Gen 18S rRNA T.equi dan B. caballi yang diasingkan daripada darah dan kutu dianalisa untuk ...... 18S rRNA genotype named genotype C (group C) novel was identified in. Jordanian equids ...... iastate.edu/ Fact sheets/pdfs/equine_piroplasmosis.pdf. Chahan, B. ...... muda forest reserve, Kedah Malaysia. Southeast Asian ...
PREVALENCE, RISK FACTORS AND GENETIC DIVERSITY OF EQUINE PIROPLASMOSIS IN KELANTAN, MALAYSIA
QAES TALB SHUKUR ALSARHAN
DOCTOR OF PHILOSOPHY
2017
8
Prevalence, Risk Factors and Genetic Diversity of Equine Piroplasmosis in Kelantan, Malaysia
by
QAES TALB SHUKUR ALSARHAN
A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy
Faculty of Veterinary Medicine UNIVERSITI MALAYSIA KELANTAN
2017
9
THESIS DECLARATION
I hereby certify that the work embodied in this thesis is the result of the original research and has not been submitted for a higher degree to any other University or Institution. OPEN ACCESS
EMBARGOES
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I agree that my thesis is to be made available as hardcopy or on-line (full text) for a period approved by the Post Graduate Committee. Dated from ___________ until ___________
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(Contains confidential information under the office Official Secret Act 1972)*
RESTRICTED
(Contains restricted information as specified by the organization where research was done) *
I acknowledge that Universiti Malaysia Kelantan reserves the right as follows. 1. The thesis is the property of Universiti Malaysia Kelantan. 2. The library of Universiti Malaysia Kelantan has the right to make copies for the purpose of research only. 3. The library has the right to make copies of the thesis for academic exchange.
_____________________ SIGNATURE _______________________
__________________________ SIGNATURE OF SUPERVISOR _________________________
IC/ PASSPORT NO.
NAME OF SUPERVISOR
Date:
Date:
10
ACKNOWLEDGMENT
First of all, my thanks are to my God. I would like to express my sincere thanks to my supervisor, Associated Professor Dr. Mohd Mokhtar Arshad for his advice, patience and enthusiasm throughout the years of work. I would also like to express my gratitude and thanks to my co-supervisors, Professor Dr. Imad Ibrahim Al-sultan, Professor Dr. Mohd Azam Khan Goriman Khan, Universiti Malaysia Kelantan (UMK) and Dr. Azlinda Abu Bakar, Universiti Sanis Malaysia (USM), for helpful discussions and comments on my thesis. I would also like to extend my gratitude and appreciation to Dr. Maizan Mohammed for helping and advising me on molecular work. I am indebted to the Faculty of Veterinary Medicine, UMK, for making this possible by providing all necessary chemicals and equipment in the laboratory. I am deeply and extremely grateful to all of the UMK laboratory assistants, especially to Mr. Nor Faizull, Mr. Badrul Hisham, Mrs Eizzati, and Miss Nani Izreen for their support in so many ways. I would also like to thank the UMK veterinary clinic staff, especially Dr. Mimi, Mr. Hamid, and Mr. Nizam for their help in the sample collections. Special acknowledgment goest to my friend, Dr. Omer Khazaal, Dr. Ali Saeed, Dr. Ahmad Mahmood, Dr. Maher Mohammed for their direct and indirect assistance during my PhD study.
Qaes
11
TABLE OF CONTENTS
PAGE THESIS DECLARATION
i
ACKNOWLEDGEMENTS
ii
TABLE OF CONTENTS
iii
LIST OF TABLES
xi
LIST OF FIGURES
xv
LIST OF ABBREIVATIONS
xx
LIST OF SYMBOLS
xxiii
LIST OF EQUATIONS
xxiv
ABSTRAK
xxv
ABSTRACT
xxvi
CHAPTER 1 INTRODUCTION 1.1
General introduction
1
1.2
Problem statement
5
1.3
Research questions
6
1.4
Hypothesis
6
1.5
The objectives of study
7
CHAPTER 2 LITERATURE REVIEW 2.1
History of equine piroplasmosis (EP)
8
2.2
Etiological agents and taxonomy
10
12
2.2.1 Theileria equi
12
2.2.1.1 Morphology
12
2.2.1.2 Life cycle
14
2.2.2 Babesia caballi
17
2.2.2.1 Morphology
17
2.2.2.2 Life cycle
18
2.3 The genes commonly targeted in the T. equi and B. caballi
21
2.4 Sequencing and genetic diversity for T. equi and B. caballi
22
2.5
Epidemiology of equine piroplasmosis
25
2.5.1 Geographic distribution
25
2.5.2 Susceptibility to the disease
33
2.6
2.7
2.5.2.1 The susceptibility related to equids factors
33
2.5.2.2 The susceptibility related to environmental and stables factors
36
Transmission of the causative agents
38
2.6.1 Biological transmission
38
2.6.2 Iatrogenic or mechanical transmission
40
2.6.3 Intrauterine or transplacental transmission
41
The tick vectors for equine piroplasms infections
42
2.7.1 Taxonomy of Ixodid ticks
46
2.7.2 Morphology of Ixodid ticks
47
2.7.2.1 Tick family identification
48
2.7.2.2 Tick genus identification
52
13
2.7.2.3 Tick species identification
53
2.7.3 Tick life cycle
53
2.7.4 Overview of the identified Ixodid ticks genera in this study
55
2.7.4.1 Genus Rhipicephalus Koch, 1844 (Soulsby, 1982)
55
2.7.4.2 Genus Haemaphysalis Koch, 1844 (Soulsby, 1982)
58
2.7.4.3 Genus Dermacentor Koch, 1844 (Soulsby, 1982)
59
2.8
Ixodid ticks in Malaysia
60
2.9
Pathogenesis of equine piroplasmosis
62
2.10 Clinical signs of equine piroplasmosis
66
2.11 Clinical pathology of equine piroplasmosis
69
2.11.1 Changes in hematological parameters
69
2.11.2 Changes in serum biochemistry parameters
73
2.11.2.1 Aspartate aminotransferase (AST)
74
2.11.2.2 Alanine aminotransferase (ALT)
74
2.11.2.3 Alkaline phosphatase (ALKP)
75
2.11.2.4 Total bilirubin
75
2.11.2.5 Total protein
76
2.11.2.6 Blood urea nitrogen (BUN)
76
2.11.2.7 Calcium
77
2.11.2.8 Glucose
78
2.11.2.9 Phosphorus
78
2.11.2.10 Creatinine
79
2.11.3 Pathological changes of equine piroplasmosis
14
79
2.11.3.1 Macroscopic findings
80
2.11.3.2 Microscopic findings
80
2.12
Immunity to equine piroplasmosis
81
2.13
Public health of significance of equine piroplasmosis
85
2.14
Diagnosis of equine piroplasmosis
86
2.14.1 Overview of the methods used for diagnosis EP in this study
87
2.14.1.1 Microscopic examination of stained blood smears
87
2.14.1.2 Competitive enzyme linked immunosorbent assay
89
2.14.1.3 Conventional and multiplex polymerase chain reaction
91
2.14.2 Other methods for diagnosis of equine piroplasmosis
94
2.14.2.1 Biological tests
94
2.14.2.2 In vitro culture technique
95
2.14.2.3 Other serological tests
96
2.14.2.4 Other molecular techniques
97
2.15 Differential Diagnosis of equine piroplasmosis
99
2.16 Prognosis of equine piroplasmosis
99
2.17 Treatment of equine piroplasmosis
100
2.18 Control of of equine piroplasmosis
103
2.18.1 Vaccination
103
2.18.2 Control of ticks
104
CHAPTER 3 DIAGNOSIS, PREVALENCE, RISK FACTORS AND VECTOR OF EQUINE PIROPLASMOSIS IN EQUIDS IN KELANTAN 3.1
Introduction
105
15
3.2
Materials and Methods
108
3.2.1 Study area
108
3.2.2 Determining the number of equids
109
3.2.3 Animals and sample collection
109
3.2.4 Epidemiological data collection
110
3.2.5 Climatic data
113
3.2.6 Laboratory analysis
113
3.2.6.1
Microscopic examination of blood smears
113
3.2.6.2
Competitive enzyme linked immunosorbent assay
115
3.2.6.3
Polymerase chain reaction techniques
120
3.2.6.3.1 DNA extraction from equids blood
120
3.2.6.3.2 Determination of DNA concentration and purity
123
3.2.6.3.3 PCR amplification of piroplasms DNA from equids blood samples
124
3.2.7 Evaluation the efficiency of different methods for diagnosis the disease 3.2.8 Statistical analysis
129
130
3.3 Results
131
3.3.1 Morphological and biometerical finding with parasitemia
131
3.3.2 Prevalence of EP by different tests
135
3.3.3 Evaluation of cELISA and multiplex PCR for detecting T. equi and B. caballi infections
137
3.3.4 Prevalence of T. equi, B. caballi and both protozoa infections by regions
16
143
3.3.5 Risk factors associated with seroprevalence of EP causative agents
150
3.3.6 Ixodid ticks: identification and infestation rate
163
3.4 Discussion
171
3.4.1 Morphological and biometerical finding with parasitemia
172
3.4.2 Determing the prevalence of EP infections using different methods
173
3.4.3 Evaluating of cELISA and multiplex PCR for detecting T. equi and B. caballi infections
177
3.4.4 Equids factors associated with T. equi, B. caballi and both protozoa
179
3.4.5 Stables factors associated with T. equi, B. caballi and both protozoa
182
3.4.6 Climatic factors associated with T. equi, B. caballi and both protozoa
185
3.4.7 Ixodid ticks: identification and infestation rate
186
3.5 Conclusions
189
CHAPTER 4 EVALUATION OF HEMATOLOGY, BIOCHEMISTRY AND ANTIBODY TITER BETWEEN EQUIDS CLINICALLY AND SUBCLINICALLY INFECTED WITH EQUINE PIROPLASMOSIS 4.1
Introduction
192
4.2
Materials and methods
194
4.2.1 Animals and study area
194
4.2.2 Recording clinical sings
194
4.2.3 Fecal samples collection
194
4.2.4 Determination of antibody titers against T. equi and B. caballi
195
4.2.5 Hematological analysis
195
17
4.2.6 Serum biochemistry analysis
196
4.2.7 Statistical analysis
197
4.3 Results
4.4
198
4.3.1 Seroprevalence of clinical and subclinical forms in equids with the relative risk
198
4.3.2 Level of antibodies titration against piroplasms in equids with clinical and subclinical forms of equine piroplasmosis
198
4.3.3 Hematological and serum biochemistry parameters in equids with clinical and subclinical form of equine piroplasmosis
199
Discussion
206
4.5 Conclusions
212
CHAPTER 5 GENETIC DIVERSITY AND PHYLOGENIC ANALYSES OF THEILERIA EQUI AND BABESIA CABALLI DETECTED IN EQUIDS AND IXODID TICKS IN KELANTAN 5.1
Introduction
214
5.2
Materials and Methods
217
5.2.1 Ticks collection from equids
217
5.2.2 DNA extraction from Ixodid ticks
217
5.2.3 PCR amplification of piroplasms DNA extracted Ixodid ticks
219
5.2.4 DNA sequencing and phylogeny analyses
219
5.2.5 Statistical analyses
221
5.3 Results
221
5.3.1 Detection rate of T. equi, B. caballi and both protozoa in Ixodid ticks using multiplex PCR
221
5.3.2 Similarity rates on detection of T. equi, B. caballi and both protozoa DNAs between equids and Ixodid ticks
222
18
5.3.3 Genotypes of T. equi and B. caballi in equids and Ixodid ticks
226
5.3.4 Similarity within and between the genotypes of T. equi and B. caballi
229
5.3.5 Phylogenic analysis of T. equi and B. caballi sequences
231
5.4 Discussion
240
5.5 Conclusions
244
CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK 6.1 General conclusions
246
6.2 Recommrndations
248
6.3 Future work
249
REFERENCES
250
APPENDIX- A
289
APPENDIX- B
293
APPENDIX- C
301
APPENDIX- D
319
LIST OF PUBLICATIONS
334
19
LIST OF TABLES
NO.
PAGE
2.1
Prevalence of T. equi, B. caballi and different countries with the references.
2.2
Hard ticks species with the geographic location, number of the hosts and confirmed or suspected to be transmitted of Theileria equi or Babesia caballi (the causative agents of EP)(Scoles & Ueti, 2015).
45
2.3
Ixodid tick species distribution in different states of Malaysia.
61
2.4
Different methods obtainable for the diagnosis of EP and their purpose (OIE, 2014b).
94
3.1
The Oligonucleotide primers used to amplify the parasites 18S rRNA genes.
125
3.2
PCR program for samples subject to conventional PCR and multiplex PCR.
125
3.3
PCR procedure for DNA extracted samples subject to multiplex PCR and conventional PCR assay (reaction volume 25 μl).
127
3.4
The epidemiological table and formulas were used to copare between tests.
130
3.5
Morphological features, biomedical data and parasitemia of T. equi, B. caballi and both protozoa base on microscopic examination of blood smears.
135
3.6
Overall prevalence of equine piroplasmosis (singly T. equi, singly B. caballi and both protozoa) in equids in Kelantan by microscopic examination, cELISA and multiplex PCR.
136
3.7
Prevalence of T. equi, B. caballi and both protozoa in equids in Kelantan by microscopic examination, cELISA and multiplex PCR.
137
20
both protozoa in the
29
3.8
The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of cELISA for ditecting T. equi antibodies in comparison to microscopic examination (Gold standard).
139
3.9
The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of multiplex PCR for detecting T. equi DNAs in comparison to microscopic examination (Gold standard).
139
3.10
The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of cELISA for detecting B. caballi antibodies in comparison to microscopic examination (Gold standard).
140
3.11
The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of multiplex PCR for detecting B. caballi DNAs in comparison to microscopic examination (Gold standard).
140
3.12
Comparison between conventional PCR and multiplex PCR techniques for detection piroplasms DNA in equids blood (n= 306).
141
3.13
Prevalence of T. equi, B. caballi and both protozoa in sampling stables in different regions and sub-regions in Kelantan state.
145
3.14
Relative risk of EP associated with the type of protozoa in equids.
154
3.15
Relative risk of equids factors associated with seropositivity of T. equi, B. caballi and both protozoa.
155
3.16
Relative risk of regional factors associated with seropositivity of T. equi, B. caballi and both protozoa.
157
3.17
Relative risk of monthly factors associated with seropositivity of T. equi, B. caballi and both protozoa.
158
3.18
Relative risk of management and ticks factors associated with seropositivity of T. equi, B. caballi and both protozoa.
160
3.19
Relative risk of climatic factors associated with seropositivity of T. equi, B. caballi and both protozoa.
161
21
3.20
Percentage of sables infested with Ixodid ticks by Kelantan regions.
166
3.21
Details and number of identified Ixodid tick species infesting equids and nearby animals related to the prevalence of EP in each field.
167
3.22
Distribution of identified Ixodid tick species and their predilection sites on equids and nearby animals.
169
3.23
The abundance and percentage of Ixodid ticks on equids and nearby animals.
170
4.1
Health status factor equids associated with seroprevalence of T. equi, B. caballi and both protozoa.
201
4.2
Relative risk of of equids health status factors associated with the T. equi, B. caballi and both protozoa.
201
4.3
The clinical symptom parameters in the equids with clinical form compared to the equids with subclinical form and healthy group.
204
4.4
Haematological changes in the equids with clinical form compared to the equids with subclinical form and healthy group.
205
4.5
Serum biochemistry changes in the equids with clinical form compared to the equids with subclinical infectedform and healthy group.
206
5.1
Detection of T. equi and B. caballi in Ixodid ticks (n= 31) using multiplex PCR.
222
5.2
Similarity on detection of T. equi and B. caballi in the equids and Ixodid ticks using multiplex PCR.
224
5.3
Detection rate of T. equi and B. caballi genotypes in equids base on individual BLASTn analysis of positive samples.
227
5.4
Detection rate of T. equi and B. caballi genotypes in Kelantan regions.
228
5.5
Detection rate of T. equi and B. caballi genotypes in Ixodid ticks (n=31) base on individual BLASTn analysis of sequences.
229
22
5.6
Similarity within and between T. equi genotypes using multiple sequence alignment- CLUSTALW (GenomeNet).
230
5.7
Similarity within and between B. caballi genotypes using multiple sequence alignment- CLUSTALW (GenomeNet).
230
5.8
GeneBank accession numbers of Kelantan T. equi genotypes in the equids and ticks.
234
5.9
GeneBank accession numbers of Kelantan B. caballi genotypes in the equids and ticks. Homology between obtained sequences (GeneBank accession numbers) of T. equi and GeneBank database using online sequence BLASTn.
235
Homology between obtained sequences (GeneBank accession numbers) of B. caballi and GeneBank database using online sequence BLASTn.
237
5.10
5.11
23
236
LIST OF FIGURES
NO.
PAGE
2.1
A) Different forms of T. equi inside the erythrocytes and the characteristic Maltese cross form in a Giemsa‘s stained blood smear 100X (S. Kumar & R. Kumar, 2007); B) Microschizontes and macroschizontes (Koch‘s blue bodies) stages of Theileria spp inside the lymphocyte in a Giemsa‘s stained lymph smear 100X (Oryan et al., 2013).
14
2.2
The life cycle of T. equi Illustration by Massaro Ueti (Wise et al., 2013).
16
2.3
Different forms of B. caballi inside the erythrocytes and the characteristic pair of joint at their posterior ends organisms forming in a Giemsa‘s stained blood smear (S. Kumar & R. Kumar, 2007).
18
2.4
The life cycle of Babesia caballi. Illustration by Massaro Uet (Wise et al., 2013).
21
2.5
The internal structure of the hard tick (40X) by Edward et al. (2009).
48
2.6
Parts of a generalized hard tick; A) Dorsal view of capitulum (mouthpart); B) Ventral view of capitulum; C) Dorsal view of female with body parts; D) Ventral view of male body parts; E) Segment of the leg; F) Ventral view of coxae (Wall & Shearer, 2001).
50
2.7
Diagrammatic dorsal view of the capitulum of seven genera of hard ticks: (A) Ixodes, (B) Hyalomma, (C) Dermacentor, (D) Amblyomma, (E) Boophilus, (F) Rhipicephalus and (G) Haemaphysalis (Wall & Shearer, 2001).
52
2.8
The life cycle of Ixodid ticks (adapted from www.life cycle of ticks family Ixodidae png.)
54
2.9
The type of ixodid ticks depending on the number of hosts attached (P. Jain & A. Jain, 2006).
55
24
3.1
Geographical map of Kelantan state showing the locations of equids sampling stables using Map Window arc GIS 10 program.
108
3.2
The clinical examination card.
112
3.3
A-F) Demonstration of thin and thick blood smears preparation (http://www.slideshare.net/drAjayAgale/05-peripheral-bloodsmear-examination).
115
3.4
Various shapes and stages with measurement of T. equi in stained blood smears with 5% Giemsa, examined under oil immersion lens (100X); A) Pyriform (pair of joint) and single pyriform ; B) Maltese cross and anaplasmoid shape ; C) Rod shape; D) The schizonte stages: microschizontes and macroschizontes (Koch‘s blue bodies) of T. equi within lymphocytes.
133
3.5
Various shapes with measurement of B.caballi in stained blood smears with 5% Giemsa, examined under oil immersion lens (100X); A) Double pear acute and obtuse angle, single pear and round shape; B) Signet ring shape; C) Amoeboid shape.
134
3.6
Gel electrophoresis image showing: lanes M) Exact Mark 1001500bp DNA ladder; Lane 1-9) Conventional PCR technique detected Theileria spp. and Babesia spp. using ‗catch-all‘ primers in approximately band size 496 bp; Lane N) DNA extracted from piroplasms-free horse used as negative control.
141
3.7
Gel electrophoresis image showing: lane M) Exact Mark 1001500bp DNA ladder; Lane P) DNA extracted from clinically infected case used as positive controls for T. equi and B. caballi; Lane 1-6) Multiplex PCR technique detected only T. equi in approximately band size 360 bp; Lane N) DNA extracted from piroplasms-free horse used as negative control.
142
3.8
Gel electrophoresis image showing: lane M) Exact Mark 1001500bp DNA ladder; Lane P) DNA extracted from clinically infected case used as positive controls for T. equi and B. caballi; Lane 1-6) Multiplex PCR technique detected only B. caballi in approximately band size 650 bp; Lane N) DNA extracted from piroplasms-free horse used as negative control.
142
25
3.9
Gel electrophoresis image showing: lane M) Exact Mark 1001500bp DNA ladder; Lane P) DNA extracted from clinically infected case used as positive controls for T. equi and B. caballi; Lane 1-6) Multiplex PCR technique detected both protozoa using specific primers in approximately band size 360 bp and 650 bp respectively; Lane N) DNA extracted from piroplasms-free horse used as negative control.
143
3.10
Geographical map of Kelantan state in Malaysia showing the distribution of T. equi infection. The different marks show infection rate in each stable at different sub-districts using Map Window arc GIS 10 program.
147
3.11
Geographical map of Kelantan state in Malaysia showing the distribution of B. caballi infection. The different marks show infection rate in each stable at different sub-districts using Map Window arc GIS 10 program.
148
3.12
Geographical map of Kelantan state in Malaysia showing the distribution of both protozoa infections. The different marks show infection rate in each stable at different sub-districts using Map Window arc GIS 10 program.
149
3.13
Seroprevalence of T. equi, B. caballi and both protozoa by months; *) Values significantly different (P < 0.05) compared to November month.
159
3.14
Seroprevalence of T. equi, B. caballi and both protozoa by means of monthly temperature (ºC); *) Values significantly different (P
