis mechanism...............................................................................28
2.6 Incubation period..................................................................................................29
2.7 Prognosis................................................................................................................29
2.8 Clinical signs..........................................................................................................29
2.8.1 Viscerotropic-velogenic type.........................................................................30
2.8.2 Neurotropic-velogenic....................................................................................32
2.8.3 Mesogenic........................................................................................................32
2.8.4 Lentogenic.......................................................................................................33
2.9 Diagnosis................................................................................................................33
2.9.1 Laboratory diagnosis.....................................................................................34
2.9.1.1 Isolation and identification of the virus................................................34
2.9.1.1.1 Collection of samples.......................................................................34
2.9.1.1.2 Virus propagation in cell culture....................................................34
2.9.1.1.3 Virus identification..........................................................................35
2.9.1.2 Differential diagnosis..............................................................................35
2.10 Control.................................................................................................................36
2.11 Newcastle disease in Sudan................................................................................39
CHAPTER TWO............................................................................................................41
MATERIALS AND METHOD.....................................................................................41
2.1. Plant material.......................................................................................................41
2.2 Preparation of the plant extracts.........................................................................41
2.2.1. Methanol and chloroform extracts..............................................................42
2.2.2. Water extract.................................................................................................43
2.3. The virus...............................................................................................................43
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2.4. Laboratory work..................................................................................................43
2.4.1 Sterilization of glassware...............................................................................43
2.4.2. Sterilization of plastic ware..........................................................................44
2.5. Vero cells...............................................................................................................44
2.6. Cytotoxicity tests..................................................................................................45
2.6.1 Preparation of stock solution of chloroform extract..................................45
2.6.2 Preparation of stock solution of methanol and water extracts..................45
2.6.3 The cytotoxicity assays..................................................................................46
2.7. Antiviral assay......................................................................................................47
2.8. Sub culture of the virus suspension....................................................................48
2.9. Hemagglutination test (HA)................................................................................48
2.9.1. Preparation of 1% red blood cells (RBCs).................................................48
2.9.2. Procedure of HA test....................................................................................49
2.10. Determination of tannin contents in the extracts............................................50
CHAPTER THREE........................................................................................................51
RESULTS........................................................................................................................51
3.1 Results of Cytotoxicity assay................................................................................51
3.2 Results of antiviral assay......................................................................................51
3.3 Results of Sub culturing the virus.......................................................................52
3.4 Results of the hemagglutination test...................................................................52
3.5 Results of tannin contents....................................................................................52
CHAPTER FOUR...........................................................................................................59
4.1 Discussion...................................................................................................................59
4.2 Future recommendations.........................................................................................63
References:.......................................................................................................................65
APPENDICES.................................................................................................................83
Appendix I.......................................................................................................................83
Tissue culture media and additives...............................................................................83
Appendix II......................................................................................................................86
Cell dispersing solution..................................................................................................86
Appendix III....................................................................................................................88
Haemagglutination test solutions..................................................................................88

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List of Figures

Fig.1: Cells with non-toxic concentration of the chloroform extract…53
Fig.2: ells with non-toxic concentration of the methanol extract…...53
Fig.3: Cells of non-toxic concentration of water extract…...54
Fig.4: Cells with the inhibitory concentration of the methanol extract…54
Fig.5: Complete CPE in cells with chloroform extract…………………55
Fig.6: CPE in cells with water extract…...55
Fig.7: Control cells with CPE after the subculture………………………56
Fig.8: Haemagglutination test……………………………………………56
Fig.9: Diagrammatic representation of Newcastle disease virus….57
Fig.10: Acacia nilotica tree…...58
Fig.11: Thorns and fruits of sub species tomentosa…………………….5

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Abstract
Methanol, chloroform and water extracts of Acacia nilotica sub
species tomentosa were tested in-vitro for their potential antiviral activity
against the Komarov vaccine strain of Newcastle disease virus (NDV). Sub
culturing the treated samples and heamagglutination test were used to
evaluate the reduction of viability in the viral growth in presence and
absence of the effective extract. The noncytotoxic concentration of each
extract was determined by the absence of the CPE. The inhibitory
concentration of the effective extract was measured. The tannin percentage
of the methanol and water extracts were evaluated by Prussian Blue assay.
The methanol extract of Acacia nilotica on vero cells showed non-
cytotoxic concentration of ≥ 40µg/ml. The methanol extract showed a
significant inhibitory effect against the tested virus at the concentration of 40
µg/ml. None of the other two extracts showed any inhibitory activity.
Subculturing of the treated samples of the methanol extract showed no CPE
compared to the control. The HA test showed zero titer of the tested samples
against 2 log 6 to the control.
The tannin percentage of the methanol extract were higher (1.6%)
than the water extract percentage (1.2%).

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ﺔﺣوﺮﻃﻻا ﺺﺨﻠﻣ

ﺎﻴﺳﺎآﻻا تﺎﺒﻨﻟ (ءﺎﻤﻟا ،مرﻮﻓورﻮﻠﻜﻟا ،لﻮﻧﺎﺜﻴﻤﻟا)تﺎﺼﻠﺨﺘﺴﻤﻟا ﻦﻣ ﺔﺛﻼﺛ رﺎﺒﺘﺧا ﻢﺗ ﺔﺳارﺪﻟا ﻩﺬه ﻰﻓ -
.ﻞﺳﺎآﻮﻴﻨﻟا ضﺮﻣ سوﺮﻴﻔﻟ ﺔﻴﺣﺎﻘﻠﻟا فورﺎﻣﻮآ ةﺮﺘﻋ ﺪﺿ تﺎﺳوﺮﻴﻔﻠﻟ دﺎﻀﻤﻟا ﺮﺛﻻا ﺪﻳﺪﺤﺘﻟ ﺎﻴﻠﻤﻌﻣ
ﻪﺘﺠﻟﺎﻌﻣ ﺪﻌﺑ سوﺮﻴﻔﻟا ﺮﺛﺎﻜﺗ ضﺎﻔﺨﻧا ﺎﺘﺒﺛا ىﻮﻣﺪﻟا نزﻼﺘﻟا رﺎﺒﺘﺧاو سوﺮﻴﻔﻟا عارﺰﺘﺳا ةدﺎﻋا-
.لﺎﻌﻔﻟا ﺺﻠﺨﺘﺴﻤﻟﺎﺑ
ﺔﻣﺪﺨﺘﺴﻤﻟا ﺎﻳﻼﺨﻠﻟ مﺎﺳ ﺮﻴﻏ ﺰﻴآﺮﺗ ﻰﻓ ﺔﻣﺪﺨﺘﺴﻤﻟا ةﺮﺘﻌﻟا ﺪﺿ ﺔﻴﻟﺎﻌﻓ ﺖﺒﺛا تﺎﺒﻨﻠﻟ لﻮﻧﺎﺜﻴﻤﻟا ﺺﻠﺨﺘﺴﻣ-
ةﺮﺘﻌﻟا ﺪﺿ ﺔﺠﻴﺘﻧ ىا ﺎﻴﻄﻌﻳ ﻢﻟ ﻦﻳﺮﺧﻻا ﻦﻴﺼﻠﺨﺘﺴﻤﻟا .ﻞﻣ/ماﺮﺟوﺮﻜﻴﻣ 40 ﺰﻴآﺮﺗ ﻮهو ﺔﺑﺮﺠﺘﻟا ﻰﻓ
.ﺎﻳﻼﺨﻠﻟ مﺎﺳ ﺮﻴﻐﻟا ﺰﻴآﺮﺘﻟا ﻰﻓ ﺔﻣﺪﺨﺘﺴﻤﻟا
ﺔﻧرﺎﻘﻣ ﺎﻳﻼﺨﻠﻟ ضﺮﻤﻣ ﺮﺛا ىا ﺮﻬﻈﻳ ﻢﻟ لﺎﻌﻔﻟا ﺺﻠﺨﺘﺴﻤﻟﺎﺑ ﺔﺠﻟﺎﻌﻤﻟا ةﺮﺘﻌﻠﻟ عارﺰﺘﺳﻻا ةدﺎﻋا -
تﺎﻨﻴﻌﻠﻟ ﺔﺒﺴﻨﻟﺎﺑ ﺮﻔﺻ ﺔﻳﺮﻬﻴﻋ ﺮﻬﻇا ىﻮﻣﺪﻟا نزﻼﺘﻟا رﺎﺒﺘﺧا .ﺔﻴﺣ ةﺮﺘﻋ ﻰﻠﻋ ىﻮﺘﺤﻤﻟا لوﺮﺘﻧﻮﻜﻟﺎﺑ
.26 ﺔﻳرﺎﻴﻋ تﺮﻬﻇا ﻰﺘﻟا و ﺺﻠﺨﺘﺴﻤﻟﺎﺑ ﺔﺠﻟﺎﻌﻣ ﺮﻴﻐﻟا تﺎﻨﻴﻌﻟﺎﺑ ﺔﻧرﺎﻘﻣ ﺺﻠﺨﺘﺴﻤﻟﺎﺑ ﺔﺠﻟﺎﻌﻤﻟا
لﻮﻧﺎﺜﻴﻤﻟا ﺺﻠﺨﺘﺴﻣ ﻰﻓ ﻰﻠﻋا ﺔﺒﺴﻨﻟا ﺖﻧﺎآو ءﺎﻤﻟاو لﻮﻧﺎﺜﻴﻤﻟا ﻰﺼﻠﺨﺘﺴﻣ ﻰﻓ ﻦﻴﻧﺎﺘﻟا ﺔﺒﺴﻧ ﺪﻳﺪﺤﺗ ﻢﺗ -
.(%1.2) ﻲﺋﺎﻤﻟا ﺺﻠﺨﺘﺴﻤﻟﺎﺑ ﺔﻧرﺎﻘﻣ (%1.6)

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