PREVALENCE, RISK FACTORS AND GENETIC
DIVERSITY OF EQUINE PIROPLASMOSIS IN KELANTAN, MALAYSIA
QAES TALB SHUKUR ALSARHAN
DOCTOR OF PHILOSOPHY
2017
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
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.
I agree that my thesis is to be made immediately available OPEN ACCESS as hardcopy or on-line open access (full text).
EMBARGOES I agree that my thesis is to be made available as hardcopy
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Dated from ______until ______
(Contains confidential information under the office CONFIDENTIAL Official Secret Act 1972)*
(Contains restricted information as specified by the RESTRICTED 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.
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Date: Date:
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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
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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
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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
2.5.2.1 The susceptibility related to equids factors 33
2.5.2.2 The susceptibility related to environmental and stables 36 factors
2.6 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
2.7 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
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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 79
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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
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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 124 blood samples
3.2.7 Evaluation the efficiency of different methods for diagnosis 129 the disease
3.2.8 Statistical analysis 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 143 by regions
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3.3.5 Risk factors associated with seroprevalence of EP causative 150 agents
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 173 methods
3.4.3 Evaluating of cELISA and multiplex PCR for detecting T. equi 177 and B. caballi infections
3.4.4 Equids factors associated with T. equi, B. caballi and both 179 protozoa
3.4.5 Stables factors associated with T. equi, B. caballi and both 182 protozoa
3.4.6 Climatic factors associated with T. equi, B. caballi and both 185 protozoa
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
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4.2.6 Serum biochemistry analysis 196
4.2.7 Statistical analysis 197
4.3 Results 198
4.3.1 Seroprevalence of clinical and subclinical forms in equids 198 with the relative risk
4.3.2 Level of antibodies titration against piroplasms in equids with 198 clinical and subclinical forms of equine piroplasmosis
4.3.3 Hematological and serum biochemistry parameters in equids 199 with clinical and subclinical form of equine piroplasmosis
4.4 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 221 ticks using multiplex PCR
5.3.2 Similarity rates on detection of T. equi, B. caballi and both 222 protozoa DNAs between equids and Ixodid ticks
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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. 229 caballi
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
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LIST OF TABLES
NO. PAGE
2.1 Prevalence of T. equi, B. caballi and both protozoa in the 29 different countries with the references.
2.2 Hard ticks species with the geographic location, number of the 45 hosts and confirmed or suspected to be transmitted of Theileria equi or Babesia caballi (the causative agents of EP)(Scoles & Ueti, 2015).
2.3 Ixodid tick species distribution in different states of Malaysia. 61
2.4 Different methods obtainable for the diagnosis of EP and their 94 purpose (OIE, 2014b).
3.1 The Oligonucleotide primers used to amplify the parasites 18S 125 rRNA genes.
3.2 PCR program for samples subject to conventional PCR and 125 multiplex PCR.
3.3 PCR procedure for DNA extracted samples subject to multiplex 127 PCR and conventional PCR assay (reaction volume 25 μl).
3.4 The epidemiological table and formulas were used to copare 130 between tests.
3.5 Morphological features, biomedical data and parasitemia of T. 135 equi, B. caballi and both protozoa base on microscopic examination of blood smears.
3.6 Overall prevalence of equine piroplasmosis (singly T. equi, 136 singly B. caballi and both protozoa) in equids in Kelantan by microscopic examination, cELISA and multiplex PCR.
3.7 Prevalence of T. equi, B. caballi and both protozoa in equids in 137 Kelantan by microscopic examination, cELISA and multiplex PCR.
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3.8 The sensitivity, specificity, accuracy, positive predictive value 139 and negative predictive value of cELISA for ditecting T. equi antibodies in comparison to microscopic examination (Gold standard).
3.9 The sensitivity, specificity, accuracy, positive predictive value 139 and negative predictive value of multiplex PCR for detecting T. equi DNAs in comparison to microscopic examination (Gold standard).
3.10 The sensitivity, specificity, accuracy, positive predictive value 140 and negative predictive value of cELISA for detecting B. caballi antibodies in comparison to microscopic examination (Gold standard).
3.11 The sensitivity, specificity, accuracy, positive predictive value 140 and negative predictive value of multiplex PCR for detecting B. caballi DNAs in comparison to microscopic examination (Gold standard).
3.12 Comparison between conventional PCR and multiplex PCR 141 techniques for detection piroplasms DNA in equids blood (n= 306).
3.13 Prevalence of T. equi, B. caballi and both protozoa in sampling 145 stables in different regions and sub-regions in Kelantan state.
3.14 Relative risk of EP associated with the type of protozoa in 154 equids.
3.15 Relative risk of equids factors associated with seropositivity of 155 T. equi, B. caballi and both protozoa.
3.16 Relative risk of regional factors associated with seropositivity of 157 T. equi, B. caballi and both protozoa.
3.17 Relative risk of monthly factors associated with seropositivity of 158 T. equi, B. caballi and both protozoa.
3.18 Relative risk of management and ticks factors associated with 160 seropositivity of T. equi, B. caballi and both protozoa.
3.19 Relative risk of climatic factors associated with seropositivity of 161 T. equi, B. caballi and both protozoa.
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3.20 Percentage of sables infested with Ixodid ticks by Kelantan 166 regions.
3.21 Details and number of identified Ixodid tick species infesting 167 equids and nearby animals related to the prevalence of EP in each field.
3.22 Distribution of identified Ixodid tick species and their 169 predilection sites on equids and nearby animals.
3.23 The abundance and percentage of Ixodid ticks on equids and 170 nearby animals.
4.1 Health status factor equids associated with seroprevalence of T. 201 equi, B. caballi and both protozoa.
4.2 Relative risk of of equids health status factors associated with 201 the T. equi, B. caballi and both protozoa.
4.3 The clinical symptom parameters in the equids with clinical 204 form compared to the equids with subclinical form and healthy group.
4.4 Haematological changes in the equids with clinical form 205 compared to the equids with subclinical form and healthy group.
4.5 Serum biochemistry changes in the equids with clinical form 206 compared to the equids with subclinical infectedform and healthy group.
5.1 Detection of T. equi and B. caballi in Ixodid ticks (n= 31) using 222 multiplex PCR.
5.2 Similarity on detection of T. equi and B. caballi in the equids 224 and Ixodid ticks using multiplex PCR.
5.3 Detection rate of T. equi and B. caballi genotypes in equids base 227 on individual BLASTn analysis of positive samples.
5.4 Detection rate of T. equi and B. caballi genotypes in Kelantan 228 regions.
5.5 Detection rate of T. equi and B. caballi genotypes in Ixodid ticks 229 (n=31) base on individual BLASTn analysis of sequences.
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5.6 Similarity within and between T. equi genotypes using multiple 230 sequence alignment- CLUSTALW (GenomeNet).
5.7 Similarity within and between B. caballi genotypes using 230 multiple sequence alignment- CLUSTALW (GenomeNet).
5.8 GeneBank accession numbers of Kelantan T. equi genotypes in 234 the equids and ticks.
5.9 GeneBank accession numbers of Kelantan B. caballi genotypes 235 in the equids and ticks. 5.10 Homology between obtained sequences (GeneBank accession 236 numbers) of T. equi and GeneBank database using online sequence BLASTn.
5.11 Homology between obtained sequences (GeneBank accession 237 numbers) of B. caballi and GeneBank database using online sequence BLASTn.
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LIST OF FIGURES
NO. PAGE
2.1 A) Different forms of T. equi inside the erythrocytes and the 14 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).
2.2 The life cycle of T. equi Illustration by Massaro Ueti (Wise et al., 16 2013).
2.3 Different forms of B. caballi inside the erythrocytes and the 18 characteristic pair of joint at their posterior ends organisms forming in a Giemsa’s stained blood smear (S. Kumar & R. Kumar, 2007).
2.4 The life cycle of Babesia caballi. Illustration by Massaro Uet 21 (Wise et al., 2013).
2.5 The internal structure of the hard tick (40X) by Edward et al. 48 (2009).
2.6 Parts of a generalized hard tick; A) Dorsal view of capitulum 50 (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).
2.7 Diagrammatic dorsal view of the capitulum of seven genera of 52 hard ticks: (A) Ixodes, (B) Hyalomma, (C) Dermacentor, (D) Amblyomma, (E) Boophilus, (F) Rhipicephalus and (G) Haemaphysalis (Wall & Shearer, 2001).
2.8 The life cycle of Ixodid ticks (adapted from www.life cycle of 54 ticks family Ixodidae png.)
2.9 The type of ixodid ticks depending on the number of hosts 55 attached (P. Jain & A. Jain, 2006).
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3.1 Geographical map of Kelantan state showing the locations of 108 equids sampling stables using Map Window arc GIS 10 program.
3.2 The clinical examination card. 112
3.3 A-F) Demonstration of thin and thick blood smears preparation 115 (http://www.slideshare.net/drAjayAgale/05-peripheral-blood- smear-examination).
3.4 Various shapes and stages with measurement of T. equi in stained 133 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.
3.5 Various shapes with measurement of B.caballi in stained blood 134 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.
3.6 Gel electrophoresis image showing: lanes M) Exact Mark 100- 141 1500bp 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.
3.7 Gel electrophoresis image showing: lane M) Exact Mark 100- 142 1500bp 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.
3.8 Gel electrophoresis image showing: lane M) Exact Mark 100- 142 1500bp 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.
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3.9 Gel electrophoresis image showing: lane M) Exact Mark 100- 143 1500bp 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.
3.10 Geographical map of Kelantan state in Malaysia showing the 147 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.
3.11 Geographical map of Kelantan state in Malaysia showing the 148 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.
3.12 Geographical map of Kelantan state in Malaysia showing the 149 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.
3.13 Seroprevalence of T. equi, B. caballi and both protozoa by 159 months; *) Values significantly different (P < 0.05) compared to November month.
3.14 Seroprevalence of T. equi, B. caballi and both protozoa by means 162 of monthly temperature (ºC); *) Values significantly different (P <0.05) compared to November month.
3.15 Seroprevalence of T. equi, B. caballi and both protozoa by 162 means of monthly rainfall amount (mm); *) Values significantly different (P < 0.05) compared to November month.
3.16 Seroprevalence of T. equi, B. caballi and both protozoa by means 163 of monthly relative humidity (%); *) Values significantly different (P < 0.05) compared to November month.
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3.17 A&B) Rhipicephalus (Boophilus) microplus dorsal & ventral 168 view male; C&D); Rhipicephalus Boophilus annulatus female; E&F) Rhipicephalus sanguineous dorsal & ventral view female; G&H) Rhipicephalus appendeculataus dorsal & ventral view male; I&J) Rhipicephalus bursa dorsal & ventral view female; K&L) Dermacentor marginatus dorsal & ventral view male; M&N) Haemaphysalis punctata dorsal & ventral view engorged female; O&P) Haemaphysalis Longicornis dorsal & ventral view engorged female; Q&S) Larval stage of H. punctata dorsal & ventral view normal female; T&U) Larval stage of H. punctata dorsal & ventral view engorged female.
3.18 Pie chart showing the percentage of Ixodid ticks infesting equids 189 and nearby animals.
3.19 The percentage of Ixodid tick species in Kelantan. 171
4.1 A) Determination the optical density of serial dilution log10 202 positive control serum for T. equi in cELISA kit, used as standerd; B) The antibody titres against T. equi in clinical and subclinical form of EP.
4.2 A) Determination the optical density of serial dilution log10 202 positive control serum for B. caballi in cELISA kit, used as standerd; B) The antibody titres against B. caballi in clinical and subclinical form of EP.
4.3 Some of the clinical signs shown in equids with clinical form of 203 EP: A) Loss of the body weight (Emaciation) and depression; B) rd Pale of mucous membrane in the 3 eye lid; C) Petechial rd hemorrhage of mucous membrane in the 3 eye lid; D) Presence of ticks under the ear.
4.4 Percentage of clinical signs on equids with clinical form (n=30). 203
5.1 Phylogenetic tree of T. equi obtained with partial sequences of 238 the 18S rRNA gene. The numbers at the branches indicate bootstrap supports (100 replications). Sequences in bold indicate to those obtained sequences in this study. Equids, ticks and locations are in between brackets. A, B, C, D and E indicate to genotypes.
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5.2 Phylogenetic tree of B. caballi obtained with partial sequences of 239 the 18S rRNA gene. The numbers at the branches indicate bootstrap supports (100 replications). Sequences in bold indicate to those obtained sequences in this study. Equids, ticks and locations are in between brackets. A, B and C indicate to genotypes.
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LIST OF ABBREIVATIONS
EP Equine Piroplasmosis
18s rRNA 18 subunit ribosomal ribonucleic acid
T. equi Theileria equi
B. caballi Babesia caballi
OIE World organization for animal health
PCR Polymerase chain reaction qPCR Quantitative polymerase chain reaction nPCR Nested polymerase chain reaction mPCR Multiplex polymerase chain reaction
LAMP Loop mediated isothermal amplification
HPLC High performance liquid chromatography
IFAT Indirect fluorescent antibody test
CFT Complement fixation test iELISA Indirect enzyme-linked immunosorbent assay cELISA Compatitive enzyme-linked immunosorbent assay
DVS Deparment of veterinary services spp Species sRNA Subunit ribosomal ribonucleic acid
16S rRNA 16 subunit ribosomal ribonucleic acid
EMA-1 Equi merozoites antigen -1
DIC Disseminated intravascular coagulation
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PCV Packed cell volume
MCV Mean corpuscular volume
MCH Mean corpuscular hemoglobin
MCHC Mean corpuscular hemoglobin concentration
ESR Erythrocyte sedimentation rate
RBCs Red blood cell count
Hb Hemoglobin concentration
WBCs White blood cell count
AST Aspartate amino transferase
ALT Alanine amino transferase
ALP Alkaline phosphatase
BUN Blood urea nitrogen
EMAs Equi merozoites antigens
GPS Global positioning system
EDTA Ethylene diamine tetra acetic acid rpm Round per minutes
HRP Horse radish peroxidase min Minutes
OD Optical density sec Second
RR Relative risk
CI Confidence interval
TECs Total erythrositic counts
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TLCs Total leukositic counts
RBA Rhipicephalus (Boophilus) annulatus
RBM Rhipicephalus (Boophilus) microplus
RB Rhipicephalus bursa
RA Rhipicephalus appendeculataus
RS Rhipicephalus sanguineus
HP Haemaphysalis punctata
HL Haemaphysalis longicornis
DM Dermacentor marginatus
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LIST OF SYMBOLS
% Percentage
ºC Celsius degree
Ml, µl Millelitter, Microlitter
ºF Fehrenhait ng Nano gram
μM Micromoles
μm Micrometre cm Centimeter mm Micrometer n Number of positive nm Nanometer
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LIST OF EQUATIONS
NO. PAGE 1/D 3.1 n= [1-(α) ] [N-(D-1) /2] 109
Number of infected RBCs
3.2 Parasitemia % = X 100 113 Number of calculated RBCs
3.3 %I=100- [(sample O.D×100) ÷ (mean negative control O.D)] 119
3.4 TP/TP+FN X 100 130
3.5 TN/TN+FP X 100 130
3.6 TP+TN/TP+FN+TN +FP X 100 130
3.7 TP/TP+FPX 100 130
3.8 TN/TN+FN X100 130
Number of reticulocytes
4.1 Reticulocyte % = X 100 196 Number of calculated reticulocytes
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Prevalen, Faktor Risiko dan Kepelbagaian Genetik Piroplasmosis Ekuin di Kelantan, Malaysia
ABSTRAK
Piroplasmosis ekuin (PE), penyakit wajib lapor di Malaysia, yang disebabkan oleh Theileria equi, Babesia caballi dan kedua-dua protozoa menyebabkan anemia, ikterus, pendarahan bintik, hemoglobinuria dan kematian dalam ekuid. Epidemiologi penyakit ini di Malaysia belum disiasat dengan teliti. Objektif kajian adalah untuk: mengenal pasti etiologi PE di Kelantan; menentukan prevalen dan faktor risiko; membandingkan keberkesanan ujian makmal untuk mengesan penyakit ini; mengenal pasti spesis kutu pada ekuid; menentukan parameter patologi klinikal dan titer antibodi dalam ekuid subklinikal; menyiasat kepelbagaian genetik dan filogenetik piroplasma dari ekuid dan kutu. Dari September 2013 hingga Mac 2014, 53 kandang telah dipilih secara mudah dari lapan Jajahan di Kelantan. Sebanyak 306 ekuid telah dipilih secara rawak dari kandang tersebut. Darah, serum dan kutu telah diambil dari ekuid tersebut. Calitan darah diwarnakan dengan Giemsa untuk mengesan T. equi, B. caballi secara mikroskopik. Darah telah dijalankan ujian multipleks PCR untuk mengesan DNA protozoa tersebut. Darah juga telah dijalankan ujian hematologi. Serum telah dijalankan ujian cELISA untuk mengesan antibody T. equi, B. caballi dan juga untuk ujian biokimia. Kutu dikenalpasti dengan menggunakan kekunci taksonomi. Data epidemiologi dikumpulkan melalui temu bual dengan pemilik kandang. Gen 18S rRNA T.equi dan B. caballi yang diasingkan daripada darah dan kutu dianalisa untuk kepelbagaian genetik dan pokok filogenetik menggunakan program talian NCBI BLAST, ClustalX (NCBI) dan Neighbor-joining (NJ). Pelbagai bentuk, saiz, peringkat dan tahap parasitemia T. equi dan B. caballi dilihat dalam pemeriksaan mikroskopik. Dengan menggunakan pemeriksaan mikroskopik darah, cELISA dan multipleks PCR prevalen keseluruhan PE adalah 32.02%, 80.06% dan 35.62%. Faktor risiko yang yang penting dan berkaitan dengan EP di Kelantan adalah terdapat kutu pada ekuid dan haiwan berdekatan; kehadiran haiwan lain di kandang kuda; ekuid yang diimpot. Enam spesies kutu Ixodid yang penting dalam menyebarkan PE telah dikenalpasti: Rhipicephalus (Boophilus) Microplus, Rh. (Boophilus) annulatus, Rh. bursa, Rh. sanguineus, Haemaphysalis longicornis dan Dermacentor marginatus. Kepelbagaian genetik yang tinggi dikesan dalam gen 18S rRNA T. equi dan B. caballi yang dikesan dari ekuid dan kutu Ixodid. Kesimpulannya, PE adalah endemik di Kelantan dan beberapa faktor risiko dan spesies kutu yang penting dalam menyebarkan protozoa telah dikenal pasti. Kawalan kutu hendaklah dijalankan untuk mencegah rebakan PE.
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Prevalence, Risk Factors and Genetic Diversity of Equine Piroplasmosis in Kelantan, Malaysia
ABSTRACT
Equine piroplasmosis (EP), a reportable disease in Malaysia, caused by Theileria equi, Babesia caballi and both protozoa can result in petechial hemorrhages, hemoglobinuria, and death in equids. The epidemiology of the disease in Malaysia has not been thoroughly investigated. The objectives of this study were to: identify the etiology of EP in Kelantan; determine the prevalence and risk factors; compare the effectiveness of laboratory tests for detecting the disease; identify ticks species on the equids; determine the clinicopathological parameters and antibody titers in equids with a clinical and subclinical form; and investigate the genetic diversity and phylogenetic of the piroplasms from equids and ticks. From September 2013 to March 2014, 53 stables were conveniently selected in eight regions in Kelantan. A total of 306 equids were randomly selected from the stables. Blood, serum and ticks were collected from the equids. Blood smear was stained with Giemsa and examined for T. equi and B. caballi microscopically. Blood was subjected for multiplex PCR to detect T. equi and B. caballi DNA. Blood were also subjected for hematology. Serum was subjected for cELISA to detect antibodies against the protozoa, and also for serum biochemistry. Ticks were identified using taxonomic keys. Epidemiological data were collected through interviews with the stables owners. The 18S rRNA gene of T. equi and B. caballi extracted from blood and ticks were analyzed for genetic diversity and phylogenetic tree using online NCBI BLAST, ClustalX (NCBI) and Neighbor-joining (NJ) programs. Various shapes, sizes, stages and parasitemia level of T. equi and B. caballi were seen in the blood smears. The overall prevalence of EP was 32.02%, 80.06% and 35.62% using microscopic examination, cELISA and multiplex PCR respectively. The risk factors that were important and significantly associated with higher prevalence of EP in Kelantan include imported equids, presence of ticks on equids and nearby animals and presence of other animals near or in the stable. Six species of Ixodid ticks that are important in spreading EP were identified: Rhipicephalus (Boophilus) microplus, Rh. (Boophilus) annulatus, Rh. bursa, Rh. sanguineus, Haemaphysalis longicornis and Dermacentor marginatus. High level of genetic diversity was detected within 18S rRNA gene of T. equi and B. caballi from equids and Ixodid ticks. In conclusion, EP is endemic in Kelantan and several risk factors and species of ticks important in spreading the protozoa were identified. Tick control program should be conducted in order to prevent the transmission of EP.
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CHAPTER 1
INTRODUCTION
1.1 General introduction
Equine Piroplasmosis (EP) is one of the most common and important
ticks-borne protozoal diseases of equids including ponies, horses, mules, donkeys,
and zebras. The disease is caused by either a single obligatory intraerythrocytic
hemoprotozoan Theileria equi (previously known as Babesia equi) or Babesia
caballi or both protozoa (Ristic et al., 1982; Mehlhorn & Schein, 1998). The
recent taxonomic classification placed T. equi and B. caballi in the phylum
Apicomplexa (protozoa) and under different families Babesiidae and Theileriidae
respectively (Taylor et al., 2007). Theileria equi is more pathogenic and widely
distributed in endemic areas than B. caballi (Katz et al., 2000; Sigg et al., 2010).
Equids infected with these parasites can remain as carriers for a long
period of time. In the infection with B. caballi, the protozoa will be completely
eliminated from blood within four years. Equids infected by T. equi may remain
as lifelong carriers and act as sources of infection for other susceptible equids via
tick vectors (de Waal & van Heerden, 1994; Friedhoff & Soule, 1996). The
protozoa are mainly transmitted by ticks of the Ixodidae family (Jongejan &
Uilenberg, 2004).
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Clinical manifestation of the disease ranges from a subclinical condition to severe and potentially fatal illness depends on the equids immune status and the virulence of the protozoa. The clinical form is characterized by fever of more than
40ºC, anemia, icterus, hemorrhages of the mucous surfaces, hemoglobinemia, and hemoglobinuria (Brüning, 1996; Ibrahim et al., 2011).
The distribution of the EP is worldwide. It is endemic in many parts of
Asia, South and Central America, Africa, and Europe (Leblong et al., 2005).
Canada, Australia, New Zealand, United Kingdom, North America, Japan,
Ireland, Netherland, and Scandinavia are considered as EP free areas
(Anonymous, 2008; Kappmeyer et al., 2012).
The geographical distribution of EP depends on the presence of tick vectors and international trade that result in increased movement of horses from endemic to non-endemic areas (Friedhoff et al., 1990; Brüning, 1996). The entry of persistently infected horses into areas in which tick vectors are dominant, can lead to outbreak of the disease (Sumbria & Singla, 2015).
There are 33 Ixodid tick species belonging to six genera that have been reported as competent tick vectors for EP. The Ixodid tick species include;
Amblyomma spp., Hyalomma spp., Rhipicephalus spp., Dermacentor spp., Ixodes spp., and Haemaphysalis spp. These ticks have been reported to transmit T. equi and B. caballi to equids (Scoles & Ueti, 2015).
The protozoa can also be accidentally transmitted through blood transfusion and contaminated needles, syringes, or any instruments used in castration or vaccination. The vertical or transplacental transmission has been
2
reported worldwide for T. equi and in suspected cases of B. caballi infection
(Georges et al., 2011; Sudan et al., 2013). Subclinically infected equids appear healthy with low parasitemia. However, they are the source of the protozoa for the tick vectors, which will transmit to a susceptible horse population (Bahrami et al.,
2014).
In epidemiological studies in different countries, there are many risk factors associated with the high prevalence of T. equi, B. caballi and both protozoa such as equids species, age, gender, breed, presence of tick, activity, regions, environmental temperature, and humidity (Kouam et al., 2010c; Garcia-
Bocanegra et al., 2013; Sumbria et al., 2016b). Controlling or removing the risk factors is important for a control program in order to prevent the spread of the disease (Kouam et al., 2010a; Dos Santos et al., 2011; Guidi et al., 2015).
Equine piroplasmosis can cause significant economic losses that include the cost of treatment, decreased equids production, abortions, loss of performance or death and inability to meet international requirements for exports or participation in equestrian sports (Friedhoff et al., 1990; Hailat et al., 1997;
García-Sanmartín et al., 2008). In addition, some countries do not allow the entry of subclinical seropositive horses (Asenzo et al., 2008).
Equine piroplasmosis is listed among the diseases of the World
Organization for Animal Health (OIE). Occurrence of the disease has to be reported to OIE within 72 hours of detection (OIE, 2013).
Long-term monitoring of the prevalence of the disease is very important in countries where EP has been reported so that the potential adverse effects on
3
the horse industry can be prevented. This is because the morbidity, mortality and case fatality rate of EP can vary from 10 to 50% can be addressed (OIE, 2013).
Besides that, EP can result in other complications such as colic, cardiac arrhythmias, pneumonia, disease of the central nervous system, laminitis, and infertility in stallions. The disease can also cause abortion and stillbirth or the birth of live foals with neonatal piroplasmosis (Bryant et al., 1969; Diana et al.,
2007; Georges et al., 2011; Chhabra et al., 2012).
A suspected case of EP can be identified based on the clinical signs of the infected equids. However, the clinical signs are often varies and non-specific (de
Waal & van Heerden, 2004; Rothschild & Knowles, 2007). To confirm a suspected case different laboratory techniques have been developed for diagnosing EP and these includes microscopic examination of blood smears, serological tests, molecular techniques, biological tests, and in vitro culture method (Sumbria & Singla, 2015).
The information of EP in Kelantan is scanty. Only two reports on EP in
Malaysia were found during a thorough literature search. Chandrawathani et al.
(1998) reported that there was no evidence of T. equi and B. caballi in Kelantan among the 91 equids sampled. Zawida et al. (2010) reported that the seropositive for T. equi was 20% and for B. caballi it was 1% in 12 states of Malaysia based on
180 equids sampled.
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1.2 Problem statement
Equine piroplasmosis had been reported in Malaysia but at low
prevalence (Chandrawathani et al., 1998 and Zawida et al., 2010). In Kelantan,
there are more than 658 equids including horses and ponies (DVSK, 2012). The
equids are used for pleasure-riding (recreation), breeding, and sport. The foals
were sold to equids owners from other states (personal communication with
equids owners).
Some of the equine in Kelantan were from Thailand (Personal
communication with equids owners). Equine piroplasmosis had been reported in
Thailand (Chungvipat & Viseshakul, 2005; Kamyingkird et al., 2014). In
Kelantan animals susceptible to EP are diversified such as horses, ponies,
ruminants and dogs. Also, the tick vectors for EP were distributed (Mariana et al.,
2005). The potential tick vectors reported in Kelantan are Boophilus microplus,
Ixodes granulatus and Dermocentor spp. (Mahmood, 1997; Mariana et al., 2005).
In Kelantan, near most equids stables have other animals such as cattle, sheep and
goats. These animals can also act as reservoir for piroplasms (Zhang et al., 2015).
The epidemiology of EP in Kelantan in term of prevalence by location,
month, equids characteristics and risk factors was not known. Without
epidemiological information, the veterinary authority in Kelantan could not
institute control strategies for EP and prioritize their resources. The prevalence of
tick infestation among equids and other animals near the equids was also not
known. This information is important for tick control as one of the strategies to
5
prevent EP. The genetic diversity of T. equi and B. caballi in Kelantan was not
known. This information is important for future study on vaccine for EP.
1.3 Research questions
4. What are the prevalence and risk factors of EP among equids in Kelantan?
5. Which laboratory test is more efficient for detecting EP?
6. What is the species of Ixodid tick that may play an important role in spreading
the disease?
7. What are the clinical, hematology and serum biochemistry parameters
alterations in the infected equids?
8. Is there any genetic diversity of the detected T. equi and B. caballi in Kelantan?
1.4 Hypothesis
1. EP is endemic in Kelantan and there are different equids, stables and climatic
risk factors associated with the high prevalence of the disease.
2. There are differences in the efficiency of the microscopic examination, cELISA
test, and PCR technique in the diagnosis of EP.
3. There are different sources of infection (ticks, equids infected with subclinical
form and imported equids) that play an important role in the spread of the disease
in Kelantan.
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4. There are different species of hard ticks that transmit the disease between equids.
5. There are changes in hematology and serum biochemistry parameters in the infected equids.
6. There is genetic diversity of T. equi and B. caballi in Kelantan.
1.5 The objectives of study
1. To identify T. equi, B. caballi and both protozoa with their parasitemia.
2. To determine the prevalence of T. equi, B. caballi and both protozoa among
equids in Kelantan and risk factors.
3. To evaluate the efficiency of different laboratory methods for detecting T. equi
and B. caballi infections.
4. To identify and determine the infestation rate for Ixodid tick species on equids
and other animals near equids in Kelantan stables.
5. To evaluate the hematology, serum biochemistry, and antibody titers in equids
with a clinical and subclinical forms of EP.
6. To investigate genetic diversity and phylogenetic analyses of the T. equi and B.
caballi detected in equids and Ixodid ticks in Kelantan.
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CHAPTER 2
LITERATURE REVIEW
2.1 History of equine piroplasmosis (EP)
The synonyms of EP are anthrax fever, biliary fever, bilious fever,
equine malaria, equine nutalliosis, equine tick fever, equine babesiosis, and
equine theileriosis (Roberts et al., 1962; Merchant & Barner, 1964; Rooney &
Robertson, 1996; OIE, 2013).
Before 1901, EP was not recognized and was often confused with other
equine diseases (Roberts et al., 1962). Wiltshire in 1883 was the first to describe
and record a case of EP in South Africa as “anthrax fever”. The condition, as in
the case in Cape Town was shown by Hutcheon and named “biliary fever” and it
was viewed as a “bilious fever” or bilious form of horse sickness by Nunn (de
Waal & van Heerden, 2004). From 1902 to 1903 in South Africa, the veterinarian
Sir Arnold Theiler mentioned that the biliary fever was not similar to African
horse sickness, but may occur at the same time and named it “equine malaria” due
to the clinical signs of the disease observed in equines in Pretone town, which
were identical to malaria disease (Plasmodiida) affecting humans. He abortively
attempted to transmit the causative agent from infected horses to uninfected native
horses by blood transfusion, Theiler guessed that he was unable to create the
infection because the local horses were previously immune to it or probably
8
because the causative agent needed a tick vector to be infective (Theiler, 1902,
1903).
Laveran, (1901) was the first to observe a parasite in the red blood cells of peripheral blood smear prepared from horses in South Africa and named it
Piroplasma equi. Later on Theiler, (1905) confirmed the result by reporting that
African horse sickness and equine piroplasms were two different diseases infecting equids. Kochs, (1904) described two species (spp.) of equine piroplasma morphological differences in Zimbabwe. Later on Nutall and Strickland, (1910,
1912) reported that the two distinguished species of the parasites were implicated in equine piroplasmosis. They observed large intra-erythrocytic protozoa, which were distinguished from Piroplasma equi and they were causing piroplasmosis in equids. They named the larger species Piroplasma caballi (Nuttall) and the small species Nutallia equi (Laveran). Later on, these parasites were classified as
Babesia equi and Babesia caballi under the genus Babesia.
Schein et al. (1981) tried to reclassify the B. equi in the genus Babesia by developing the cycle of the infection in horses and in lymphocyte culture demonstrated that the B. equi showed the characteristic of the genus Theileria in their morphological features, and their development in the horse host and in the tick vectors.
Mehlhorn & Schein, (1998) reclassified the Babesia equi (Laveran, 1901) as Theileria equi and transferred from one valid genus to another. This changed was required since it worked out that this equids parasite observed the pertinent characteristics of Theilerians with morphological features, biochemical properties,
9
molecular biological relationship and biological data. Kappmeyer et al. (2012)
who support the connotation of reclassified B. equi to T. equi during newly
genomic analysis of T. equi. Nevertheless, the taxonomy of the piroplasms agents
remains controversial for B. equi since their discovery.
2.2 Etiological agents and taxonomy
Equine piroplasmosis is a disease that is spread by ticks and caused by
two different protozoan species, T. equi and B. caballi of the genus Theileria and
Babesia respectively. The protozoa infected all equids including horse, pony,
donkey, mule and zebra (Laveran, 1901; Schein, 1988).
Historically, the classification of Babesia spp has been on the basis of
their size and their vertebrate host (Ristic et al., 1982; Euzeby, 1987; Levine,
1988; Ristic, 1988). The large Babesia is typically greater than 3 microns in
length while the small Babesia generally measures 1-3 microns in length (Ristic,
1988). Over 100 species of Babesia have been described in different hosts
(Levine, 1988).
The absence of a pre-erythrocytic stage in the life cycle is viewed as a
significant phenotypic characteristic of the genus Babesia. Thus far, genetic data
for Babesia that was listed in the gene bank was only available for 15 species
(Benson et al., 2003). The classification of the family Babesiidae is likely to
undergo considerable revision in the future due to the availability of molecular
10
biology and the use of DNA sequences to conduct phylogenetic classification of more than 100 described Babesia spp.
Earlier taxonomic classification placed Babesia spp in the phylum
Apicomplexa, class Piroplasmea, order Piroplasmoridea, family Babesiidae, genus
Babesia and species B. equi and B. caballi (Riek, 1968; Mahoney et al., 1977).
The recent taxonomic classification placed T. equi and B. caballi in the phylum
Apicomplexa (protozoa), class Sporozoasida (Piroplasmea), subclass
Coccidiasina, order Eucoccidiorida (Piroplasmida), suborder Piroplasmorina, B. caballi in the family Babesiidae (de Waal & van Heerden, 2004, Taylor et al.,
2007) and T. equi in the family Theileriidae species (Mehlhorn & Schein, 1998;
Taylor et al., 2007).
In general, there are many causes that resulted in the reclassification of B. equi to T. equi which include: the sporozoites of B. equi enter lymphocytes then start a schizogonic phase resulting in motile merozoites (Schein et al., 1981; de
Waal & van Heerden, 2004), the reproductive development of B. equi occurs in the salivary glands of ticks and not in the other organs like the ovaries of the tick; it similarly occurs in the Theileria spp. (Shaw & Young, 1995; Guimaraes et al.,
1998; Zapf & Schein, 1994a), and the sexual phases of B. equi is different from that of Babesia spp (Zapf & Schein, 1994b; Mehlhorn et al., 1980).
Morphologically the Babesia spp. hss been reclassified into small Babesia (T. equi) and the trophozoites measure 1.0-2.5 μm while the large Babesia (B. caballi) the trophozoites measure 2.5-5 μm (Homer et al., 2000).
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In phylogenetic studies Brüning, (1996) suggested a distinct paraphyletic
group of T. equi by using small subunit ribosomal RNA (sRNA). The B. equi
surface protein (encoding gene) was symmetrically homologous with that in
Theileria spp. (Kappmeyer et al., 1993). The reclassification of B. equi in the
genus Theileria was due to the multiplication entra-erythrocytic, with the results
presenting four merozoites and only transstadial transmission occurs (Uilenberg,
2006). Kuttler, (1988) found that the schizont stages of B. equi in the lymphocytes
were very sensitive to parvaquone and halofuginone, which are influential against
Theileria schizonts.
The other piroplasms spp. Babesia bovis in cattle and Babesia canis of
dogs had been reported in horses (Criado-Fornelio et al., 2003; Hornok et al.,
2007).
2.2.1 Theileria equi
2.2.1.1 Morphology
Synonyms: Nuttallia equi, N. minor, N. asini, Nicollia equi, Piroplasma equi,
Babesia equi.
The Theileriidae family comprises relatively small organisms. They are
ovoid, round, irregular form or rod-like, are usually present in the lymphocytes,
erythrocytes and histiocytes. They do not produce pigments (Soulsby, 1982). The
protozoa appear either singly, in pairs or in tetrad (Schein, 1988).
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Theileria equi (Fig. 2.1A) is a small Theileria spp. The sporozoites stages of this species are 3 to 4 micrometre (μm) in length and 1 to 2 μm in diameter; they are ovoid or spindle-shaped in the tick’s salivary glands (Mehlhorn & Schein,
1998). Besides, the T. equi merozoites stages within erythrocytes are Anaplasma like shaped or spherical when they have just entered the red blood cells. Then they develop into round or ovoid shapes, are 2 to 3 μm in diameter which makes them look like ring form. After the first binary fission, they are pear-shaped. 2 μm in length or pyriform and 2 to 3 μm in length. Following the second fission, they are four pear-shaped and develop a tetrad form called “Maltese cross” an arrangement which is a characteristic form of this species (Levine, 1985; Mehlhorn & Schein,
1998; Jain, 2000). The schizont stages of T. equi (Fig. 2.1B) within lymphocytes are developed to two forms; microschizontes and macroschizontes, which are 1.5 to 2 μm in length (Schein et al., 1981; Mehlhorn & Schein, 1998).
There are different shapes of T. equi, most of them are the spherical shape (69%) and round (20%), while other shapes include ovoid, pear-shape and
“Maltese cross” (20%) (Kumar et al., 2002a). On the other hand, Selim and
Abdelgawaad, (1982) observed that the percentage of Maltese cross shape was up to 1.9%) while the Anaplasmoid shape was 96% in the blood smears of infected donkeys.
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A B
Figure 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).
2.2.1.2 Life cycle
The life cycle (Fig. 2.2) of T. equi can be divided into three stages: i) the
sporogony (sporozoites forming) is an asexual transmission stage, which occurs
in the tick; ii) the merogony (merozoites forming) is an asexual blood stage,
which occurs in the equids host erythrocytes, and iii) the gamogony (gametocytes
forming) is a sexual blood stage, which occurs inside the midgut of the tick (Vial
et al., 2006). There is one more stage observed in the Theileria spp. called
schizogony (schizonts forming), which is an asexual blood stage occurring in the
equids host lymphocytes and also occurring between sporogony and merogony
stages (Mehlhorn & Schein, 1998). These developments depend on the tick
species involved (Schein et al., 1981; Moltmann et al., 1983b).
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During the infection, adult ticks are attached to equids host and suck the blood which results in the transstadial transmission of T. equi sporozoites through their saliva. These parasites penetrate the lymphocytes and develop into two large microschizontes and microschizontes (schizogony stage). Then these schizonts form about 200 merozoites per infected cell (Schein et al., 1981; de Waal & van
Heerden, 2004). After around nine days the merozoites are liberated to invade several erythrocytes then multiply by binary fission (merogony stage). After first division, a pyriform shape is formed within erythrocytes. After the second division, a four pear-shaped called “Maltese cross” shape is formed (Schein, 1988;
Moltmann et al., 1983a). When the infected erythrocytes rupture the merozoites are released and penetrate new red blood cells and continue to reproduce
(Uilenberg, 2006). Eventually, some merozoites are developed to form gamonts in the equids peripheral blood (Zapf et al., 1994a; Guimaraes et al., 1998).
The schizogony and merogony are the asexual stages that occur in the equids host, where another sexual stage is started when the ticks become infected by feeding on infected equids blood (gamonts). The gamonts develop in the midgut of the tick and begin nuclear replication, forming protrusions called “ray bodies”. After 4-6 days of feeding, the ray bodies divide to form thread-like microgamonts and spherical or ring-shaped macrogamonts, which combine to become zygotes (gamogony stage). Within the zygotes large club-shaped “kinete bodies” are formed, which are 14 μm in length and 4 to 7 μm in diameter after 5-7 days of tick feeding. The kinetes perforate the tick’s midgut into the hemolymph,
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and in the last 6-9 days the kinetes invade the tick’s salivary gland cells type III and develop into large multinuclear sporont forms, then divide into many spherical multinuclear sporoblast forms. Finally, sporozoites are formed, which are ovoid to spindle shaped, with complete development between 6 and 24 days after the tick feeding (sporogony stage) (Mehlhorn & Schein, 1998; de Waal & van Heerden, 2004).
Lymphocytes
Figure 2.2: The life cycle of T. equi Illustration by Massaro Ueti (Wise et al., 2013).
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2.2.2 Babesia caballi
2.2.2.1 Morphology
Synonyms: Piroplasma caballi
The Babesiidae family comprises large organisms. They are round or
oval shapes and pyriform or amoeboid shapes and found in the erythrocytes singly
or in pairs of organisms (Soulsby, 1982; Kuttler, 1988). Babesia caballi is larger
Babesia spp, which is morphologically similar to B. bigemina. The merozoites in
the RBC are 2 to 5 μm in length and 1.3 to 3.0 μm in diameter. The pear-shaped
and pyriform in a pair of joints at their posterior ends is the diagnostic feature
(Mehlhorn & Schein, 1984; Levine, 1985) (Fig. 2.3). Babesia caballi appears as
pears to a spherical or amoeboid shape, which are 2.5 to 4 μm or sometimes reach
6 μm in length and 2 μm in diameter (Ueti et al., 2008). Besides, Ueti et al. (2005)
recorded that the Anaplasma like-shape, which are the smaller shape of B. caballi
are 1 μm in size. They are found in the erythrocytes and more common in the
blood smears. On the other hand, B. caballi are also found as round or rod-shaped
intra-erythrocytes and the pear-shape in acute angle is observed more in the blood
smears stained with Giemsa’s (Uilenberg, 2006).
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Figure 2.3: Different forms of B. caballi inside the erythrocytes and the characteristic pair of joints at their posterior ends organisms forming in a Giemsa’s stained blood smear100X (S. Kumar & R. Kumar, 2007).
2.2.2.2 Life cycle
The life cycle (Fig. 2.4) of B. caballi is also divided into three stages:
sporogony and gamogony are asexual and sexual stages respectively that occur in
the tick and schizogony is a sexual blood stage, which occurs in the equids host.
The schizogony stage starts when the infected ticks attached to the equids
host. The sporozoites in the salivary gland of the ticks are transmitted during
feeding via the saliva and directly invades the equids erythrocytes by the
invagination process (Homer et al., 2000). The sporozoites in the erythrocytes
develop by a budding process from trophozoites which are a small anaplasmoid
body to the larger spherical or ameboid body that is divided by binary fission to
18
produce merozoites which are larger paired bodies joined at the posterior ends, and when the infected erythrocytes lyse or rupture , the merozoites are liberated and go on to invade new erythrocytes (Homer et al., 2000). Some of the erythrocytes have multiple infections due to multiple binary fissions of trophozoites, rather than a multiple infection of the erythrocytes and this is the common blood form of B. caballi (Soulsby, 1982). Some of the trophozoites within the cells do not multiply. They only increase in size and form gamonts.
Subsequently, these gamonts will develop into gametes within the cells (Homer et al., 2000). Following 10 hours after the ticks start to feed on the infected equids host all B. caballi stages can be detected in the consumed cells.
The gamogony stage begins when the tick is feeding a blood meal from an infected equids, in the tick’s midgut the arguments develop in ray bodies or
Strahlenkorper, containing an arrowhead structure. These are divided to form gametes, and through asexually coupling produce zygotes called kinete bodies.
These parasites use their arrowhead structure to enter the epithelial cell of the tick’s midgut. After 80 hours from the time the tick begins feeding, the kinetes penetrate the tick gut and invade various organs via the tick hemolymph, including the ovaries, but not, at first, the salivary acini (Koch, 1906; Uite et al.,
2003; Uilenberg, 2006). The B. caballi can be transovarially transmitted to the next tick and the growing of the sporozoites occurs in the salivary acini of a nymph, larvae and adult ticks (Rothschild & Knowles, 2007). On the contrary, T. equi kinete bodies, initially, invade the tick’s salivary gland cells and transstadial
19
transmission of the infective stage occurs (Mehlhorn & Schein, 1998; Uilenberg,
2006).
The sporogony stage starts when the sporozoites in the tick’s salivary acini develop and divide into three stages: the first is the formation of a sporoblast, which is a multinucleated form, relatively undifferentiated and with branching meshwork, which by budding produces sporozoites (Karakashian et al.,
1983). The second, after the tick hosts start feeding again, the rhoptries, micronemes and the double membrane segments beneath the plasma membrane
(the specialized organelles of future sporozoites) grow in the meshwork (Kakoma
9. Mehlhorn, 1993). Finally, by the budding process the mature sporozoites form is produced, with its pyriform shape measuring 2.2 μm length and 0.8 μm in diameter and containing free ribosome, several micronemes, a smooth endoplasmic reticulum, mitochondrion-like organelle, and a single anterior rhoptry (Karakashian et al., 1983; Kakoma & Mehlhorn, 1993). Homer et al.
(2000) mentions that a single sporoblast can produce approximately 5,000-10,000 sporozoites.
20
Erythrocytes
Figure 2.4: The life cycle of Babesia caballi. Illustration by Massaro Ueti (Wise et al., 2013).
2.3 The genes commonly targeted in the T. equi and B. caballi
There are various genes in the T. equi and B. caballi that have been used
for identification, characterization and differentiation of these parasites. The 18S
ribosomal ribonucleic acid (rRNA) genes of T. equi and B. caballi were the more
commonly used genes (Nagore et al., 2004; Hall et al., 2013; Qablan et al., 2013),
because the DNA sequences of this gene at species level were mostly conserved.
It contains highly frequented sequences existing in diverse copies in the genome
and the sequences of this gene are available in molecular databases (Prichard,
1997; Adam et al., 2000; Alhassan et al., 2005; Bhoora et al., 2009). Moreover,
the 18S rRNA gene is highly important in the epidemiological and genetic
21
diversity studies of these parasites (Bhoora et al., 2009; Ros-Garcia et al., 2013;
Gallusová et al., 2014).
The other genes have been targets for T. equi, including the 16S rRNA
gene (Bashiruddin et al., 1999), the equi merozoites antigen -1, 2 and 3 (EMA-1,
EMA-2, EMA-3) genes (Xuan et al. 2001a; Kappmeyer et al., 2012; Vianna et al.,
2014), Babesia equi 82 (Be82) gene (Hirata et al., 2002, 2003) and Babesia equi
158 (Be158) gene (Hirata et al., 2005). On the other hand, the genes have been
targets of B. caballi, including the 16S rRNA gene (Vargas et al., 2004), the
Babesia caballi 48 (BC48) gene (Alhassan et al., 2005; Zhang et al., 2015),
Babesia caballi 134-kilodalton protein (BC134) gene (Tamaki et al., 2004), and
the rhoptry associated protein-1 (RAP-1) gene (Suarez et al., 2003).
2.4 Sequencing and genetic diversity for T. equi and B. caballi
The basis for using 18S rRNA gene, including this gene is the more
suitable genetic marker for T. equi and B. caballi diagnosis and genetic diversity
studies of piroplasms (Hunfeld et al., 2008). Different PCR techniques followed
by sequencing have been used to study the genetic diversity of these protozoa
(Kouam et al., 2010b; Ros-García et al., 2013; Alanazi et al., 2014). The first
genetic diversity of T. equi was Spain 1 and Spain 2, notified in Spanish horses,
with similarity of 99% to the African genotype (Criad- Fornelio et al., 2003). Two
other T. equi genotypes (T. equi and T. equi-like) have been reported in Spanish
22
horses and Grecian equids, showing 96.8% similarity between them (Nagore et al., 2004; Kouam et al., 2010b).
Three genotypes of T. equi 18S rRNA gene including A, B and C genotypes have been identified in South Africa (Bhoora et al., 2009). Genotype A
(group A) contains isolates from horses, similar to previous T. equi isolate in
Africa and all T. equi genotypes isolate in Spain. Genotype B (group B) encompassed new isolates from Zebra, similar to T. equi-like isolate from horses.
Genotype C (group C) contains the new African T. equi isolate (Nagore et al.,
2004; Bhoora et al., 2009). Recently, other T. equi 18S rRNA genotypes including genotype D (group D) and genotype E (group E) have been reported with the aforementioned genotypes that have been reported in different countries such as in
Mongolian horses by using nested PCR (Munkhjargal et al., 2013), in Jordanian equids by using multiplex PCR (Qablan et al., 2013), in Saudi Arabia by using real-time PCR (Alanazi et al., 2014), in Sudan by using primary PCR (Salim et al., 2010). In North America, T. equi genotypes A, B, C and D have been reported using quantitative PCR (qPCR) assay (Hall et al., 2013), and in Tunisia three genotypes A, C and D of T. equi have been identified by using reverse line blot
(RLB) hybridization assay (Ros-García et al., 2013).
The first genetic diversity of B. caballi 18S rRNA gene was reported in
Spanish horses including (Spain 1 and Spain 2) using semi-nested PCR and RLB-
PCR (Criad-Fornelio et al., 2004; Nagore et al., 2004). The isolate Spain1 showed lower similarity of 97% with the African genotype, but isolate Spain 2 showed a high genetic similarity (97.7%-100%) with the B. caballi genotype in Africa
23
(Nagore et al., 2004). Another B. caballi genotype (B. caballi-like) has been reported in Spanish horses and Grecian equids, showing lower similarity of 97.4% with B. caballi genotypes in South Africa (Nagore et al., 2004; Kouam et al.,
2010b). Two others of B. caballi 18S rRNA genotypes (A and B) have been reported in South Africa by using RLB-PCR assay (Bhoora et al., 2009); genotype
A sharing B. caballi Spain 1 and B. caballi-like genotypes in Spain (Criad-
Fornelio et al., 2004; Nagore et al., 2004), as well as B. caballi genotype USDA reference strain (Kappmeyer et al., 1999). Genotype B encompassed new isolates
B. caballi genotype from South Africa, which is subdivided into sub-groups B1 and B2, with these sub-groups containing the original African B. caballi sequence
(accession number: Z15104) (Allsopp et al., 1994). Recently, another B. caballi
18S rRNA genotype named genotype C (group C) novel was identified in
Jordanian equids using a multiplex technique (Qablan et al., 2013) and in
Mongolian horses using nested PCR assay (Munkhjargal et al., 2013).
There is a high level of genetic variation in the T. equi and B. caballi 18S rRNA gene and these DNA sequence variances between different strains of single species have been identified as an eventual cause of incomplete immunization after vaccination trial and poor therapeutic results or impaired immunologic diagnosis of the disease (Criad-Fornelio et al., 2004). This happens, especially in countries that have reported genetic diversities of T. equi and B. caballi.
24
2.5 Epidemiology of equine piroplasmosis
2.5.1 Geographic distribution
The geographical distribution of EP is depend on the presente of potential
tick vectors, the international trade related to increased movement of horses from
endemic to non-endemic areas, and the variance of T. equi and B. caballi
persistence period in equids host (Brüning, 1996; Salim et al., 2008; Pitel et al.,
2010; Gallusová et al., 2014).
Equine piroplasmosis is considered a global problem that affects the
horse industry and given rise to the need for stringent international requirements
in relation to the export or participation in equestrian sporting events (Friedhoff et
al., 1990). Equine piroplasmosis is distributed worldwide but is endemic in the
majority of tropical and subtropical areas of the world, in many parts of Asia,
South and Central America, Cuba, Africa and Europe (Leblong et al., 2005;
Karatepe et al., 2009), and also in some temperate climatic zones (Brüning, 1996).
On the other hand, countries such as Canada, Japan, Australia, New Zealand,
United Kingdom, North America, Ireland and the Scandinavian countries are
considered as disease free areas (Phipps, 1996; Kappmeyer et al., 2012). Despite
the fact that Australia is considerably genereally free from EP, T. equi had entered
the country on several occasions. Firstly, infected horses entered Australia in the
1950s and 1960s having been imported from Texas. Andalusian infected horses
also entered in the 1970s, being imported from Spain. However, EP had never
25
been recognized due to the absence of appropriate tick vectors that transmit the disease (Martin, 1999).
The distribution of the disease depends on the type of serological test used for screening and the measures taken to prevent the introduction of B. caballi and T. equi into disease-free areas (Brüning, 1996; Friedhoff et al., 1990). The distributions also depend on equids exported from countries where the disease is endemic, without proper quarantine. It has a significant impact on the geographical distribution of disease, like equids exporteded from France (Fritz,
2010).
The causative agents of EP always share the same tick vectors. However,
T. equi is more common and distributed than B. caballi (Kouam et al., 2010a;
Ribeiro et al., 2013; Garcia-Bocanegra et al., 2013). Other reports have indicated that the prevalence of B. caballi was significantly higher than T. equi. This result may be due to change in the epidemiological pattern of EP (Kerber et al., 2009;
Moretti et al., 2010; Mujica et al., 2011; Munkhjargal et al., 2013).
The disease is geographically distributed and endemic in many countries of Asia like Saudi Arabia (Alanazi et al., 2012, 2014), Oman (Donnelly et al.,
1980a; Fadya et al., 2008), Jordan (Abutarbush et al., 2012; Qablan et al., 2013),
Iraq (Alsaad et al., 2012; Alsaad & Mussa, 2012), Kuwait (Donnelly et al.,
1980b), Turkey (Acici et al., 2008; Karatepe et al., 2009), Iran (Bahrami et al.,
2014; Abedi et al., 2014a), Pakistan (Hussaina et al., 2014), India (Sumbria et al.,
2016a), Mongolia (Rüegg et al., 2007; Munkhjargal et al., 2013), Japan (Ikadai et al., 2002), China (Wang et al., 2014), Korea (Seo et al., 2011), Philippines (Cruz-
26
Flores et al., 2010), and Thailand (Tantaswasdi et al., 1998; Chungvipat &
Viseshakul, 2005).
Both causative agents of EP are recorded in African countries like South
Africa (Bhoora et al., 2010), Morocco (Rhalem et al., 2001), Kenya (Oduori et al., 2015), Sudan (Salim et al., 2013), Egypt (Salib et al., 2013), Nigeria (Garba et al., 2011), Tunisia (Ros-Garcia et al., 2013), and Central Ethiopia (Gizachew et al., 2013).
The disease is also present in European countries such as Greece (Kouam et al., 2010a), Belgium (Mantran et al., 2004), France (Fritz, 2010; Guidi et al.,
2105), Spain (Garcia-Bocanegra et al., 2013), Italy (Laus et al., 2013, 2015),
Portugal (Baptista et al., 2013; Ribeiro et al., 2013), Romania (Gallusovà et al.,
2014), Hungary (Farkas et al., 2013), Germany (Boch, 1985), Switzerland (Sigg et al., 2010), Poland (Adaszek et al., 2011), Netherlands (Butler et al., 2012),
Commonwealth of Independent State (CIS) such as Ukraine, Moldavia and in the
Central and Southern parts of Russia (Friedhoff & Soulé, 1996).
Equine piroplasmosis is also presente in some countries of South and
Central America like Brazil (Kerber et al., 2009; Prochno et al., 2014), Trinidad
(Asgarali et al., 2007), Venezuela (Rosales et al., 2013), Argentina (Asenzo et al.,
2008), Colombia (Tenter et al., 1988), North Eastern Mexico (Cantú-Martıúnez et al., 2012), United States of America (Taylor et al., 1969; APHIS, 2009), Florida and southern Texas, and the Caribbean (Rampersad et al., 2003) (Table 2.1).
In Malaysia, EP is a notifiable disease. The occurrence of the disease must be reported to the Department of Veterinary Services (DVS). The disease
27
has been reported in Johor and the other twelve states in Malaysia
(Chandrawathani et al., 1998; Zawida et al., 2010).
28
Table 2.1: Prevalence of T. equi, B. caballi and both infections in different countries with the references.
Number & type of Type of Continent Country Type & prevalence of infections References equids tests a- 242 (equids) - T. equi, B. caballi 1.2% Blood smears Chandrawathani et al. Malaysia b-180 (horse) -T. equi 20%, B. caballi 1% cELISA (1998); Zawida et al. (2010) a- 158 (horses) -T. equi 49.83%, B. caballi 5% IFAT Chungvipat and b- 240 (horses & mules) T. equi 5.42%, B. caballi 2.50% cELISA Viseshakul (2005); Thailand -T. equi 8.75%, B. caballi 5.00% IFAT Kamyingkird et al. T. equi 1.25%, B. caballi 0.0% mPCR (2014) Korea 184 (horses) T. equi 1.1%, B. caballi 0.0% cELISA Seo et al . (2011) a- 93 (donkeys) -T. equi 9.6%, B. caballi 38, Both 2.2% ELISA Chahan et al. (2006); China b- 1990 (horses) -T. equi 11.51%, B. caballi 51.16, Both 7.64% iELISA Wang et al. (2014) 104 (horses) T. equi 10.0%, B. caballi 71.0%, Both 19.0% ICT Cruz-Flores et al. Philippines (2010) Asia 2,019 (horses) -T. equi 2.2%, B. caballi 5.4% ELISA Ikadai et al. (2002) Japan -T. equi 2/44 (27.52%), B. caballi 30/109 WBT (27.52%) a-510 (horses) -T. equi 78.8%, B. caballi 65.7% IFAT Rüegg et al. (2007); T. equi 66.5%, B. caballi 19.1% mPCR b-250 (horses) -Overall: 81.6%, T. equi 19.9%, B. caballi ELISA Munkhjargal et al. Mongolia 51.6%, Both 10.4%. mPCR (2013) Overall: 51.2%, T. equi 6.4%, B. caballi 42.4%, Both 2.4% 180 (horses, donkeys, T. equi 73.89%, B. caballi 1.11% cELISA Sumbria et al. (2016a) India mules) T. equi 14.14%, B. caballi 0.0% mPCR 430 (horses, donkeys, Overall: 52.6%, T. equi 41.2%, B. caballi cELISA Hussaina et al. (2014) Pakistan mules) 21.6%, Both 10.2%
29
Number & type of Continent Country Type & prevalence of infections Type of tests References equids a-165 (horses) -T. equi 28.5% PCR Bahrami et al. ( 2014); Iran b-100 (horses) -T. equi 48%, B. caballi 2%, Both 3%. IFAT Abedi et al. (2014a); Both 5% Blood smears -T. equi 45%, B. caballi 0.0% mPCR Abedi et al. (2014b) c-194 (horses) B. caballi 4.12% IFAT a- 90 (horses) -T. equi 86.58%, B. caballi 54.39%, Both Alsaad et al. (2010); b- 46 (horses) 16.10% cELISA Alsaad et al. (2012) Iraq 45 (donkeys) - T. equi 71.73%, B. caballi 19.56% in horses T. equi 42.22%, B. caballi 4.44% in donkeys a- 125 (horses) - Overall: 18.4%, T. equi 12.8%, B. caballi IFAT Karatepe et al. (2009); 9.6%, Both 6.2%. qPCR Asia Turkey b- 203 (horses) - Overall: 4.93%, T. equi 2.96%, B. caballi PCR Kizilarslan et al 1.97% T. equi 66.7%, B. caballi .(2015) 25.0% Oman 481(horses, donkeys) Overall: 9% in horses, 3.71% in donkeys Blood smears Fadya et al. (2008) 35 (horses) T. equi 51.4%, B. caballi 11.4% CFT Donnelly et al. (1980b) Kuwait T. equi 77.1%, B. caballi 11.4% IFAT a- 253 (horses) T. equi 14.6%, B. caballi 0.0% cELISA Abutarbush et al Jordan b- 288 (horses, Overall: 27.1%, T. equi 18.8%, B. caballi 7.3% mPCR (2012); donkeys, mules) Qablan et al. (2013) Saudi 241 (horses) T. equi 10.4%, B. caballi 7.5%, Both 3% IFAT Alanazi et al. ( 2012) Arabia South 176 (horses) T. equi 45%, B. caballi 33% IFAT Gummow et al (1996) Africa Kenya 314 ( donkeys) T. equi 81.2%, B. caballi 0.0% cELISA Oduori et al. (2015) a-158 (horses) -T. equi 25.2%, B. caballi 0.0% PCR Salim et al. (2008); T. equi 63.5%, B. caballi 4.4% ELISA Africa Sudan b-499(horses, -T. equi 35.95%, B. caballi 0.0% PCR Salim et al. (2013) donkeys) a- 100 (horses) -T. equi 18%, 30%, 26% Blood smears, Ibrahim et al. (2011); Egypt ELISA, PCR b-149 (horses) -T. equi 41.61% Blood smears Salib et al. (2013)
30
Number & type of Continent Country Type & prevalence of infections Tests types References equids 104 (horses) T. equi 12.5%, B. caballi 1.92% qPCR Ros-Garcia et al. Tunis (2013) Africa Nigeria 254 (horses) Overall: 20.1%, T. equi 80.4%, B. caballi 19.6% Blood smears Garba et al.( 2011) Central 395 (donkeys) T. equi 12.2%, B. caballi 1.8% Blood smears Gizachew et al. Ethiopia T. equi 55.7%, B. caballi 13.2% IFAT (2013) 544 (horses, mules, Overall: 11.6%, T. equi 11%, B. caballi 2.2% cELISA Kouam et al. (2010a) Greece ponies) 142(horses) T. equi 22.5%, B. caballi 2.1% PCR Davitkov et al. Serbia (2016) a- 111 (horses) -T. equi 80%%, B. caballi 1.2% PCR Fritz, (2010); France 166 (dogs) T. equi 19%, B. caballi 0.6% b-443 (horses) -T. equi 58%, B. caballi 12.9% CFT Guidi et al. (2015) a-60 (horses) -T. equi 40%, B. caballi 0.8%, Both 20% IFAT Camacho et al. b-537 (horses, - Overall: 58.4%, T. equi 56.1%, B. caballi cELISA (2005) Spain donkeys, mules) 13.2%, Both 10.8% Garcia-Bocanegra et al. (2013) European a- 294 (horses) - Overall: 8.5%, T. equi 8.2%, B. caballi 0.3%, IFAT Grandi et al. (2011); Both 0.0%. T. equi 33%, B. caballi 0.0% PCR Laus et al. (2013); Italy b- 300 (horses) - T. equi 41.0%, B. caballi 26.0%, Both 14.7% IFAT T. equi 11.7%, B. caballi 6.0% PCR Laus et al. (2015) c- 138 (donkeys) - T. equi 40.6%, B. caballi 47.8%, Both 19.6% IFAT T. equi 17.4%, B. caballi 3.6%, Both 0.0% qPCR a-305 (horses) -T. equi 9.3%, cELISA, Baptista et al. b-250 (horses) T. equi 1.9% nPCR (2013); Portugal -T. equi 3.1%, B. caballi 1.9% Blood smears Ribeiro et al. (2013) T. equi 17.9%, B. caballi 11.1% cELISA 193 (horses, donkeys) Overall: 25.4%, T. equi 20.3%, B. caballi 2.2%, mPCR Gallusovà et al. Romania Both 3.0%. (2014)
31
Number & type Type of Continent Country Type and prevalence of infections References of equids tests 324 (101 PCR) T. equi 32% cELISA, Farkas et al. (2013) Hungary (horses) T. equi 49% IFAT-PCR
Germany 321(horses) T. equi 5.6%, B. caballi 1.2% CFT Boch, (1985) 689 (horses) Overall: 7.3%, T. equi 4.4%, B. caballi 1.5%, IFAT Sigg et al. (2010) Switzerland Both 1.5% 300 (horses), 12 T. equi 25%, B. caballi 75% IFAT Butler et al. (2012) Netherland positive T. equi 1.6%, B. caballi 1.3% qPCR Southern Texas 063 (horses) T. equi 81.1% cELISA Scoles et al. (2011) a- 198 (horses) -Overall: 97.5%, T. equi 78.3%, B. caballi cELISA Vieira et al. (2013); Brazil b- 400 (horses) 69.2%, Both 50%. Prochno et al. - T. equi 61% ELISA (2014) 93 (horses) Overall: 82.8%, T. equi 33.3%, B. caballi 68.8%, IFAT Asgarali et al. Trinidad Both 19.4% (2007) a- 360 (horses) -T. equi 50.3%, B. caballi 70.6%, Both 35.5% ELISA Mujica et al. ( 2011); b- 694(163PCR) - Overall: 50.2%, T. equi 14%, B. caballi 23.2%, cELISA Rosales et al. (2013) America Venezuela (horses) Both 13% mPCR -Overall: 66.2%, T. equi 61.4%, B. caballi 0.0% 82 (horses) T. equi 65%, B. caballi 41% CFT Tenter et al. (1988) Colombia T. equi 94%, B. caballi 90% IFAT 248 (horses) Overall: 61.7%, T. equi 45.2%, B. caballi 27.4% iELISA Cantú-Martıúnez et Mexico al. (2012) North America 37 (horses) T. equi 73% qPCR Hall et al. (2013) 285 (horses) -T. equi 88.5%, B. caballi 69.2%, Both 62.3% cELISA Posada-Guzmán et Costa Rica -T. equi 46%, B. caballi 20%, Both 7% nPCR al. (2015)
cELISA) Competitive-enzyme linked immunosorbent assay; iELISA) Indirect enzyme linked immunosorbent assay; IFAT) Indirect fluorescent antibody test; CFT) Complement fixation test; ICT) Immunochromatographic test; WBT) Western blot test; mPCR) Multiplex polymerase chain reaction; nPCR) Nested polymerase chain reaction; qPCR) Quantitative real time polymerase chain reaction; and PCR) Conventional polymerase chain reaction.
32
2.5.2 Susceptibility to the disease
2.5.2.1 Susceptibility related to equids factors
The susceptiblity of the equids to the disease depends on many factors
including: type of equids, gender, age, breed, health status, origin, activity,
pregnancy, the stage of pregnancy, and presence of ticks on the animal.
Equine piroplasmosis is a protozoal disease affecting all equids including
ponies (Laveran, 1901; Kouam et al., 2010a), horses, donkeys, mules and zebras
(Kumar et al., 2002a). However, horses are more susceptible to the disease than
donkeys. Mules are less susceptible than horses and donkeys (Uilenberg, 2006).
There was no significant difference in susceptibility to T. equi and B. caballi
between ponies and horses and between ponies and mules (Kouam et al., 2010a).
After recovery from the disease, the infected horses remain carriers as
subclinical infection (persistent infection) to the parasites for an indefinite period
of time before they are cleared of the parasites. Equids are infected with T. equi
for many years or for life. Equids infected with B. caballi remain carriers up to 12
months or more. Reinfection occurs when the animal is exposed to stress factors
such as hunger, malnutrition, injury and other diseases which lowered the
immunity status (Friedhoff et al., 1990; Kumar et al., 2009; Kumar et al., 2002b).
There is no difference in susceptibility to the causative agents of EP
between male and female equids (Uilenberg, 2006; Asgarali et al., 2007). In
addition, Karatepe et al. (2009) noted that the infection rates between male and
33
female were similar when exposed to the same conditions, whereas Moretti et al.
(2010) and Dos Santos et al. (2011) observed that the females were more susceptible to the infection than the males. Similarly, Shakp et al. (1998) found that stallion equids were less susceptible than mares and geldings. This may be because males are maintained under hygienic breeding and they are less exposed to ticks. Gelding equids are more susceptible to T. equi and B. caballi than the males and females. This finding may be due to accidental transmission of the protozoa through contaminated surgical instruments during castration (Barros,
2008; Rüegg et al., 2007). Salib et al. (2013) observed that the stallions were more susceptible than mare equids to the infections. All equids breeds are susceptible to the causative agents of EP and the alteration in the susceptibility depends on the animals ages (Asgarali, 2007; Rüegg et al., 2007).
Ages of the equids figure significantly in the epidemiology of the disease. The infection rate in newborn and young growing equids (less than one year) is lower than in horses aged one to five years. This may be due to the presence of passive immunity through the colostrum, the activity of hemopoietic system and the activity of the thymus, which make the newborn and young aged equids more resistant to EP. Foal born from non-vaccinated mare or did not have antibodies against the parasites is more susceptible to the infections (Soulsby,
1982; Mark, 2010). Older equids (over five years) are more affected than newborn and younger animals (Asgarali et al., 2007; Garcia-Bocanegra et al., 2013). This finding could reflect the higher exposure to ticks and persistence of antibodies (de
Waal & van Heerden, 1994; Rüegg et al., 2007). On the other hand, there is no
34
difference in the susceptibility to the infections for all ages of equids (Farkas et al., 2013; Malekifard et al., 2014; Acici et al., 2008) perhaps due to the high number of tick vectors in the regions and continuous exposure of young and old animals to infected ticks (Razmi et al., 2002).
The native equids are more susceptible to the infections than imported equids when not both infections are registered in the imported equids (Dos Santos et al., 2011). Otherwise, there is a difference in the susceptibility to the infections between the native and imported equids with regard to both infections (Kouam et al., 2010a). The imported horses in enzootic areas may have a low case fatality rate ranging between 5% and 10% (Rothschild & Knowles, 2007). Nevertheless, the case fatality rate may increase significantly among native mature horses
(Maurer, 1962; Rothschild & Knowles, 2007).
There is a difference in the susceptibility of T. equi and B. caballi infections among the types of equids activity. The endurance and sport equids were more susceptible to the infection than those breeding activities. This may be due to strenuous exercise (stress), moving for training and competitions and spleen contraction during stress leads to the release of the parasites from spleen, which is predisposed to the disease (Hailat et al., 1997). Baldani et al. (2008) found no significant difference in the infection rates of T. equi and B. caballi in sports horses at rest and after strenuous exercise or stress.
Lewis, (1999) found that the pregnant mares in the third stage were more susceptible to the infections than non-pregnant. This may be due to subclinical infection and reactivation of the latent parasites during parturition stress.
35
2.5.2.2 The susceptibility related to environmental and stables factors
There is a difference in the susceptibility of equids to the infections in
various countries. This may be due to the difference in the management practice
of equids: the sensitivity of the diagnostic methods used in the screening of the
disease, the occurrence of competent tick vectors, the existence and efficacy of
any ticks control program and the variations in the climatic conditions (Kouam et
al., 2010a; Grandi et al., 2011). Similarly, the causes of the difference in the
susceptibility of equids among the regions or districts might also be attributed to
the management practice, sampling size, equids activity, and presence of
competent tick vectors (Kouam et al., 2010a).
Various climatic factors such as temperature, relative humidity and the
amount of rainfall influence the ticks habitat (Garcia- Bocanegra et al., 2013). The
susceptibility of equids to the infections varies between the seasons: it is increased
in the summer and begins to decline with the advent of autumn and in the winter
(Phipps, 1996; Niven, 2002; Salib et al., 2013). In spite of that, maximum increase
of the causative agents of the disease have been recorded in the rainy seasons
(Rothschild & Knowles, 2007). On the other hand, a study did not find a
significant difference of infections between the various seasons and the incidence
of EP (Moretti et al., 2010).
The equids kept in grazing are more susceptible to T. equi and B. caballi
infections than those kept in a stable, probably due to more exposure to external
environmental conditions and being directly in contact with the tick vectors
36
(Abutarbush et al., 2012; Ribeiro et al., 2013). Equids mixed with other animals in the same stable are more susceptible to the causative agents of an EP than equids kept by themselves without other animals. This may be due to the presence of predisposing factors that transmit the infections from animals by direct or indirect contact with the equids and also the absence of ticks control program in the stables (Kerber et al., 2009; Kouam et al., 2010a).
Labruna et al. (2001) observed that cattle were the primary host for
Rhipicephalus (Boophilus) microplus and the infestation of the horses with this tick species depends on the presence of cattle in the same area. The existence of the competent tick vector species Rhipicephalus bursa and Rhipicephalus sanguineus for transmission of the T. equi and B. caballi were collected from horses and dogs, respectively, in the same stables (Kouam et al., 2010a).
The presence of ticks in the stables increases the susceptibility of the equids to the infections compared to the absence of ticks in stables because the ticks are considered the main biological vectors of the causative agents of EP
(Garcia- Bocanegra et al., 2013).
37
2.6 Transmission of the causative agents
2.6.1 Biological transmission
Equine piroplasmosis is a hemoprotozoal tick-borne disease, transmitted
biologically between equids via tick vectors. Therefore, the worldwide
distribution of EP has a close relation with the distribution of competent tick
vectors (Schein, 1988). There are three exclusive forms of tick-borne
transmission: first form is a intrastadial transmission which occurs when the tick
acquires the parasite from infected host and transmits it to another uninfected one
within same tick life stage or without development (no stage transition before
transmission) (Stiller et al., 2002; Ueti et al., 2008).
The second form is a transstadial transmission which occurs when the
tick stages (larval or nymphal) acquire the pathogen from an infected or reservoir
animal and then transmits the parasite to another uninfected animal in the next
stage of the life cycle (stage transition before transmission) (Knowles et al., 1992;
Ueti et al., 2005, 2008).
The third form is a transovarial transmission which happens when the
female tick acquires the pathogen, which is passed to the ovaries, resulting in
infected eggs, then the offspring, of the parasites crosses tick generations
permitting maintenance of the pathogens cross tick development stages (one or
more stages in the next generation can transmit pathogens) (Howell et al., 2007;
Ueti et al., 2008).
38
Because of variations in the T. equi and B. caballi life cycles, they have a different form of transmission. Theileria equi is generally either intrastadial or transstadial transmitted (The parasites move from tick midgut directly to the salivary glands) (Uilenberg, 2006; Ueti et al., 2008). Theileria equi are also transovarial transmitted, but the accurate role in epidemiology has not been detailed (Moltmann et al., 1983b; Ikadai et al., 2007). On the other hand, B. caballi transovarial transmission usually occurs when the parasite moves from the tick midgut directly to the ovaries then enters the eggs and thereafter the offspring. Babesia caballi is also transstadial transmitted (Brüning, 1996; Ueti et al., 2008).
Ticks that feed on persistently infected horses with low T. equi 3 6 -1 parasitemia (2 ×10 - 10 ml of blood) have been known to be successful in transmitting the parasites to non-infected horses (Ueti et al., 2005, 2008).
There are 33 Ixodid tick species that belong to six genera reported as competent tick vectors of EP, including Amblyomma (1 species), Dermacentor (9 species), Haemaphysalis (1 species), Hyalomma (13 species), Ixodes (1 species), and Rhipicephalus (8 species) (Scoles & Ueti, 2015). Twenty-seven tick species belonging to these six genera, can be transmitted: T. equi includes Amblyomma mixtum, Dermacentor marginatus, D. niveus, D. li, D. reticulatus, D. silvarum, D. variabilis, Haemaphysalis longicornis, Hyalomma aegyptium, H. marginatum, H. excavatum, H. dromedarii, H. anatolicum, H. lusitanicum, H. detritum, H. scupense, H. turancatum, H. plumbeum, H. uralense, Ixodes ricinus,
39
Rhipicephalus bursa, Rh. evertsi evertsi, Rh. microplus, Rh. evertsi mimeticus, Rh.
pulcbellus, Rh. sanguineus, and Rh. turanicus (Scoles & Ueti, 2015).
Twenty-two tick species belonging to four genera can transmit B. caballi
including Dermacentor albipictus, D. marginatus, D. nitens, D. niveus, D. nuttalli,
D. pictus, D. reticulatus, D. silvarum, D. variabilis, Haemaphysalis longicornis,
Hyalomma aegyptium, H. anatolicum, H. dromedarii, H. excavatum, H.
marginatum, H. plumbeum, H. turancatum, H. Vegans, Rhipicephalus annulus,
Rh. bursa, Rh. evertsi evertsi, and Rh. sanguineus (Scoles & Ueti, 2015).
Sixteen tick species belonging to four genera can transmit T. equi and B.
caballi simultaneously, including Dermacentor marginatus, D. niveus, D. nuttalli,
D. reticulatus, D. silvarum, D. variabilis, Haemaphysalis Longicornis, Hyalomma
aegyptium, Hyalomma marginatum, H. anatolicum, H. dromedarii, H. excavatum,
H. turanicum, Rhipicephalus bursa, Rh. evertsi evertsi, and Rh. sanguineus
(Scoles & Ueti, 2015) .
2.6.2 Iatrogenic or mechanical transmission
Equine piroplasmosis is a disease that can be transmitted accidentally
through the blood by contaminated syringes, hypodermic needles or instruments
used in any surgery or vaccination procedure (Iatrogenic transmission). It also
transmits by transfusion of infected blood or experimentally induced infection into
the susceptible equids via intramuscular, intravenous or subcutaneous inoculation
40
of infected blood (Nafie et al., 1986; Donnellan et al., 2003b; de Waal & van
Heerden, 2004).
Some researchers have raised the possibility of mechanical transmission
of T. equi and B. caballi by mosquitoes and flies, by observeing the spread of the
disease in various regions worldwide, according to the proliferation of these
vectors (Tutt, 1968; de Waal & van Heerden, 2004). In addition, blood sucking
(haematophagous) insects have been reported as a transmitter of T. equi and B.
caballi (Donnellan et al. 2003b; de Waal & van Heerden, 2004).
2.6.3 Intrauterine or transplacental transmission
Equine piroplasmosis can be transmitted intrauterine to the unborn fetus
during carrier (persistently) infected mares resulting in abortion due to the
potential infection of their fetus during pregnancy (de Waal, 1992; Lewis et al.,
1999). The infection of the fetus in uterus either causes abortion with observed
characteristic lesions of the EP or stillbirth, or the birth of the live foals with
neonatal piroplasmosis (Phipps & otter, 2004; Allsopp et al., 2007). This type of
transmission may occur due to placental damage or occurrence of reverse
erythroblastosis foetalis (Erbsloh, 1975). However, Allsopp et al. (2007) indicated
that the born foals can be infected throughout the pregnancy when the mare's
placenta is normal.
The vertical or transplacental transmission is a more common occurrence
in T. equi infection and suspected evidence in B. caballi infection (Georges et al.,
41
2011; Sudan et al., 2013). Transplacental transmission of T. equi has been
reported in different countries such as the United Kingdom (Phipps & Otter,
2004), the United States (Georges et al., 2011) and India (Chhabra & Ranjan,
2012).
2.7 The tick vectors for equine piroplasms infections
Since more than 3,500 years ago, both the Ancient Greeks and Ancient
Egyptians, already knew about the ticks and their medical importance (Varma,
1993). Among other arthropod groups the ticks vectors are very important in the
medical and veterinary aspects (Norval et al., 1992). It is considered a second
important vector after mosquitoes, worldwide, able to transmit mammalian
infectious disease agents (Dalgic et al., 2010). The importance of ticks is
highlighted by the fast development of molecular biological techniques, which can
easily examine blood samples and ticks for disease pathogens (Criado-Fornelio et
al., 2003; Monis et al., 2005; Ikadai et al., 2007). The increased knowledge of
animals’ ticks is due to some of them being able to transmit serious zoonotic
pathogens to humans such as Ehrlichia spp., Rickettsia conorii, Babesia spp. etc.
(Shaw et al., 2001; Beugnet, 2002).
Ticks belong to the family Ixodidae, called hard or Ixodid ticks (Acari:
Arachnida), which obligate blood sucking ectoparasites of various mammals,
vertebrates, birds, amphibians, and reptiles (Schmidt & Roberts, 1989). It
transmits a greater variety of pathogens such as bacteria, virus, spirochaetta,
42
protozoa and rickettsia to livestock, humans and their companion animals
(Jongejang & Uilenberg, 2004). Each female tick can be fed for several days and it takes 1 to 5 ml of the host's blood depending on their species and size
(Randolph, 2005). In addition, ticks feeding on domestic animals can cause direct deterioration and mechanical injury, excitement, inflammation and when found with heavy infestation or biting might cause anemia, decrease productivity, and cause poor health (Wall & Shearer, 2001). In some species of ticks, secretion of saliva causes paralysis and toxicoses to the host (Mans et al., 2004).
There are two types of pathogen transmission through tick vectors; mechanical when the mouthparts are contaminated, and biological when the pathogen can develop or proliferate itself in the tick vector. Various factors play important roles in the effectivity and activity of the tick vectors such as vectorial competence (measure of the overall efficiency of transmission), a great number of ticks, the behavior of tick and host, tick life span, fundamental physiological characteristics of the tick compared with the exterior incubation period of the pathogen, etc. (Scoles & Ueti, 2015). Tick vectors are more active through the warm season as long as there is adequate rainfall (Urquhart et al., 1996).
Ixodid tick species adapt to certain temperatures and humidity. It adapts to warm temperatures with moderate humidity, while other species adapt to low temperatures in the winter and can be effective in dry conditions. These changes in the temperature and humidity lead to changes in the dynamic life of ticks.
Doube and Kemp, (1979) found that the optimal temperature for adhesion and
º feeding of Boophilus microplus on the host ranged between 31 to 38 C, while the
43
relative humidity was between 70 to 80%. The larvae stage remains adherent on º the host during those circumstances and when the temperature goes up to 38 C and low humidity reaches 20 - 45%, the larvae drop to the ground before they complement feeding and many die within 24 hours.
There is an obligatory relationship between piroplasms and their Ixodid tick vectors (Gou et al., 2013). Out of the 867 tick species currently known
(Jongejang & Uilenberg, 2004), there are 33 hard ticks species belonging to six genera that transmit naturally or experimentally equine piroplasmosis (Table 2.2).
The stable cannot maintain either piroplasm in the absence of competent tick vectors as a tick-borne transporter, because the piroplasms require the ticks for completion of their life cycle. As a result, the universal distribution of EP is closely related to the spread of competent tick vectors (Schein, 1988).
Ixodid ticks are the final (definitive) hosts as well as transporters for T. equi and B. caballi, due to the former parasites which undergo sexual-stage development in the tick vectors to complete their life cycle (Zapf & Schein,
1994a, b).
44
Table 2.2: Hard tick species with the geographic location, number of the hosts and confirmed or suspected to transmit Theileria equi or Babesia caballi (Scoles & Ueti, 2015).
No. of Transmit Transmit Mode of Tick genus / species Geographic location or region hosts T. equi B. caballi transmission Amblyomma spp A. cajennense North America (Texas, US) 3 + – IS Dermacentor spp D. albipictus North America 1 – + TO D. marginatus Europe, North Africa, Middle 2-3 + + TS, IS, TO East, Southern Russia D. nitens North and South America 1 – + IS, TO D. niveus Russia 3 + + ? D. nuttalli Asia (Mongolia) 3 + + TS,TO D. pictus Eastern Europe 3 – + TS D. reticulatus Europe, Russia 3 + + TS,TO D. silvarum Russia 3 + + TS, TO D. variabilis United States 3 + + IS Haemaphysalis spp H. longicornis Japan 3 + + TS, TO Hyalomma H. aegyptium Russia, India 3 + + TS, ? North Africa, Mediterranean, H. anatolicum 2-3 + + TS, TO India Western Europe, North Africa H. detritum 2 + – IS and Eastern China H. dromedarii Europe 2-3 + + TS, TO H. excavatum North Africa 3 + + TS H. lusitanicum Mediterranean 3 + – TS H. marginatum North Africa, Mediterranean 3 + + IS, TO H. plumbeum Russia 3 – + TO Western Europe, North Africa H. scupense 1 + – TS and Eastern China Africa (Namibia), Asia, H. truncatum 3 – + TO Middle East North Africa, Mediterranean, H. turanicum 3 + + IS, TO Russia H. uralense Western Europe 1-2 + – TS H. volgense Eastern Russia 1-2 – + TO Ixodes I. ricinus Europe (Italy) 3 + – TS Rhipicephalus spp Rh. annulatus Italy, North Africa 1 + – IS, TS Rh. bursa Italy, North Africa 2-3 + + IS, TS Rh. evertsi evertsi Africa (southeastern) 2 + + TS Rh. microplus South America 1 + – TS, IS Rh. evertsi mimeticus Africa (southwestern) 2 + – TS Rh. pulchellus East Africa 3 + – TS, IS? Cosmopolitan, Middle East, Rh. sanguineus 3 + + TS Africa Rh. turanicus Southern Europe, Africa 3 + – TS?
IS) Intrastadial; TS) Transstadial; TO) Transovarian; ?) Indicates that the mode of transmission is suspected.
45
2.7.1 Taxonomy of Ixodid ticks
In general, the tick vectors include three families: Ixodidae, known as
hard ticks, comprising nearly the complete range of species of the ticka that are
veterinarlyy important. The next family is argasidae, known as soft ticks,
comprising a relatively few species that are significant in veterinary science. The
third family is Nuttalliellidae, compsising merely one little-known species which
is present in South Africa (Wall & Shearer, 2001; Horak et al., 2002). Ixodid tick
vectors of EP are classified as reported by Taylor et al. (2007) and Wikipedia,
(2010):
Kingdom: Animalia
Phylum: Arthropoda
Subphylum: Chelicerata
Class: Acarina
Subclass: Arachnida
Superorder: Parasitiformes
Order: Ixodida
Superfamily: Ixodidoidea
Family: Ixodidae (Hard tick)
46
The family Ixodidae (hard tick) comprises seven genera: Ixodes,
Amblyomma, Hyalomma, Boophilus, Dermacentor, Haemaphysalis and
Rhipicephalus, each genus has different species (Taylor et al., 2007).
2.7.2 Morphology of Ixodid ticks
Edward et al. (2009) described the internal structure of the Ixodid tick's
body (Fig. 2.5). It contains neuron nodules (synganglion) representing the brain in
ticks called primitive brain, which connects with eyes and a pair of salivary
glands, a grape-like structure extended along the sides of the tick's body and
nearby the gut. In the ticks the gut is a dark red structure because of the blood
absorption. It is spider-shaped, consisting of interior-gut, posterior-gut, mid-gut
extending along the mid-line and it is characterized by the presence of a jagged
edge.
The respiratory system of the tick consists of tracheae or bronchus
clustered at one point called respiratory opening, which is a pair present in the
lateral ventral surface, and malpighian tubules, which are clearly a thin tubule,
white-colored show (William, 2001). In addition, the male and female have
genital tract and muscular system, while some species have festoons which are a
chitinous structure on the posterior margin of the body (Mohamed et al., 2007;
Faisal, 2010).
47
Figure 2.5: The internal structure of the hard tick (40X) by Edward et al. (2009).
2.7.2.1 Tick family identification
The morphological characteristic of the Ixodidae family has been described
by Soulsby, (1982); Urquhart et al. (1996) and Richard, (1997). Ixodid ticks are
dorsoventrally flatted when unfed and relatively large ranging between 2 to 20 mm in
length when feeding and are structurally similar to that of mites. They are wingless
and without antennae. The body is oval and divided into two sections, the anterior
gnathosoma (capitulum), which bears the mouthparts and posterior idiosoma which
bears the legs (William, 2001; Roberts & Janovy, 2005).
The capitulum or pseudo head structurally consists of: a pair of four
segmented palps which are simple sensory organs that help to locate its host. A
pair of heavy sclerotized, two segmented appendages called chelicerae are placed
48
in cheliceral sheaths between the palps, which can move back and forth and its tooth-like digits are for cutting and piercing the skin of the host when feeding. A basis capituli is the expanded, fused coxae of the palps, which vary in shape in the different genera from rectangular or hexagonal or triangular or other shapes (Fig.
2.6A). A hypostome is the anterior and ventral extension of the lower wall of the basis capituli, which lies beneath the chelicerae, and which does not move , but in larvae, nymphs, adult females have rows of backwardly pointed ventral teeth for attaching securely to its host (Fig. 2.6B), (Wall & Shearer, 2001).
The immature stages of ticks are morphologically very identical to the adults, the larvae sometimes named seed ticks have three pairs of legs, but the adults have four pairs of legs (Fig. 2.6C and 2.6D). The posterior idiosoma which carries the legs is named podosoma and the region behind the legs is named opisthosoma. Attachment of the legs to the body is at coxa. Following the coxa are the trochanter, femur, genu, tibia, and tarsus (Fig. 2.6E). The coxa could be equipped with internal and external ventral spurs and their number, size and shape are dependent on tick species (Fig. 2.6F). On the first pairs of legs, the tarsus has a pit named Haller’s organm which is packed with chemoreceptor setae used for locating the host. The chemoreceptors are also present on the scutum, chelicerae and palps (Wall & Shearer, 2001).
49
F i g u r e
2 . 6 :
P a r t s o f a g e neralized 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
Hard ticks (Ixodid ticks) have a rigid, chitinous dorsal shield or plate on the posterior idiosoma named scutum which covers the whole dorsal surface of adult male, whereas in adult female, larvae, and nymphs, it extends to cover only small areas to facilitate the swelling of the abdomen after feeding. Some of the hard ticks have a pattern (gray and white on dark background) on the scutum which is an ornate tick, but if not is inornate tick. Female hard ticks are larger than males, which take relatively little blood feed and show little in size (Wall &
Shearer, 2001).
Ixodid ticks have no antennae, and when eyes are found, they are simple and consist of one pair located on the lateral margin of the scutum. There are a series of grooves on the scutum and body, their presence and the number are important in identifying tick species. Other distinct features are the presence of festoons which comprise a number of rectangular areas (a row of notches) on the posterior border of the body (Fig. 2.6C). Sometimes chitinous plates are also found on the ventral surface of male ticks (Wall & Shearer, 2001).
The genital opening is typically not obvious in the larva and nymph ticks, but it is present in the ventral mid-line; the juniper is a transverse slit positioned at the level of the second pair of legs in the adult ticks (sexual differentiation). The anus opening is present in the ventral surface posterior to the fourth pair of legs, which is found in all tick stages. A pair of genital grooves starts from the anus and extends backwards towards the anal groove. The respiratory opening (stigmata) is large and situated posterior to the case of the fourth pair of legs, and
51
shown in the adult and nymph, but lacking in larvae ticks (Fig. 2.6D) (Wall &
Shearer, 2001).
2.7.2.2 Tick genus identification
One of the distinguishing characteristics of the Ixodid tick at the genus level is based on the morphology of the capitulum parts (gnathosoma) (Fig. 2.7)
(Wall & Shearer, 2001).
Figure 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
2.7.2.3 Tick species identification
The morphological characterization of the Ixodid ticks at the species
level is carried out either using stereoscopic microscopes (Sajid et al., 2008) or by
electron microscope (Abdel-Shafy et al., 2013) for identification based on
taxonomies and structural difference of the species, according to taxonomic keys
(Keirans & Litwak, 1989; Petney & Keirans, 1995, 1996; Walker et al., 2003;
Estrada-Peňa et al., 2004). In addition, the molecular methods are used for
identification of the hard ticks species such as a quantitive real time PCR and
reverse line blotting methods (Kara et al., 1999; Gou et al., 2013; Rodríguez-
Bautist et al., 2014).
2.7.3 Tick life cycle
The complete life cycle commonly lasts less than one year, but some
Ixodid ticks like Ixodes ricinus may take more than two to three years for
completion of their whole life cycle (Gary & Lance, 2009), also it may take more
than six years (Anderson & Magnarelli, 1993). Once Ixodid ticks infest a suitable
host, they feed blood for 2-15 days to complete engorgement; this duration may
depend on several factors like tick species and stage, type of host, and attachment
site (Parola & Raoult, 2001).
The life cycle of Ixodid ticks consists of four phases: egg, larvae have
six-legs, nymphs have eight-legs and adulta have eight-legs, differentiated into
53
male or female (Fig. 2.8). During the passage through these phases, the hard ticks
feed many times interspersed by prolonged free-living and as little as 10% of their
life is spent on the hosts (Wall & Shearer, 2001).
Figure 2.8: The life cycle of Ixodid ticks (adapted from www.life cycle of ticks family Ixodidae png.).
Depending on the number of hosts (1 to 3 hosts) attached by the Ixodid ticks
through their life cycle; they are classified into three types:
4. One host tick; direct life cycle, in this type the tick development from larvae to
adult, takes place on one host such as Boophilus spp.
5. Two host tick; in this type the larvae and nymph are found on one host and the
adult on another such as Hyalomma spp and Rhipicephalus spp.
6. Three host tick:in this type each stage of tick is found on different hosts such as
Ixodes spp (Figure 2.9) (P. Jain & A. Jain, 2006).
54
Figure 2.9: The type of Ixodid ticks depending on the number of hosts attached (P. Jain & A. Jain, 2006).
2.7.4 Overview of the identified Ixodid ticks genera in this study
2.7.4.1 Genus Rhipicephalus Koch, 1844 (Soulsby, 1982)
Rhipicephalus means Rhipi: fan, cephalus: head. This genus includes 70
species, geographically distributed in Africa, Southern Europe, South America and
the Middle East. It is mainly named “dog tick” and infests a variety of mammals.
Morphologically, the species of this genus are usually inornate, have eyes and
festoons. The basic anterior gnathosoma is hexagonal and the mouthparts
55
are short. The coxa-1 has two strong spurs. Spiracles are comma-shaped in both sexes (long in the male and short in the female). In the male, on each side of the anus, two plates are found (P. Jain & A. Jain, 2006; Taylor et al., 2007). The common species of this genus are:
7. Rhipicephalus (Boophilus) microplus: previously classified as belonging to genus Boophilus; it is a one-host tick (Battsetseg et al., 2002; Ueti et al., 2008).
These species are rarely present on horses in the stable unless they are grazed with cattle (Teglas et al., 2005). It has been reported in the frequent outbreaks of the T. equi infection and transstadial or intrastadial transmission of T. equi (Kerber et al.,
2009).
8. Rhipicephalus (Boophilus) annulatus: previously classified as belonging to genus Boophilus (CFSPH, 2007). It is usually present on cattle and occasionally on horses, deer, sheep, goats and dogs, and also attached to humans (CFSPH,
2007; Lohmeyer et al., 2011). This species has been reported as a transmitter for both piroplasms. However, it has only been related to the epidemiological outbreaks with B. caballi (Laus et al., 2013). It is a one-host tick; all stages of this tick infest one host (Estrada-Pena et al., 2004). On the other hand, Scoles and Ueti
(2015) they considered this Ixodid tick as a suspected transmitter for both piroplasms.
9. Rhipicephalus bursa: known as “Anatolian brown tick” (Raele et al.,
2015). It is a major vector of tick-borne diseases in horses, cattle, sheep, dogs and birds. Classified as a three-host tick it is distributed in North Africa and Southern
Europe (Taylor et al., 2007). It is an important vector for both piroplasms: T. equi
56
by intrastadial transmitted and B. caballi by transstadial transmitted (Kouam et al., 2010a; Ros-Garcia et al., 2013; Abedi et al., 2014a).
4. Rhipicephalus sanguineus: known as “Brown dog tick or kennel tick”. It originated in East Africa, however, it is now considered to be the most widely spreading tick species in the world. This species is classified as a three-host tick, fundamentally found on dogs, also present on other mammals as well as birds
(Taylor et al., 2007). It is reported as a vector for T. equi and B. caballi by transstadial transmission of both (P. Jain & A. Jain, 2006; Kouam et al., 2010a).
7. Rhipicephalus appendiculatus: known as “brown ear tick”. This species can feed on cattle, sheep, goats, horses, deer, dogs, antelope and rodents, as well as birds. It is classified as a three-host tick. The mating occurs on the host.
Geographical distribution is in South Sahara, Africa and the areas without deserts or with significant rainfall. Tick salivary gland toxins and fluids causes toxoicosis and tick paralysis to the hosts; it can also be a vector of T. parva (east coast fever) in cattle, T. lawrencei, Hepatozoon canis, Ehrlichia bovis, Nairobi virus, Thogoto virus and Rickettsia conorii (Taylor et al., 2007). It is not reported as a vector for
T. equi and B. caballi yet, although, it can attach on horses (P. Jain & A. Jain,
2006).
57
2.7.4.2 Genus Haemaphysalis Koch, 1844 (Soulsby, 1982)
There are about 155 species distributed throughout the world, of which
44 species are reported in India. Other species inhabit humid, well-vegetated
habitats in tropical Africa and Eurasia. It is classified as a three-host tick with the
larvae and nymph infesting birds and small animals, while, the adults feed on
large animals such as horses, buffaloes, cattle, sheep, goats, etc.
This genus is morphologically characterized by small size, short
mouthpart with conical palps and rectangular basis capitulum. These ticks are
eyeless, and festoons and posterior anal groove are found. In the male the ventral
plates are absent and the spiracular plates are comma shaped or ovoid but in
female oval or rounded (P. Jain & A. Jain, 2006). The common species of this
genus are:
a) Haemaphysalis longicornis: this species usually affects horses, donkeys,
deers, cattle, sheep, goats and dogs, also infests humans and birds. It is a three-
host tick species, the larvae and nymph infests birds and small animals, while, the
adults feed on large animals. It is widely spread in Australasia and the Far East.
The tick’s bite causes tick worry and skin damage (Wall & Shearer, 2001; Taylor
et al., 2007). In Japan this species has been reported as a vector for both protozoa;
T. equi by transstadial transmission and B. caballi by transovarial transmission
(Rodríguez-Bautista et al., 2001).
b) Haemaphysalis punctata (H. cinnabarina punctata): this species can
infest horses, deer, cattle, sheep, goats, rabbit, bears, wolf, bats, birds, reptiles
58
(snakes and lizards) and rodents. It is a three-host tick, distributed in North Africa,
Central Asia, and Europe. It has been reported as a vector for Babesia bigemina,
B. major, Theileria mutans, Anaplasma centrale and A. marginale in cattle. It also
transmits Babesia motasi and Theileria ovis in sheep. This species causes tick
paralysis, transmitting Tribec virus, Crimean-Congo hemorrhagic fever virus and
Bhanja virus (Taylor et al., 2007). It is not reported as a vector for T. equi and B.
caballi yet, although, it can attach on horses (P. Jain & A. Jain, 2006).
2.7.4.3 Genus Dermacentor Koch, 1844 (Soulsby, 1982)
The species of this genus are medium to large size ticks. There are about
30 known species mostly distributed in the New World. Morphologically
characterized by ornate patterning, the mouthparts are short with the rectangular
shape of basic capituli and with eyes and festoons. The male has no ventral plates
and the fourth coxae are greatly enlarged with the first pair divided into two
sections in the male and female ticks.
Most members of this genus are classified as three-host ticks, while a few
are one-host ticks. Many species are directly associated with Q fever, Colorado
tick fever, tularaemia, and Rocky Mountain spotted fever. The salivary fluids of
some species cause tick paralysis (P. Jain & A. Jain, 2006; Taylor et al., 2007). In
addition, nine species have been reported as vectors for T. equi and B. caballi or
both, including Dermacentor albipictus (Duell et al., 2013), D. marginatus (Iori et
al., 2010), D. nitens (Kerber et al., 2009), D. niveus (Zasukhan, 1935), D.
59
nuttalli (Battsetseg et al., 2001), D. pictus (Knuth et al., 1917), D. reticulatus, D.
silvarum (Enigk, 1944), and D. variabilis (Stiller et al., 1980).
a) Dermacentor marginatus: known as “sheep tick”. It is a three-host tick.
The adult ticks feed on sheep, hare, deer, cattle, dogs, hedgehogs and humans. The
larva and nymph feedon, small mammals, birds, and insectivores. Geographically
it is widely-distributed in Spain, Morocco, Italy, Switzerland, Southern France,
Poland, Western Germany, and East to Central Asia. This species has been
reported as vectors of both protozoa: T. equi by transstadial transmission and B.
caballi by transstadial and transovarial transmission (Enigk, 1944; Iori et al.,
2010).
2.8 Ixodid ticks in Malaysia
Peninsular Malaysia has a tropical climate characterized by moderate
temperature, higher humidity and rainfall amount. These climatic factors have an
influence on the tick habitat (MMD, 2009). There have been many studies
conducted on the distribution of Ixodid ticks which infest rodents and wild
animals in different Malaysian states, while a few studies have been done on
Ixodid ticks infesting domestic animals (Table 2.3).
60
Table 2.3: Ixodid tick species distribution in different states of Malaysia.
Ixodid ticks Type of animals State of Malaysia Reference
Amblyomma spp. Boophilus autrallis Dermacentor spp. Domestic animals Kedah Lancaster, (1939) Haemaphysalis spp. Rhipicephalus spp. Ixodes granulatus Rats Borneo and Malaya Kohls, (I957)
Amblyomma testudinarium Domrow & Dermacentor auratus Wild animals Kedah Nadchatram, Haemaphysalis hystricis (1963) Ixodes granulatus Haemaphysalis spp. Rats Peninsular Malaysia Lim, (1972) Dermacentor spp. Dermacentor (Indocentor) Peninsular Malaysia Hoogstraal & Pigs compacts Wassef, (1984) Wild pigs, carnivores, Dermacentor (Indocentor) Hoogstraal & monkeys, deer, Peninsular Malaysia auratus Wassef, 1985 domestic chickens and other hosts Dermacentor (Indocentor) Wassef & Pigs Peninsular Malaysia steini Hoogstraal, (1988) Cattle Boophilus microplus Kedah and Kelantan Mahmood, (1997)
Ixodes granulatus small mammals as Mariana et al. Kelantan Dermacentor spp. well as Rats (2005) Amblyomma testudinarium Dermacentor spp. Birds and small Mariana et al. Haemaphysalis spp. Kedah mammals (2008) Ixodes granulatus Ixodes spp. Amblyomma spp. Dermacentor spp. Bats, rodents, shrew Mariana et al. Johor Haemaphysalis spp. and myriapods (2011) Ixodes granulatus Rhipicephalus (Boophilus) Cattle Selangor state Tay et al. (2014) microplus Selangor, Pahang and Che Lah et al. Ixodes granulatus Wild rodents Terengganu (2014) Ixodes spp. Wild rodents, Amblyomma spp. Selangor, Pahang, and Che Lah et al. Shrews and large Haemaphysalis spp. Terengganu (2015) mammals Dermacentor spp. Rhipicephalus sanguineous Stray dogs Peninsular Malaysia Koh et al. (2016)
Haemaphysalis wellingtoni Dogs, cats, Haemaphysalis hystricis Perak Khoo et al. (2016) chickens Haemaphysalis bispinosa.
61
2.9 Pathogenesis of equine piroplasmosis
Equine piroplasmosis has a severe and complicated pathogenic effect on
the tissue and organs of infected equids, including mechanical effect represented
by the destruction and the hemolysis of the red blood cells (RBCs) (hemolytic
anemia) or the release of vaso-active substances such as kinin and kallikrein,
followed by hypotension, shock and the death of affected equids (Donnellan et al.,
2003b; Hunfeld et al., 2008). Theileria equi is considered more pathogenic than B.
caballi. It can infect up to 80% of equids red blood cells (Mehlhorn & Schein, 1998).
The advancement of anemia in equids is a distinct clinical sign of T. equi
infections (de Waal & van Heerden, 2004). Three mechanisms for hemolytic anemia
include mechanical hemolysis by parasites intra erythrocyte binary fission, auto-
immunity of the anti-erythrocytic autoantibodies enhancing more
erythrophagocytosis (infected and uninfected erythrocytes) and the toxic effect of
producing the hemolytic factor by the parasites or bone marrow depression (de
Gopegui et al., 2007; Zygner et al., 2007; Zobba et al., 2008).
The severity of hypophosphatemia, hemoglobinemia, hypoferronamia,
thrombocytopenia and hyperbilirubinamia, which occurs in the infected equids with
EP, depends on the level of parasitemia (de Waal et al., 1988; de Waal & van
Heerden, 2004). Theileria equi and B. caballi are dependent on erythrocytes as an
energy source and the increased intake of phosphorus by parasites may be in charge
for progressive hypophosphatemia and fragility of infected erythrocytes, resulting in
erythrocyte destruction (de Waal & van Heerden, 2004).
62
There are many reasons for the destruction of RBCs as a result of infections including the increased intracellular pressure of infected cells during the multiplication of the protozoa, the toxic mechanism by hemolytic factor produced by the parasite and increased erythrocyte fragility due to protozoal consumption of the phosphorus component or disturbance in the accumulation of proteins and fats of cell wall, which lead to more destruction (de Gopegui et al.,
2007; Zobba et al., 2008). Moreover, in T. equi infected horses, the changes in the biochemical structure of RBCs membranes lead to the deformation of the cells and aggregation of oxidative ions, subsequently resulting in more erythrocytes destruction (Ambawat et al., 1999).
In the severely infected horses, the hemolysis or destruction of RBCs is followed by liberation of hemoglobin (hemoglobinemia), eventually anemic anoxia (hypoxia), which may cause death of the animal (Niven, 2002; Rothschild
& Knowles, 2007). The parasite will again be liberated into the blood stream to invade new RBCs, which lead to more destruction, causing the depression of the total erythrocyte count, hemoglobin concentration, and packed cell volume
(Alsaad et al., 2010). An increase in the level of hemoglobinemia may be followed by hemoglobinaemic nephrosis, hemoglobinuria, and the appearance of coffee-colored urine (de Waal et al., 1988; Ambawat et al., 1999).
Equine piroplasmosis leads to the systemic inflammatory response syndrome (SIRS) and hypercoagulability, following multi-organs dysfunction (i.e. hepatopathy, pancreatitis, etc.) (Donnellan & Marais, 2009). The clumping
(aggregation) of infected erythrocytes resulting in microthrobus formation might
63
block small blood vessels, leading to subsequent venous blood stasis and an acute hypotensive condition, which may cause the death of the animal (Schein, 1988; de
Waal & van Heerden, 2004).
Thrombocytopenia and prolonged clotting times have been reported in horses infected with EP (Alsaad & Al-Mola, 2006; Alsaad, 2009). It has been observed in 39.1%, 80%, and 100% of infected horses with T. equi, B. caballi and co-infection respectively (de Waal et al., 1988; Camacho et al., 2005). The reasons for the platelet decrease are immune mediated erythrocyte destruction, systemic and local disseminated intravascular coagulation (DIC), and splenic sequestration or excessive consumption of thrombocytes (Ambawat et al., 1999;
Boozer & Macintire, 2003).
Hyperbilirubinemia, hypoprotinemia, hypoferronamia and hypocalcemia have already been described by many researchers in natural EP infected equids
(Zobba et al., 2008; Takeet et al., 2009; Alsaad et al., 2010).
Equids infected with clinical form of EP may suffer from metabolic acidosis due to the accumulation of lactic acid, a decrease in blood PH and lactic acidemia (hyperlactemia), resulting in anaerobic metabolism (Vannier & Krause,
2009; Alsaad & Mussa, 2012).
Equids infected with T. equi and B. caballi experience imbalance between the oxidant processes (increase in malondialdehyde and nitrate) and antioxidant process (decrease in glutathione and vitamin E). These alterations may play an important role in the pathogenesis of EP (Deger et al., 2009).
64
Hanafusa et al. (1998) found that horses experimentally infected with B. caballi showed overproduction of cytokine products like tumour necrosis factor-alpha
(TNF-α), nitric oxide (NO) and other cytokines.
The appearance of colicky signs in an infected horse may occur due to hepatic insufficiency leading to deficit of bile salts secretion and resulting in digestive disturbances, either in the form of diarrhea or constipation, while frequent hemoglobinuria might cause glomerulonephrosis and renal damage as well as microthrombosis in intestinal capillaries (Hailat et al., 1997; Radostitis et al., 2008; Zobba, 2008; Alsaad, 2009).
The fetlock joint oedema was also observed in equids infected with EP.
It could be due to the differences between arterial hydrostatic pressure and venous osmotic pressure causing fluids to escape from vessels and accumulate in the distal parts of the body. Hypoprotienemia may also play a role in the dialysis and accumulation of oedematous fluids (Romerrio & Dyson, 1997; Alsaad et al.,
2010).
Dehydration appears in equids infected with EP. This may be due to the lack of body fluids, resulting in urea or oliguria, increased thirst, and rough hair coat (Alsaad et al., 2010).
Many complications occur due to EP including gastrointestinal stasis, catarrhal enteritis, colic, infertility in stallions, cardiac arrhythmias, pneumonia, lung edema, acute renal failure, laminitis, and disease of the central nervous system
(Bryant et al., 1969; de Waal, 1992; de Waal & van Heerden, 2004; Diana et al.,
2007). In addition, abortion or stillbirth occurs in carrier mares infected with
65
T. equi and B. caballi due to transplacental transmission of these protozoa to foals
(Lewis et al., 1999; Georges et al., 2011; Chhabra et al., 2012).
2.10 Clinical signs of equine piroplasmosis
Equine piroplasmosis varies from a subclinical form to severe and
potentially fatal illness, depending on several factors such as equids immunity,
virulence of the single or mixed infective pathogens, stage of the disease, animal
susceptibility to the infections, equids activities, the species of the pathogen, and
the environmental and ecological system (Kuttler, 1988; Brüning, 1996; Radostitis
et al., 2008).
The clinical manifestations of the disease are variable or non-specific and
are often confused with a variety of other illnesses such as trypanosomiasis,
equine granulocytic anaplasmosis, equine infectious anaemia, etc. (Rothschild &
Knowles, 2007; Radostitis et al., 2008). Infected horses with T. equi and B.
caballi have similar clinical signs. However, the signs associated with B. caballi
are milder or not so obvious (Rothschild & Knowles, 2007).
The incubation period of EP depends on whether the disease is an
experimental or natural infection (Zobba et al., 2008). In experimentally infected
equids, the incubation period ranges from 5 to 9 days relative to the method of
infection (Sippel et al., 1962). In naturally infected equids the incubation period
ranges from 8 to 10 days (Soulsby, 1982; Khalf et al., 1991). When blood is
transferred from infected to uninfected animals, the incubation period ranges from
66
9 to 10 days (Mahoney, 1977). The incubation period also depends on the virulence of the parasite and can range from 6 to 12 days (Hartwigk & Gerber,
1986). Another opinion suggests that the incubation period in equids naturally infected with T. equi ranges from 12 to 19 days, while, it ranges from 10 to 30 days when equids are naturally infected with B. caballi (de Waal, 1992; Friedhoff
& Soulé, 1996; CFSPH, 2008).
There are different clinical forms of EP: it may be peracute, acute, subacute and chronic or subclinical (Zobba et al., 2008). In a rarely peracute form the disease is manifested by sudden onset of signs, which lead to collapse and sudden death. The death mainly occurs due to multiple organ dysfunctions, which are related to systemic formation of DIC (Donnellan & Marais, 2009). Hemolytic anemia causes icteric or pale mucous membranes, tachycardia, tachypnea, weakness and pigmenturia (Ambawat et al., 1999; Diana et al., 2007).
More common, acute forms of disease are often characterized by fever exceeding 40°C, loss of appetite and weight, severe sweating, congested mucous
rd membranes with petechial hemorrhages in the 3 eyelid, incoordination, nervous signs, pale and/or icteric mucous membranes, peripheral edema, panting, depression, colic with signs of diarrhea and/or constipation, hemoglobinuria, muscle tremor, coughing, dehydration with a rough coat, and the presence of ticks on different body parts. Moreover, there is also increase in the respiratory rate, heart rate and capillary refilling time in the infected equids (Alsaad et al., 2010;
Ibrahim et al., 2011). Abortion or neonatal infections can occur in pregnant mares
(Allsopp et al., 2007).
67
In the subacute form of EP the clinical signs are characterized by intermittent fever, anorexia, weight loss, tachycardia and tachypnea, with variable degrees of icterus, hemoglobinuria, and constipation frequently followed by diarrhea and mild edema of the distal part of the limbs, which sometimes occur
(Friedhoff & Soulé, 1996).
In the chronic form of the disease the clinical signs are variable and nonspecific such as mild loss of appetite, poor performance, loss of body weight and enlarged spleen during the rectal examination (Irby, 2002; OIE, 2013).
Furthermore, subclinically (persistently) infected equids appear healthy with the absent of clinical signs and low parasitemia (Alsaad et al., 2012; Kappmeyer et al., 2012; Alanazi et al., 2014). These persistently infected animals can act as reservoirs and sources of the parasites to the ticks and horse population (Bahrami et al., 2014). The carrier animals show clinical signs when exposed to stress factors (OIE, 2013).
Other non-specific clinical signs may also be seen on horses suffering from EP, such as severe colic and constipation, followed by diarrhea and hemoglobinuria with passing of dark yellow to brown or coffee-like urine (de
Waal, 1992; Alsaad & Al-Mola., 2006; Zobba et al., 2008).
EP may also affect young, growing foals and the clinical manifestations similar to that in adults and in which the weakness, inability to stand and suck, fever, anemia, and icterus are more prominent signs (de Waal, 1992; Alsaad,
2009; Chhabra & Ranjan, 2012).
68
The nervous signs recorded include an acute form of EP manifested by
walking in a circle, myalgia, ataxia, and mild tonic-clonic spasms, and seizures
and paralysis of the hind limbs (Soulsby, 1982; Radostitis et al., 2008)
.
2.11 Clinical pathology of equine piroplasmosis
2.11.1 Changes in hematological parameters
Hematological finding varies according to the virulence of the parasitic
species, the host health status, the presence of concurrent infections, and previous
exposure to the infection (Boozer & Macintire, 2003). The direct effect of the
parasites in the infected erythrocytes may be incriminating or decrease the life
span of RBCs, suppression of hemopoitic system and also lead to decrease the
number of RBCs resulting in anemia (Murase et al., 1996; Sellon, 1997). The
severity of anemia varies according to the type of piroplasm infections. The most
severe anemia is associated with T. equi infection resulting in severe hemolytic
anemia due to intra-erythrocytic binary fission of trophozoites or secondary
immune mediated hemolytic anemia or oxidative damage to the erythrocytes
(Murase et al., 1996).
The hematological changes in the equids infected with EP include
decrease in red blood cell count (RBCs), packed cell volume (PCV) and
hemoglobin concentration (Hb) (Alsaad & Al-Mola 2006; Zobba et al., 2008;
Alsaad et al., 2010). The percentage of erythrocytes hemolysis may depend on the
69
virulence of the parasites. The highest level of hemolysis occurs within 10-15 days after the observation of the parasite in the blood smears (Sellon, 1997).
Therefore, the time in which anemia starts and reaches the maximum stage is different depending on the degree of parasitemia, erythrocytes hemolysis and the activity of reticulo-endothelial phagocytes in removing infected erythrocytes from the blood stream (Mehlhorn & Schein, 1984; Hailat et al., 1997).
In acute EP infection, severe anemia occurs with the PCV as low as 10% and disturbances in values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)
(de Waal et al., 1988; Ambawat et al., 1999; Rothschild & Knowles, 2007).
Different types of anemia are recognized in EP infection, which mostly depends on the virulence of the parasite and the stage of the disease (Alsaad,
2009; Ibrahim et al., 2011). The normocytic normochromic type of the anemia was observed by Hailat et al. (1997); Alsaad and Al-Mola, (2006) and Alsaad et al. (2010). Nevertheless, macrocytic hypochromic type of anemia was reported by some studies (Nafie et al., 1986; Ibrahim et al., 2011; Alsaad & Mussa, 2012). In
T. equi infection, microcytic hypochromic type of anemia was observed by Khlaf,
(1988) and Guimaraes et al. (1998). In neonatal foals infected with T. equi, the macrocytic normochromic type of anemia has been observed (Chhabra & Ranjan,
2012).
The appearance of reticulocytes in the blood stream during EP infection indicate regenerative anemia and the activity of bone marrow in producing immature erythrocytes to compensate for the destroyed erythrocytes (Schalm et
70
al., 2003). These reticulocytes are observed within 10 to 15 days of infection, and the highest level recorded has been within 23 to 25 days of the infection (Maxis,
2011).
In horses infected with both T. equi and B. caballi, an increase in the erythrocyte sedimentation rate value occurs (Alsaad, 2009; Alsaad et al., 2010).
There is a correlation between the ESR value and intensity of anemia due to infection. The increase in the sedimentation of RBCs will occur when anemia is severe (Jain, 1993), while Allen, (1988) noticed that ESR may increase when PCV values decrease. Bush, (1975) mentioned that increased Rolex phenomenon in a horse's blood may lead to increased ESR values during piroplasmosis infection.
Different changes in clotting factor indices have been observed during
EP infection such as thrombocytopenia, hypofibrinogemia, increase in clotting time, and prothrombin time of the blood, which reflects petechial hemorrhages may be detected on the mucus membranes of infected horses (Alsaad & Al-Mola,
2006; Zobba et al., 2008; Alsaad et al., 2010). In addition, there is also depression of Proaccelerin )Factor V), Proconvertin (factor VII), Stuart factor power (Factor
X) and hypovitaminosis K (Ali et al., 1996).
The thrombocytopenia was observed during 7 to 10 days of the infection and reported in 30.1% and 80% of horses infected with T equi and B. caballi respectively (Zobba et al., 2008). Moreover, Collatos, (1997) found that EP has an adverse effect on blood clotting indices, which may disturb the hemostatic mechanism, resulting in DIC which may affect some vital organs such as brain,
71
lung, intestine, and kidney causing ischemic necrosis of tissue and may be considered as primary or secondary causes of death in infected animals.
The leukocyte count was varied depending on the disease stage and the severity of infections (Rudolph et al., 1975). In early EP infection, depression of leukocyte count (leukopenia) occurs, followed by a sharp increase in leukocyte count (leukocytosis) during the progression of the disease, which is accompanied by increase in the lymphocytes (Alsaad & Al-Mola, 2006; Zobba et al., 2008).
The Leukogram in an experimental study of EP observed a reduction in the total number of WBCs on the fourth day of the disease, even before seeing the parasite in blood smears; this decline is due to decrease in the lymphocytes numbers as a result of the multiplication of the parasite inside it at the beginning of infection and destroying it, and subsequent WBCs begin to increase after the sixth day of the infection to reach the highest level the tenth day so as to increase neutrophils and monocytes, which may indicate the role of these cells in the phagocytosis process (Khalf et al., 1991). On the other hand, Hailat et al. (1997) and Jain (2000) mentioned that the increase in white blood cells (WBCs) is due to the stimulation of lymphoid tissues and stem cells in the bone marrow by the parasites and their toxins. The significant increase in total WBCs is due to neutrophilia and lymphocytes in the infected equids (Alsaad et al., 2010; Ibrahim et al., 2011; Javed et al., 2014).
In neonatal foals suffering from T. equi infection is shown leukopenia with an increase in the lymphocytes (lymphocytosis) and decrease in the
72
neutrophil (neutropenia) with mild left shift degenerative (Chhabra & Ranjan,
2012).
The differential leukocytes count in the equids infected with EP, was
lymphocytosis (Alsaad, 2009; Rashid et al., 2009), monocytosis, and decrease in
basophils during acute infection with T. equi (Purnell, 1981) and neutrophilia in
splenectomized donkeys infected with piroplasmosis (Selim & Abdel-gawaad,
1982). On the other hand, Alsaad et al. (2010) found no significant difference in
the number of monocytes, eosinophils and basophils and all values within the
normal range.
2.11.2 Changes in biochemical parameters
The evaluation of the biochemical changes is an important method used
to assist in the diagnosis of some diseases and to give an accurate prognosis
(Ricketts, 1981). A different biochemical test is done on EP to evaluate the main
effect of the disease on different body tissues such as aspartate amino transferase
(AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), blood urea
nitrogen (BUN), total bilirubin, total protein, albumins, globulins, calcium,
phosphorous, glucose, and creatinine.
73
2.11.2.1 Aspartate aminotransferase (AST)
An aspartate aminotransferase is normally found in red blood cells, liver,
heart, muscle tissue, pancreas, and kidneys. Formerly it was called serum glutamic
oxaloacetic transaminase (SGOT) (Mullen et al., 1979). The AST test measures
the amount of this enzyme in the serum. It is raised in an acute liver damage when
hepatocytes are damaged, myocardial infarction, and muscular dystrophy
(Kramer, 1980; Jensen, 2008). The AST level is increased during EP infection
(Zobba et al., 2008; Alsaad et al., 2010). The high level of AST occurs during
piroplasmosis due to the indirect effect of parasites on the hepatic tissues, the
excessive destruction of erythrocytes and nephrosis when the renal tissue becomes
affected (Salem et al., 1986; Hailat et al., 1997; Pitel et al., 2010).
2.11.2.2 Alanine aminotransferase (ALT)
An alanine aminotransferase is present fundamentally in the liver, and
also in minimal amounts in the muscles, heart, pancreas, and kidneys. ALT
previously was named serum glutamic pyruvic transaminase (SGPT) (Doxey,
2006). ALT test measures the quantity of this enzyme in the serum. Low levels of
ALT are normally present in the blood, while when the liver is diseased, it is
liberated into the blood stream (Meyer & Harvey, 1998). The ALT level has been
increased during severely affected equids with EP, as well as in vaccinated horses
74
against babesiosis (Salem et al., 1986; Zobba et al., 2008; Alsaad et al., 2010;
Pitel et al., 2010).
2.11.2.3 Alkaline phosphatase (ALP)
Alkaline phosphatase is an enzyme synthesis in the bone, liver and
placenta and normally found in high amounts in growing bone and bile (Smith,
1996). It is liberated into the blood during bone or liver or placenta damage and in
normal activities as bone growth (Coles, 1986). The abnormally high levels of
ALP may indicate damage in bone or liver, bile duct obstruction, degeneration
and coaggulative necrosis that occur in the central lobules of the liver, or certain
malignancies. The enzyme is often elevated in the leukemic cells (Jensen, 2008).
High level of the enzyme occurs during acute and chronic forms of EP (Wright,
1981; Hailat et al., 1997; Alsaad et al., 2010).
2.11.2.4 Total bilirubin
Total bilirubin was formerly named hematoidin. It is the yellow
breakdown product of normal heme catabolism (Meyer & Harvey, 1998). Heme is
found in hemoglobin, a principal component of red blood cells and bilirubin is
excreted in bile. It is responsible for the yellowish discoloration in jaundice
(Jensen, 2008). Two types of bilirubin: unconjugated bilirubin (insoluble in water)
are present in the RBCs and macrophages (Meyer & Harvey, 1998). The
75
conjugated bilirubin (soluble in water) is present in the liver as glucuronic acid
(Ziemer et al., 1987). The equids infected with EP show an increase of the total
bilirubin especially when the excessive destruction of erythrocytes occurs (Selim
& Abdel-gawaad, 1982; Hailat et al., 1997; Zobba et al., 2008).
2.11.2.5 Total protein
The protein in the plasma consists of albumin and globulin. The globulin
consists of α1, α2, β, and γ globulins (Jensen, 2008). The depression of total
serum protein (Hypoprotenemia) may occur during malnutrition, malabsorption,
and renal diseases and fever (Taira et al., 1992). A significant decrease in the total
protein (hypoprotenemia), albumin and globulin is recorded in horses infected
with piroplasms species (Hailat et al., 1997; Zobba et al., 2008; Takeet et al.,
2009; Alsaad et al., 2010).
2.11.2.6 Blood urea nitrogen (BUN)
The urea constitutes about 50% of non-protein nitrogen, which is a
product of the demolition of protein in the body, which is mainly removed from
the blood by the kidneys. The BUN test is a measure of nitrogen amount in the
blood (Ziemer et al., 1987). A greatly elevated BUN may indicate the moderate or
severe degree of renal failure, azotemia and the impaired renal excretion of urea
may be due to temporary conditions such as dehydration or shock, or acute and
76
chronic disease of the kidneys (Meyer & Harvey, 1998). The increase of BUN
amount occurs during acute infection with T. equi and B. caballi of equids (Niven,
2002; Alsaad & Al-Mola, 2006; Alsaad, 2009).
2.11.2.7 Calcium
The calcium in the body is present in two forms, the ionized (active
form) and the nonionized (non-active form) (Merey & Harvey, 1998). It is
essential for living organisms, particularly in cell physiology, where movement of
2+ the calcium ion Ca into and out of the cytoplasm functions as a signal for many
cellular processes. It is considered as a major material used in mineralization of
bones, contractions of muscles and clotting processes (Doxey., 2006). The lower
level of calcium (hypocalcemia) has been seen in malabsorption, starvation, some
metabolic disorders, deficiency of vitamin D and hypomagnesimic tetanies
(Radostitis et al., 2008). Hypocalcemia has been recorded in the equids suffering
from EP and the animals shows slight clonic convulsions, incoordination, muscles
tremors and recumbencey (Wright, 1981; Nel et al., 2004; Alsaad et al., 2010;
Ibrahim et al., 2011).
77
2.11.2.8 Glucose
Glucose is used as a source of energy and metabolic intermediate. It is
critical in the production of proteins and lipid metabolism (Doxey, 2006). The
liver can produce the glucose by gluconeogenesis process. Hypoglycemia occurs
due to normal hepatic glucose output with increased peripheral uptakes or a
decrease in hepatic glycogens combined with normal peripheral utilization of
glucose or a combination of both mechanisms (Coles, 1986; Jensen.,2008).
Hypoglycemia is observed in severe complicated EP cases (Nel et al., 2004;
Alsaad et al., 2010). Whereas, Ibrahim et al. (2011) found an increase in glucose
level in horses infected with T. equi.
2.11.2.9 Phosphorus
Phosphorus is an inorganic phosphate, very important in many
metabolic pathways, especially when high energy compounds are involved like
calcium; it is a major component of bone (Kaneko et al., 2008). Phosphate plasma
concentrations are controlled by vitamin D and parathyroid hormone and the
increase in the level (hyperphosphataemia) is commonly seen in chronic renal
failure (Radostitis et al., 2008). In horses, the increase of phosphate concentration
may be due to recent stress or excitement (Kerr, 2002). Hypophosphatemia is
common in equids infected with T. equi and B. caballi, due to alteriation of RBCs
78
metabolism (Zobba et al., 2008). Ibrahim et al. (2011) found hyperphosphataemia
in horses infected with T. equi.
2.11.2.10 Creatinine
Creatinine is a nitrogenous waste product of the kidneys, it is a product
of the breakdown of creatine (a substance present in the muscle) (Kaneko et al.,
2008). The changes in plasma creatinine concentration are due to changes in the
creatinine excretion, i.e., they reflect renal function. It is used to investigate
kidney disease. The decreases in the plasma creatinine level are not clinically
significant, while, its increament are indicators of dehydration, heart failure,
bladder rupture, urethral obstruction, and acute or chronic renal failure (Kerr,
2002). Furthermore, no significant change in creatinine level is observed in equids
infected with T. equi (Takeet et al., 2009; Ibrahim et al., 2011).
2.11.3 Pathological changes of equine piroplasmosis
The pathological changes associated with EP are similar to those
occurring in other diseases as a result of anemia (Howe, 1973). The hemopoietic
tissues like liver and spleen are more exposed to pathological changes resulting
from the infections (T. equi and B. caballi). The severity of the lesions in different
tissues depends on the type of parasites (T. equi is more pathogenic than B.
caballi), the parasitemia level, the degree of anemia, stage of the disease, age of
79
the infected animal, and presence of the disease complications (de Waal, 1992;
Rooney & Robertson, 1996).
2.11.3.1 Macroscopic findings
The macroscopic examination, at necropsy of equids infected with EP
showed general body weakness, varying degrees of anemia, jaundice, thin blood
and watery, cardiac hemorrhage, hydropericardium, lung oedema and congestion,
hydrothorax, splenomegaly, hepatomegaly, pale or dark orange-brown, ascites and
enlargement of the lymph nodes (lymphadenopathy). In addition, the kidneys
might be flabby and pale or dark red with petechial hemorrhages and the urinary
bladder is distended, full of dark color urine (coffee like). The secondary
infections lead to oedema, pulmonary emphysema, signs of pneumonia, catarrhal
enteritis and the brain is soft, oedematous and slightly congested (Alsaad & Al-
Mola, 2006; Radostitis et al., 2008; OIE, 2013).
2.11.3.2 Microscopic findings of EP
Histopathological examination of the tissues sections prepared from the
postmortem cases infected with EP noted congestion, oedema and many
hemosiderin-containing macrophages in the pulmonary alveolar walls and
hemosiderinosis could be seen in the spleen and liver due to erythrolysis and
micro thrombi within the lungs and liver. The hepatic tissue sections also
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demonstrated hypertrophy of hepatic cells, congestion of lobular vien with
activation of kupffer cells and necrosis in the centrilobular with bile stasis, while
in the kidney there was fatty degeneration, hydropic and necrosis of the renal
tubular epithelium and hemoglobin casts and protein in many tubules of the renal
medulla and cortex. Furthermore, there is an increase of the reticuloendothelial
cell system in the lungs, liver, lymph nodes and kidneys (de Waal, 1992; de Waal
& van Heerden, 2004).
The macroschizontes and microschizontes (Koch’s blue bodies) are seen
within the cytoplasms of the lymphocytes and macrophages in the lymph nodes
sections. In intestinal tissue sections there is extravasation of erythrocytes in the
mucosa, submucosa and subserosa with mononuclear inflammatory cell
infiltration at mucosal villi indicating catarrhal enteritis (Mahoney et al., 1977;
Alsaad & Al-Mola, 2006; Taylor et al., 2007).
2.12 Immunity to equine piroplasmosis
Equids are able to develop immunity against piroplasms species, either
after an occurrence of disease and recovery or after preventive immunization. The
immunity of EP infections is not thoroughly defined; it appears to be multifaceted
and complex (Wise et al., 2013).
The immune response is incapable of clearing T. equi. The infected
equids are documented to bear infection for life, while the infections with B.
caballi are self-curing, lasting more than four years after the onset of the disease
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(de Waal, 1992; Zweygarth et al., 2002). These carrier equids act as a parasite reservoir for ticks and play a significant role in the epidemiology of EP (de Waal
& van Heerden, 2004).
In general, horses born and originated in endemic regions are more resistant to the disease than those from EP-free areas. Those horses are protected by premunition immunity, which depends on the parasite presence in the body and lasts eight months to one year, but if the infected tick should bite the pre- immune animal during this period, a further pre-immunity period is conferred and the equids becomes a lifelong pre-immune carrier (Maslin et al., 2004).
Foals in endemic regions have reported premunity to EP for the first one year of life (Retief, 1964), probably due to resistance by maternal antibodies from colostrum taken. The protection is for five or six months of age (Phipps, 1996; de
Waal & van Heerden, 2004). Non-specific factors protect foals through the first six to nine months of age (Waal & van Heerden, 2004). If infected ticks infest foals during this immunity period, it usually produces persistent diseases with latent infections leading to a constant immunity (Phipps, 1996). Gummow et al.
(1996) reported that colts have higher antibodies against B. caballi than fillies.
Both humoral and cellular factors are implicated in the immunity against piroplasms (Homer et al., 2000). There is no attested cross-immunity between T. equi and B. caballi infections, but co-infections with both parasites may occur
(Donnelly et al., 1980b; Donnellan et al., 2003b; Waal & van Heerden, 2004).
The control of parasitemia is correlated with the antibody production orimmune responses (Cunha et al., 2006).
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Equids infected with T. equi produce antibodies against merozoites antigen named equi merozoites antigens (EMAs), while in B. caballi infection the antibodies are produced against rhoptry associated protein-1 (RAP-1) (Knowles et al., 1994; Yokoyama et al., 2006). In the acute form of T. equi disease, a high level of IgGa (now IgG1) and IgGb (now IgG4 and IgG7) is related to the control of the infection, while in chronic infection there are high IgG (T) levels (now
IgG5 and IgG3) (Cunha et al., 2006). These antibody levels rise to an early peak and thereafter decline during the chronic stage of infection, these antibodies remain at low level all the time and the animal is still a carrier, and the infection can be detected in the first 7 to 11 days via experimental inoculation infection into ponies and reach a peak 30-45 days after inoculation (Potgieter et al., 1992).
The spleen plays an important role in immunity against piroplasms. A horse with intact spleen is typically competent to control the acute form of T. equi-induced infection, but in splenectomized horses probably the disease occurs with parasitemia reaching 80% (Kuttler et al., 1986; Ambawat et al., 1999).
Experimental inoculation of B. caballi into splenectomized horses, may or may not induce the occurrence of acute clinical disease, however, death due to infection has been reported (de Waal et al., 1988). This contradiction may be due to differences in piroplasms strain, the overall health condition of the horse, infective dose or all these factors.
Although, the function of cell-mediated immunity is important in the control of other protozoal infections such as Babesia bovis, but in piroplasmosis infection, it is still not fully specified (Banerjee et al., 1977; Kumar et al., 2002b).
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In experimental B. caballi infection, producing nitric oxide (NO) by macrophages may play an important role in immune control of disease (Hanafusa et al., 1998). Importantly, innate immunity is responsible for preventing the development of the parasite as well as the parasite expanding with higher parasitemia which happens in a shorter time in the absence of the macrophages and Natural killer cells (NK cells). The inhibition of the piroplasms is most likely accomplished by the production of many factors like gamma interferon (IFN-γ) by NK cells and tumor necrosis factor alpha (TNF-α), as well as nitric oxide by macrophages (Homer et al., 2000).
The presence of innate immunity and spleen are inadequate to protect against T. equi infection, perhaps with the spleen intact with severe combined immunodeficiency (SCID) and incapable of producing functional T and B lymphocytes, mounting an antigen specific antibody and T. equi parasitemia controlling (Knowles et al., 1994). An immune-mediated hemolytic anemia is one of the most common manifestations of piroplasmosis in animals (Farwell et al.,
1982); the exact mechanism by which Babesia spp. induces immune mediated hemolytic anemia is unknown, and the most commonly accepted theory is that the immune mediated hemolytic anemia is due to soluble parasite antigens binding to the red blood cell surface either through antibody or complement mediated hemolysis or complex autoimmunity (Homer et al., 2000).
Rarely, in apparent T. equi carriers can relapses of the disease related to strenuous exercise, exhibit immunosuppression, steroid therapy and stress (Hailat et al., 1997).
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2.13 Public health significance of equine piroplasmosis
In 1957, the first diagnosis of human babesiosis was reported in
Yugoslavia, and the infecting piroplasms were presumed to be Babesia divergens
(Skrababl & Deanovic, 1957). Some people have been infected with B. bovis and
B. divergens which are reported in cattle, and these peoples die due to plural form
illness (Adam et al., 1971). Babesia microti and Babesia divergence are
confirmed to infect humans as cases have been reported in Europe (Homer et al.,
2000; Krause, 2002; Gelfand & Callahan, 2003).
People usually obtain piroplasms infection through transfusion of infected
blood or by tick vectors (CFSPH, 2008). Other Babesia species have been
detected in humans, such as B. duncani, B. divergence-like organisms and B.
venatorum can also be shown (Vannier & Krause, 2009).
Human infection with the EP is not well documented and very rare.
However, cases of T. equi and B. caballi infection have been reported in human
beings in North and South America (Ash & Orinel, 1990). Human piroplasmosis
(Babesiosis) is manifested by fever, anemia, headache, fatigue, hemoglobinuria,
jaundice, and nervous signs, while, the complications of the disease are
characterized by DIC, congestive heart failure (CHF), kidney failure, dyspnea,
and death. Mixed infection with piroplasms causes increase in the seriousness of
diseases like Lyme disease. The antibiotics such as clindamycin and atovaquone
or azithromycin can be used for treating human piroplasmosis (CFSPH, 2008).
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Recently, T. equi and B. caballi infections were detected in camels
(Qablan et al., 2012a), and dogs (Qablan et al., 2012b; Gabrielli et al., 2015).
Theleria equi infection has also been detected in cattle, sheep and goats (Zhang et
al., 2015). In contrast, these parasites cannot infect pigs and other experimental
laboratory animals (Soulsby, 1982).
2.14 Diagnosis of equine piroplasmosis
The diagnosis of EP can be based on demonstration of clinical signs on
the infected equids. Although the clinical signs are often variable or non-specific,
the disease is being confused with a variety of other illnesses. It is not possible to
differentiate between T. equi and B. caballi infections based on clinical signs
alone (de Waal & van Heerden, 2004; Rothschild & Knowles, 2007). Therefore,
different laboratory techniques have been developed for the diagnosis of EP,
including the direct observation of parasites in blood smears, serological tests,
molecular techniques (Table 2.4), biological tests and in vitro culture method
(Sumbria & Singla, 2015).
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2.14.1 Overview of the methods used for diagnosis of EP in this study
2.14.1.1 Microscopic examination of stained blood smears
Microscopic examination of stained blood smears is a simple and rapid
traditionally diagnostic method of EP. It is generally the method of choice for the
clinically infected equids that need instant veterinary interference (Kouam et al.,
2010a). The direct observation of the parasites in the staining blood smears is
based on their morphometric features, the biometric data and the technique for
preparing blood film (OIE, 2014b). There are two types of blood smear
obtainable: thin and thick blood smears. The first is the method of choice for the
identification of the parasite species, the second is more efficient for detection of
the parasite in cases where the parasitemias are low (de Waal & van Heerden,
2004). T. equi and B. caballi are better observed in thin or thick blood smears
stained with a Giemsa solution 10% using a light microscope (Nagore et al.,
2004).
In chronic infection of B. caballi it is virtually unfeasible to observe the
parasite in blood smear, while in chronic T. equi infection sometimes can
successfully observe the parasite. Generally, apparent or chronic infections can
only be assured after transfusion of about 500 ml of blood in susceptible equids
(Ali et al., 1996). The precision of microscopic identification of parasites relies on
the experience and skills of the laboratory technician in inspecting the blood
smears. Furthermore, it is very difficult to observe the blood protozoa from a
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persistent infected animal (carrier state) due to low parasitemia (<0.01-0.001%, 4 5 means one infected erythrocyte out of 10 -10 erythrocytes). The persistent condition in EP usually presents a particular problem of diagnosis because no clinical signs appear on infected animals (Knowles, 1988).
Diagnosis of T. equi and B. caballi principally depends on traditional
® Romanowsky stains (Giemsa’s, Wright’s, Leishmann’s, or Diff-Quik stains).
The disadvantages of these stains are missing the samples with low parasitemias and occasionally being confused with the staining debris. Staining of blood smears with Acridine Orange stain is a more rapid method for identification of the parasites, because it is a fluorochrome stain and gives a green fluorescence of deoxy ribonucleic acid (DNA) and red fluorescence of ribonucleic acid (RNA).
Therefore, this method is considered more sensitive than Romanowsky staining
7 due to the detection of one parasite per 10 RBCs and it stains the DNA of the organisms which prevent the confusion with the staining particles demonstrated in traditional staining (Sumbria & Singla, 2015). The microscopic examination of staining blood film in prepared horses infected with EP showed that the parasitaemia seldom exceeds 1% in the horses infected with B. caballi, whereas, in the horses infected with T. equi the parasitemia commonly ranges from 1% to
7% and may reach 80% in some horses (Friedhoff et al., 1990).
In clinically infected equids, the preparation of smears from the peripheral blood of the animal ear vein is observed to have a higher number of parasites (Soulsby, 1982). Additionally, the preparation of smears from blood
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sample collected via jugular venepuncture for detection of piroplasms is
frequently reported (Servinc et al., 2008).
In spite of the fact that microscopic method is low sensitivity, it continues
to be used, but should be supplemented in the case of regular screening by other
more accurate and sensitive tests like serological tests and molecular techniques.
2.14.1.2 Competitive enzyme linked immunosorbent assay
It seems to be unsatisfactory to diagnose an organism in a carrier animal
by only the microscopic examination of blood smears and it is not practical on a
large scale. Therefore, serological tests are recommended as a preferred method of
diagnosis, especially when horses are destined to be imported into countries that
are free of the disease, while the tick vectors are present (Huang et al., 2004). The
serological diagnosis of the disease is based on the detection of the circulating
antibodies against the current and previous infectious agents.
A cELISA was developed in 1991 to detect T. equi using T. equi equine
merozoites antigen-1 (EMA-1) and specific monoclonal antibodies (MAb) that
define the merozoites surface protein epitope (Knowles et al., 1991). Later on, this
cELISA was improved by using recombinant protein rather than the culture of
whole parasites (Knowles et al., 1992). In 1999, the recombinant rhoptry
associated protein -1 (RAP-1) of B. caballi antigen was used in a cELISA
(Kappmeyer et al., 1999). The recombinant proteins help the standardization of
the diagnostic assay and if it is used there is no need for culturing of the parasite
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or the experimental infection of equids for the production of antigen, thus making cELISA a perfect screening test for piroplasmosis infection (Rothschild &
Knowles, 2007). Further, Brüning, (1996) found that cELISA could detect the chronic infection of an EP which is not detected by the CFT.
In 2004, the OIE approved the cELISA for detection of both T. equi and
B. caballi, and as a specified test for international horse movement (OIE, 2013).
Subsequently, development of two kits by veterinary medical research and development (VMRD) manufactured one test kit for detection of T. equi antibodies, and a second kit for B. caballi antibodies. These VMRD kits were licensed by the Center for Veterinary Biologics (CVB) (USDA, 2010).
The cELISA is prepared by coating plates with known amount of antigen and it needs a small amount of recombinant protein and monoclonal antibody in contrast to IFAT. The result is recorded automatically, permitting the testing of a large number of samples at the same time (OIE, 2013).
The cELISA is highly valued (Shkap et al., 1998), and is supported and recommended by the OIE (Goff et al., 2006; OIE, 2013). The test has been confirmed to be better than CFT in the detection of the previous infection with T. equi and B. caballi. Furthermore, it has been demonstrated to have a higher specificity for these parasites compared with indirect ELISA (Shkap et al., 1998;
Kappmeyer et al., 1999). The sensitivity and specificity of the cELISA to detect
T. equi are 96%, 95% respectively, compared with CFT which with 47% and 94% respectively, while for detecting B. caballi the sensitivity of the cELISA is 91%
90
and of the CFT is 88%; the specificity of the cELISA and CFT is 98% and 70%
respectively (Knowles et al., 1991; Kappmeyer et al., 1999; Katz et al., 2000).
The cELISA has been an alternative method of detection of acute,
chronic and latent infection of T. equi and B. caballi (Salim et al., 2008 and
Servinc et al., 2008). This assay is reported to be an effective test in detection of
piroplasms species in many countries such as Malaysia (Zawida et al., 2010),
Greece (Kouam et al., 2010a), Iraq (Alsaad et al., 2012), Spain (Garcia-
Bocanegra et al., 2013), and Venezuela (Rosales et al., 2013), among others.
2.14.1.3 Conventional and multiplex polymerase chain reaction
Traditional methods, including microscopy and serology, do not
consistently meet the demand, as low parasitaemia cannot be shown by
microscopic examination of blood smears. In addition, utilizing the serological
tests has some impediments since the observation of the antibodies against the
parasite gives little information on whether the parasite is still present in the
animal’s body or not and if the parasite is in essential infection, the disease will be
created before the antibodies are distinguishable. Therefore, the need to be able to
reveal the parasites immediately has led to the development of highly specific and
sensitive techniques based on the demonstration of parasites DNA.
In 1985, PCR was used to detect and identify the pathogenic
microorganisms in water, sediments and soil (Montandon, 1994). Earlier, using
DNA probes for detection of T. equi showed a higher number of carrier animals
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infected with EP (Posnett & Ambrosio, 1989). These probes were demonstrated to be specific and did not hybridize to DNA from horses infected with B. caballi.
Furthermore, they found it to be equal in sensitivity to the microscopic examination of thin blood smears (Posnett et al., 1991). The primary or conventional PCR has a higher specificity and sensitivity for detection of the parasite DNA in blood than DNA probe technique (Saiki et al., 1988), real time
PCR assay (Kizilarslan et al., 2015), microscopic examination (Bashiruddin et al.,
1999; Rampersad et al., 2003; Bahrami et al., 2014), and serological tests (Buling et al., 2007; Jefferies et al., 2007).
Primary PCR techniques based on the design of species specific oligonucleotide primers from the 16S rRNA gene were developed for detecting T. equi genome in blood with estimated parasitaemia of 0.0083% and for B. caballi genome with estimated parasitaemia of 0.017% (Bashiruddin et al., 1999).
Moreover, PCR assay has sufficient sensitivity to detect the parasite DNA from
2.5μl of blood with an estimated parasitaemia of 0.000001% (Xuan et al., 2001a;
Alhassan et al., 2007a). The 18S rRNA gene mainly targets use in the PCR assay for epidemiological studies of piroplasms infection (Moretti et al., 2010; Farkas et al., 2013). Primary PCR and nested PCR have been constructed for the routine detection of T. equi in horses (Rampersad et al., 2003).
Another shortcoming of PCR is that it is specific multiplex PCR. In this
PCR assay T. equi and B. caballi can be detected at the same time with higher sensitivity, more simply and more rapidly than the nested PCR (Alhassan et al.,
2005). In addition, multiplex PCR can be used to detect the Theileria and Babesia
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parasites in one reaction, identify the species of these parasites and propose a simple tool in the routine diagnostic laboratory and in epidemiological studies
(Alhassan et al., 2005; Rosales et al., 2013). This assay is more sensitive than microscopic examination methods for detection of T. equi and B. caballi
(Malekifard et al., 2014). The PCR assay has been recommended for detection of a latent infection with T. equi and B. caballi and also can be used to identify the genetic diversity of parasites and the morphologically identical species that cannot be detected microscopically (Moretti et al., 2010). In addition, PCR assay has provided worthy data on the biogeographical distribution of piroplasms species
(Kouam et al., 2010b). The PCR techniques are applied to amplify a sequence of the parasite DNA, at first, parasite DNA is extracted, and the resulting purified
DNA is used as a DNA template for following PCR amplification, two specific primers (forward and reverse) for each parasite species are important to test for the existence of parasites DNA; subsequently, the existence and quantity of purified DNA is detected using PCR thermocycler or real time PCR system, gel electrophoresis, and PCR imager (Alhassan et al., 2005; Farkas et al., 2013).
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Table 2.4: Different methods obtainable for the diagnosis of EP and their purpose (OIE, 2014b).
Method Purpose Population Individual Contribute to Confirmation Prevalence Immune status freedom Animal eradication of clinical of infection in individual from freedom policies cases surveillance animals or infection from populations infection post-vaccination Agent identification Microscopic – + – ++ + n/a examination PCR +++ +++ +++ +++ +++ n/a Detection of immune response IFAT ++ ++ ++ +++ ++ n/a C-ELISA +++ +++ +++ +++ +++ n/a CFT + + + + + n/a
Key: +++) recommended method; ++ ) suitable method; + ) may be used in some situations, but cost, reliability, or other factors severely limit its application; – ) not appropriate for this purpose; n/a) not applicable. Although not all of the tests listed as category +++ or ++ have undergone formal validation, their routine nature and the fact that they have been used widely without dubious results, makes them acceptable; PCR) Polymerase chain reaction; IFAT) Indirect fluorescent antibody test;c-ELISA ) Competitive enzyme-linked immunosorbent assay; CFT) Complement fixation test.
2.14.2 Other methods for diagnosis of equine piroplasmosis
2.14.2.1 Biological tests
The biological tests include: the isotest which is done by inoculation of
500 ml of blood or better, washed RBCs transfused from a dubious animal into a
splenectomized susceptible horse or donkey, then conserved under close
monitoring for the observation and recording of the clinical signs of infection.
Diagnosis is confirmed by the existence of parasites in the RBCs (OIE, 2013;
Schwint et al., 2009).
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The xenotest, which is applied by feeding of tick vectors on suspected
infected horse; then can either identify the parasite in the ticks or through feeding
the vector ticks on the susceptible horse (de Waal & van Heerden, 2004; Schwint
et al., 2009). However, these tests are used for a research purpose for the isolation
of parasite, especially when the parasitaemia is low. The disadvantages are
obviously the high cost, cumbersome and not used as diagnostic tests.
2.14.2.2 In vitro culture technique
There are many types of medium used in the in vitro culture methods to
identify T. equi and B. caballi such as H-Y medium (Baldani et al., 2008),
microaerophilous stationary phase (MASP) medium (Brok et al., 2003), RPMI
1640 medium (Kawai et al., 1999), HL-1 medium (Holman et al., 1993), and
medium 199 with Hanks salts (Zweygarth et al., 1997). Improvements in the in
vitro culture techniques for EP have enabled researchers to identify both parasites
in carrier horses or subclinical infected, which were negative in microscopic
method and serological test (Holman et al., 1994; Zweygarth et al., 1995).
Therefore, there is no need to use experimental equids for isolation of parasites in
carrier animals. The culture method is very important for serological tests; and can
be applied for verification of standard sera, obtained large amounts of antigen and
evaluation of other diagnostic tests (Ali et al., 1996). Despite the high sensitivity
and specificity of in vitro culture method, it needs professional personnel and
expensive culture medium and medium supplements. Moreover, the
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culture techniques are only used for individual cases, must be performed on fresh
blood samples, is time-consuming, and therefore cannot be use as a screening test
(Bhoora et al., 2010).
2.14.2.3 Other serological tests
In addition to cELISA, there are several other serological tests used for
detection the antibodies for T. equi and B. caballi. Previously, the main techniques
used in serological diagnosis and seroepidemiological studies, which were
recommended by the Office International des Epizooties (OIE) and the United
States Department of Agriculture (USDA) were indirect enzyme-linked
immunosorbent assay (iELISA), indirect fluorescent antibody test (IFAT), and
complement fixation test (CFT), but there is restriction for use due to cross
reaction between the parasite species, which has been recorded in these tests
(Weiland, 1986; Papabopoulos et al., 1996).
Latex agglutination test (LAT) is one of the serological tests used for
detection of T. equi infection in horses (Xuan et al., 2001c), which has been used
in expression of EMA-1 in insect cells by recombinant baculovirus as described
by Xuan et al., (2001a, 2001b). Recently, immunoblot assay or western blot test
has been used firstly in a research setting for the detection of T. equi and B.
caballi infections (Ikadai et al., 2002). The procedure for this test has been
explained by Schwint et al., (2009). It is now displayed by the National
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Veterinary Services Laboratory (NVSL) in Ames, Iowa, as a routine
serodiagnostic tool (APHIS, 2012).
Immunochromatographic test (ICT) is a new, simple, rapid, more
accurate and cheaper method for detection of T. equi and B. caballi antibodies
based on recombinant EMA-2 and BC48 proteins, respectively (Huang et al.,
2006; Curz-Flores et al., 2010). It has the capability in the serodiagnosis of both
acute and subacute piroplasmosis in horses (Huang et al., 2003, 2004).
2.14.2.4 Other molecular techniques
In addition to primary and multiplex polymerase chain reaction (mPCR),
there are other PCR techniques used for detection of T. equi and B. caballi species
such as the nested PCR technique (nPCR), which has been subsequently
developed for specific detection of T. equi using EMA-1gene sequence with
estimated parasitaemia of 0.000006% (Nicolaiewsky et al., 2001). It is able to
diagnose subclinical infection, determining the efficiency of medical treatment
and controlling the exportation of infected horses (Rampersad et al., 2003). The
reverse line blot (RLB) hybridization is highly sensitive and a specific technique
has been developed for improved diagnosis of EP in equids (Nagore et al., 2004).
It has been confirmed to be a very sturdy test in detecting carrier infection,
identification of co-infection and novel species or genotypes of T. equi and B.
caballi (Nagore et al., 2004; Nijgof et al., 2005; Bhoora et al., 2009).
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Recently, developments of quantitative real time PCR (qPCR) have greatly improved the molecular detection, diagnosis of many organisms of veterinary and medical importance and determining the quality of experimental samples (Wengi et al., 2008). The qPCR techniques have higher significantly confirmed sensitivity and specificity of parasite detection, based on the 18S rRNA gene for detection of T. equi and B. caballi infections in horses (Bhoora et al.,
2010).
The last PCR assay is loop mediated isothermal amplification (LAMP), which is a simple, low cost, rapid and powerful new generation of novel gene amplification assay with great sensitivity and specific for early detection and identification of piroplasms infections in horses (Alhassan et al., 2007b; Parida et al., 2008). It has been reported as a more sensitive technique than PCR assays, and in vitro culture methods for diagnosis of EP in horses and can also be applied as a supplementary technique at postmortems. (Alhassan et al., 2007a), This technique does have disadvantages: it is laboratory-founded, complicated primer design (six primers), two long primers of high performance liquid chromatography (HPLC) grade purity, and the equipment and reagents are restricted in some countries (Parida et al., 2008).
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2.15 Differential diagnosis of equine piroplasmosis
There are a number of diseases similar to EP, that should be
differentiated such as: trypanosomiasis (Surra caused by T. evansi and dourine
caused by T. equiperdum), equine granulocytic anaplasmosis caused by
Anaplasma phagocytophilum, equine viral diseases (African horse sickness caused
by Orbivirus, equine infectious anaemia caused by retrovirus, equine influenza
caused by influenza A viruses: H7N7 and H3N8, equids herpesvirus-1 caused by
herpesvirus-1, encephalitis virus infection caused by herpesvirus), purpura
haemorrhagic (associated with reduction of platelets counts), chemical toxicities
(phenothiazine), poisonous plants (wild onions, red maple leaves), and diseases
that cause mares abortion and liver failure (de Waal & van Heerden, 2004;
CFSPH, 2008; James & Thomas, 2008; Radostitis et al., 2008).
2.16 Prognosis of equine piroplasmosis
The favorable prognosis is during the early stage of disease and effective
treatment, especially in foals at the first time suffering from an acute form of an
EP, whereas, it becomes unfavorable or bad in older equids observed with severe
clinical signs and exhibiting other diseases such as renal failure or pneumonia,
etc. (Maslin et al., 2004; OIE, 2013).
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2.17 Treatment of equine piroplasmosis
It is not easy to eliminate the hemoparasites from horses or other animals
infected with the disease (Bruning, 1996). However, there are different drugs
available for the treatment of EP, each with different degrees of success. T. equi
parasites are mostly more resistant to antiprotozoal drugs than B. caballi (Lindsay
& Blagburn, 2001). Therefore, treatment of B. caballi is effectively done by
different drugs, while the elimination of T. equi is very difficult (de Waal & van
Heerden, 2004). Successfully treating the end of EP depends on protozoal species
and susceptibility of the diseased equids (Brüning 1996).
Previously, various drugs such as quinuronium derivatives (acaprin),
acridine derivatives (acriflavine Hcl) and bizaso dyes (trypan blue) had been
reported to be effective on B. caballi, but resistant to T. equi (Kuttler 1988; de
Waal & van Heerden, 2004). The side effects of those aforementioned drugs like
acaprin which is effective on sympathetic nervous system and signs of poisoning
appear on animals; drooling, sweating, urination, diarrhea and hypotension, which
may lead to death. Also, acriflavine Hcl and trypan blue drugs cause discoloration
of the equids tissues (Howe, 1973; Kuttler, 1981). Hence, those drugs are not used
at present. Other drugs are also reported to be less effective for treatments of T.
equi like oxytetracycline hydrochloride, tetracycline and anti Theilerial
compounds including parvaquone and buparvaquone (de Waal & van Heerden,
2004; Alsaad & Al-Mola, 2006).
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Another approach in treatment using Aromatic diamidines derivatives such as amicarbalide diisethionate, diminazene diaceturate, and imidocarb dipropionate have been observed to be more efficient for the treatment of EP infection (Brüning 1996; de Waal & van Heerden, 2004). The use of amicarbalide
(diampron) at a dose 10-9 mg/kg intramuscular (IM) is sufficient to hide the clinical symptoms from the infected animals and it completely eliminates B. caballi when used in two doses of 8.8 mg/kg of body weight within 24 hours, but
T. equi is resistant to the drug even if given at four doses 11 mg/kg (Kuttler,
1981). Successful elimination of B. caballi in naturally infected horses and donkeys is achieved when using diminazine aceturate at two doses of 3.5 mg/kg of body weight IM within 24 hours, but it also does not completely remove the T. equi (Singh et al., 1980). Although, there are various antipiroplasms drugs, imidocarb dipropionate is the drug of choice for treating EP infection (Vial &
Gorenflot, 2006; Donnellan et al., 2003b). The imidocarb dipropionate proved its efficacy in the treatment of EP infection in horses when used at two doses of 4 mg/kg body weight IM within 48 hours and treated animals were observed to recover completely, appeared normal and the clinical signs returned to normal on the 6th day (Alsaad & Al-Mola, 2006). This drug has a widespread distribution in body fluids, tissue and can also cross equids placenta to reach in the fetus with similar concentrations to the mares (Lewis, 1999; Vial & Gorenflot, 2006). The toxicity of imidocarb dipropionate with regards to its cholinergic is direct hepatotoxic and nephrotoxic effects (Donnellan et al. 2003a; Vial & Gorenflot,
2006). Hence, to decrease the side effects there should be prior treatment of either
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glycopyrrolate at a dose 0.0025 mg/kg intravenously (IV) or atropine sulphate at a dose 0.035 mg/kg IV (Kutscha et al., 2012).
In experimental inoculation of T. equi isolated from an imported horse into normal native horses, these horses were treated with imidocarb dipropionate at four doses of 4 mg/kg body weight IM within 72 hours after showing the acute form of the disease, elimination of the T. equi from treated animals based on serological and nPCR negativity. Therefore, this result supports the use of imidocarb dipropionate as a drug of choice in controlling T. equi outbreaks
(Grause et al., 2013). On the other hand, another study made a comparison between pentamidine methunsulphate at a dose rate of 3 mg/kg body weight IV, imidocarb dipropionate at a dose rate of 2.4 mg/kg body weight IM and diminazene diaceturate at a dose rate of 3.5mg/kg body weight IM, and demonstrated 100%, 90%, 80% respectively, the efficacy of drugs against equine babesiosis (Chaudhry et al., 2014). Moreover, triclosan (broad-spectrum microbiotical activity), fusidic acid (antimalarial, antibacterial and antifungal activities), and allicin (antiprotozoal, antifungal, antibacterial, and antiviral activities), are inhibitors with inhibition effect on the in vitro growth of T. equi and B. caballi (Vial & Gorenflot, 2006; Salama et al., 2013, 2014).
Chemotherapy is rarely recommended in endemic regions, but may be indicated when horses are to be moved from endemic regions to a disease-free region (de Waal & van Heerden, 2004). Depending on the severity of the EP infection, there should be supportive care that includes blood, glucose and polyionic electrolyte infusions (Hailat et al., 1997), vitamins administration, good
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nutrition, antibiotics, essential phospholipids, and oral laxatives (Phipps, 1996; de
Waal & van Heerden, 2004). In addition, using the aspirin at a dose of 10 mg/kg
of body weight IM, repeated after 48 and 72 hours and heparin at a dose of 100
IU/kg of body weight subcutaneously, repeated after 48 and 72 hours, can be a
supportive treatment for EP, treated with imidocarb dipropionate (Alsaad &
Mohammad, 2011).
2.18 Control of equine piroplasmosis
In 1962 a control program was implemented in South Florida for the
eradication of EP. The program included quarantine, testing and chemotherapy for
carriers and diseased equids, spraying the tick on the infected and exposed equids,
and controls of infected animals movement to block disease propagation. As a
result of this control program, the United States was announced free from the
disease in 1988 (APHIS, 2009).
2.18.1 Vaccination
Although, there are reports of successful immunization of donkeys
against T. equi infection using killed merozoites vaccine (Singh et al., 1981;
Kumar et al., 2002), there are still no effictive vaccines currently available against
EP (de Waal & van Heerden, 2004; OIE, 2014b). Hence, chemoprophylaxis using
imidocarb dipropionate at a dose of 2 mg/kg body weight IM conferred protection
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for 4 to 6 weeks in cattle and equids (Maslin et al., 2004). This prophylaxis
therapy should be repeated during or at risk seasons.
2.18.2 Control of ticks
In general, efficient control over ticks is a vital part in the fight against
tick-borne disease and the elimination of ticks would prevent transmission of
pathogens to an animal; moreover it would lead to disease eradication. On equids
ticks can be seen everywhere on the body surface, but commonly found on the
face, in the axillary, at the base of the tail or around the anus. The control of ticks
is based on:
1. A complete examination of a horse’s body to search for ticks and if any horse is
found to be ticks infested, isolated it from the other animals (Sumbria & Singla,
2015).
2. Regular application of chemical acaricides either by total inundation in a dipping
bath or in the form of a spray, shower or spot-on. Nowadays, because of random
use of these chemical compounds for ticks control, the problems are resistance in
ticks, animal products pollution, and environmental remains have arisen which are
toxic to abundant water organisms, bees, waterfowl and human fatal asthma has
been reported (Aldridge, 1990; Wagner, 2000).
3. Nowadays, due to the aforementioned problems, the development of vaccine
against ticks (anti-ticks vaccine) is a new method for arthropod control (Sathaporn
et al., 2006; Imamura et al., 2008; Matthias, 2013; Merino et al., 2013).
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CHAPTER 3
DIAGNOSIS, PREVALENCE, RISK FACTORS AND VECTOR OF
EQUINE PIROPLASMOSIS IN EQUIDS IN KELANTAN
3.1 Introduction
Equine piroplasms is a listed disease by the World Organization for
Animal Health (OIE) because it affects the horse industry causing high morbidity
and mortality rate. In the endemic areas the mortality rate ranges from 10% to
50% (OIE, 2013). The disease can also result in abortion, stillbirth and birth of a
live foal with neonatal piroplasmosis (de Waal & van Heerden, 1994; Allsopp et
al., 2007).
In Malaysia, EP is a notifiable disease where suspected and confirmed
cases must be reported to the Department of Veterinary Services (DVS). The disease
had been reported by Chandrawathani et al., (1998) and Zawida et al., (2010)
However, there is no report in OIE (OIE, 2014a).
The distribution of EP depends on the presence of vectors and increased
movement of horses, especially in international trade and sports activity from
endemic to non-endemic areas (Brüning, 1996). The susceptibility of the equids to
EP infections, depends on many risk factors such as equids species, gender, age,
breed, region, activity, tick found, management, and environmental temperature
and humidity (Kouam et al., 2010c; Sumbria et al., 2016b). The susceptibility of
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equids varies among the regions or districts and might be due to differenced in the management practice, sampling size, equids activity, and presence of convenient tick vectors (Kouam et al., 2010a). The various climatic factors (temperature, relative humidity, and rainfall amount) influence the ticks habitat (Garcia-
Bocanegra et al., 2013).
There are 33 Ixodid tick species belonging to six genera that have been reported as competent vectors for EP, which include Amblyomma spp.,
Dermacentor spp., Haemaphysalis spp., Hyalomma spp., Ixodes spp., and
Rhipicephalus spp. (Scoles & Ueti, 2015).
The clinical signs of the EP are not specific and may be confused with other illnesses (Rothschild & Knowles, 2007). Therefore, suspected cases based on clinical signs should be confirmed by laboratory methods which include the observation of intraerythrocytic forms of the protozoa in Giemsa‟s-stained blood or organ smears and by polymerase chain reaction (PCR) technique (Moretti et al., 2010; Ribeiro et al., 2013). The disease can also be confirmed by several serological tests such as complement fixation test CFT, immunofluorescence antibody test (IFAT), indirect enzyme linked immunosorbent assay (iELISA), as well as the competitive ELISA (cELISA). Serological tests are suitable for large scale studies and to monitor the infection during the latent stage when parasitemia can not be detected by microscopic examination (Ogunremi et al., 2008; Alsaad et al., 2012). The cELISA is the advisable sero-epidemiological assay for detection of antibodies against B. caballi and T. equi in equids and for certification purposes
(OIE, 2013).
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Various molecular techniques can be used to confirm the EP infections such as conventional PCR, nested PCR, multiplex PCR, real time PCR, and loop mediated isothermal amplification (LAMP) technique. Multiplex PCR assays can be used to detect the Theileria and Babesia parasites in one reaction, identify the species of these parasites and be offered as an easy tool in the routine diagnostic laboratory and epidemiological studies (Alhassan et al., 2005). This assay is more sensitive than microscopic examination methods for detection of T. equi and B. caballi (Ibrahim et al., 2011; Malekifard et al., 2014).
Base on a thorough literature search, information on the epidemiology of
EP in Kelantan was not available . Therefore, the aims of this study were to:
10. Identify T. equi, B. caballi and both protozoa with their parasitemia.
11. Determine the prevalence of T. equi, B. caballi and both protozoa in Kelantan.
12. Evaluate the efficiency of different laboratory methods for detecting T. equi
and B. caballi infections.
13. Identify the risk factors associated with seroprevalence of T. equi, B. caballi
and both protozoa infections.
14. Identify and determine infestation rate of Ixodid tick species on equids and
other animals near equids in Kelantan stables.
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3.2 Materials and Methods
3.2.1 Study areas
The locations of the study encompassed eight regions in Kelantan
including Kota Bharu, Pasir Mas, Machang, Bachok, Pasir Puteh, Tumpat, Tanah
Merah and Gua Musang, using the Global Positioning System (GPS) data
(4º30'0"N-6º0'0"N latitude -101º30'0"E-102º30'0"E longitude). Kuala Krai and
Jeli regions were not include in this study because there is no equids in these
regions. A Total of 53 stables were visited in the course of the study (Fig. 3.1).
Figure 3.1: Geographical map of Kelantan state showing the locations of equids sampling stables using Map Window arc GIS 10 program.
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3.2.2 Determining the number of equids
The number of equids for this study was based on expected prevalence of
1%, confidence level 95% and population of 658 equids in Kelantan (DVSK,
2012). In order to detect at least one equid infected the following equation was
used to calculate the required number of equids:
1/D n = [1-(α) ] [N-(D-1) /2] (3.1)
where: n = required samples, α= 1- confidence level (0.05), D= estimated
minimum number of diseased animals in population (population size x the
minimum expected prevalence), N= population size (Stevenson, 2008; Dohoo et
al., 2010). The number of equids needed for this study was 258.
3.2.3 Animals and sample collection
After getting permission from the owners, 50% of the equids in the
visited stables were sampled. The health status of the equids was recorded at the
time of sampling. From September 2013 to March 2014, a total of 306 equids
(148 horses and 158 ponies) were sampled. The sex, age and breed of the equids
were recorded. Blood samples were collected from the jugular vein using 18G
® needle into two sterile vacutainers tubes (5ml each), one with anticoagulant
ethylene diamine tetraacetic acid (EDTA) and another without any anti-coagulant.
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A total of 612 blood smears, comprising thick and thin smears from each
case, were prepared from the blood with anticoagulant upon arrival at the
laboratory and the remaining blood was stored at -80°C for molecular analysis.
Serum were separated from clotted blood in tubes without anti-coagulant
by centrifuging at 2500 rpm (280 G-force) for 15 min and stored at -20°C for
cELISA (Kouam et al., 2010a).
At the time of sampling, equids and other domestic animals (cattle,
sheep, goats and dogs) near the stable were also inspected on the head, neck,
pectoral, armpit, inguinal and under tail areas, in order to determine whether the
animals were infested with ticks or not. A total of 533 ticks of both sexes (male,
female) and stages (larvae, nymph and adult) were collected and stored in 70%
ethanol at 4ºC until identification of ticks according to taxonomic keys (Walker et
al., 2000; Bouattour, 2002; Pavlidou et al., 2008).
3.2.4 Epidemiological data collection
During samples collection, epidemiological data were collected through
interviews with the owners of the stables using standard clinical examination
cards.
7. Equids data that were collected included type of equid (horse or pony),
gender (male, female or gelding), age (<1year, >1-2years, >2-5years or >5years),
breed of horse (Arab breed, crossbred or thoroughbred), breed of pony (pony A,
pony B, pony C or crossbred), origin of the equids (local or imported), equids
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exporting countries, purpose of keeping the equids (breeding, recreation or sports), pregnancy (yes or no), stages of pregnancy (<3months, 3-7months or
>7months), and presence of ticks on equids (yes or no).
10. Stables data that were collected included regions, sampling months, species of animals in stables (only equids or mixed with other animals), management (in stable or grazing), presence of ticks in stables (yes or no), ticks found on animals (on nearby animals, on at least one equid or on equids and nearby animals).
11. The equids case history, a general examination of animal, type of sample (blood sample and blood smears) and the results of laboratory analysis were recorded in clinical examination card (Fig. 3.2). The stages of pregnancy were determined in the mares using Ultrasound scans (IMAGO, ECM-
1211MG05, France).
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Figure 3.2: The clinical examination card.
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3.2.5 Climatic data
Kelantan‟s climatic data was collected from the Department of
Meteorology Malaysia in Selangor. The data includes amount of daily rainfall
(millimeter), mean temperature (ºC) and mean relative humidity (%), from
September 2013 to March 2014. Refer to document No. JMM.COM 31/599/07
JId. 158 (92) on 17 October 2014, the detailed in (Appendix- A).
3.2.6 Laboratory analysis
3.2.6.1 Microscopic examination of blood smears
A total of 612 staining blood smears comprising 306 thin smears and 306
thick smears were prepared from blood samples. The instrument and chemicals
used in this method are detailed in (Appendix- B).
The thin blood smears were examined to determinate the parasitemia and
evidence of intracellular morphological and biometrical compatible with the shape
and size of T. equi and B. caball, whereas, thick smears were examined to confirm
the parasitemia especially when the parasitemia was low in the thin blood smear.
Parasitemia was calculated following the equation of Fritsche and Smith, (2001):
Number of infected RBCs Parasitemia % = X 100 (3.2) Number of calculated RBCs
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Briefly, the procedure to prepare and stain thin and thick blood smears was as follows:
5. Blood in a tube with EDTA was gently flicked for a few minutes to mix the whole blood.
6. To obtain a thin blood smear, a small droplet (approximately 5μl) of blood was placed near the label end of the microscope slide by using capillary tube (Fig.
3.3A)
7. A second slide (spreader slide) was positioned at an angle of about 30-40º and spreader tip was allowed to contact the drop of blood (Fig. 3.3B)
8. The blood was allowed to spread across the spreader (Fig. 3.3C)
9. The drop of blood was slowly drawn, leaving a thin layer of blood in its wake
(Fig. 3.3D)
10. The complete blood smear was allowed to dry as rapidly as possible (Fig. 3.3E)
11. To prepare thick smear, a small blood droplet (approximately 100 μl) was placed near the label end of the slide and spread moderately on a flat surface of the slide, then mixed by using toothpicks, then exposed to rapid drying (Fig.
3.3F).
12. After complete drying of the thick and thin smears, the slides were fixed in absolute methanol in the staining jar for 3-5 min and air dried.
13. Stock Giemsa stain was diluted (1:20) with distilled water and the fresh stain was prepared every two days. Then the blood smears were stained for 30-35 min by placing the slides in the staining jar with a suitable amount of diluted Giemsa, then washed with tap water for 30 - 40s.
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8. The smears were air-dried and examined under a light microscope for at least
b) fields per smear using the 100X (oil immersion) objective. The
morphological and biometrical features of T. equi and B. caballi were recorded
and photographed using image analysis software. These steps were adapted from
Hendrix and Robinson, (2006).
A B C
D E F
Figure 3.3: A-F) Demonstration of thin and thick blood smears preparation (http://www.slideshare.net/drAjayAgale/05-peripheral- blood-smear-examination).
3.2.6.2 Competitive enzyme linked immunosorbent assay
In this assay, recombinant T. equi equine merozoites antigen 1 (EMA-1)
and a specific monoclonal antibody (MAb) that defines this merozoites surface
protein epitope were used. A similar, recombinant B. caballi rhoptry-associated
protein 1 (RAP-1) and a MAb reactive with a peptide epitope of a 60 kDa B.
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caballi antigen were used. The binding of the primary monoclonal antibody to the antigen coated plate was observed in the binding of horse radish peroxidase
(HRP) labeled secondary antibody.
Lastly, binding of the HRP-labelled secondary antibody is quantified by the addition of enzyme substrate and subsequent color product development. The strong color developments indicated little or no inhibition of primary monoclonal antibody was binding, hence, the absence of T. equi or B. caballi antibodies in the sample serum. The weak color development due to inhibition of primary monoclonal antibody binding to an antigen on the solid phase indicates the presence of T. equi or B. caballi antibodies in the sample serum. Before conducting the test, chemicals were prepared as follows: a. In the beginning the serum samples, reagent and plates were put at room temperature (23 ± 2ºC). b. The positive and negative controls (B & C) and test serum samples were diluted
1:2 with serum diluting buffer (G). The dilution was carried out on a non-antigen- coated transfer plate. The positive and negative controls were run in duplicate and triplicate respectively. The control and serum sample IDs were entered on a copy of the following set up record.
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Setup record
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
c. The plates were removed from the foil pouch (A). d. 1X primary antibody was prepared by diluting 1 part of the 100X primary anti body (D) with 99 parts of antibody diluting buffer (F). For 96 wells , 60µl of
100X primary anti body (D) was mixed with 5.940 ml of antibody diluting buffer
(F) to yield 6 ml of 1X primary antibody . 50µl was needed per well. e. 1X secondary antibody-peroxidase conjugate was prepared by diluting 1 part of the 100 X secondary antibody-peroxidase conjugate (E) with 99 parts of antibody diluting buffer (F). f. 1X wash solution was prepared by diluting 1 part of the 10 X wash solution concentration (H) with 9 parts of deionized or distilled water. (10 ml of 10 X wash solution concentration was diluted with 190 ml of distilled water). Approximately
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1.8 ml was needed per well. Extra quantity was allowed for the reservoir, tubing, pipetting, etc.
The procedure of ELISA test proceeded as follows: c) An amount of 50µl from diluted controls and serum samples was transferred to the antigen-coated plate (A) according to the set up recorded. The side of the loaded assay plate was tapped several times to ensure that the wells bottom was well coated by the samples. This was followed by incubating the plate for 30 min at room temperature (21-25 ºC, 70-77 ºF). d) After incubation, the plate was washed three times. Manual washing was used, and the plate contents were dumped into a sink while the remaining sera and controls were discarded by sharply striking the inverted plate four times on a clean paper towel. Immediately after, each well was filled with 300µl of washing solution by multichannal pipettor. Then the plate was dried as above. The washing procedure was repeated three times. e) An amount of 50µl of diluted (1X) primary antibody was added to every well.
The side of the loaded assay plate was tapped many times, followed by incubation for 30 min at room temperature. f) Following the incubation, the plate was washed three times as in step 2. g) An amount of 50µl of the diluted (1X) secondary antibody-peroxidase conjugate was added to every well. The side of the loaded assay plate was tapped many times followed by incubation for 30 min at room temperature. h) Following the incubation, the plate was washed three times as in step 2.
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b) An amount of 50µl of substrate solution (I) was added to every well. The side of the loaded assay plate was tapped several times and then incubated in a dark place for 15 min at room temperature. c) An amount of 50µl of stop solution (J) was added to evrty well. The side of the loaded assay plate was tapped several times to thoroughly mix the solutions. d) As soon as the stop solution was added, the plate was fixed on a plate reader and the optical density (O.D) was read at wavelength 630nm. An automatic plate ® reader (BioTek Elx808, USA) was used in this study. e) The remaining kit chemicals were returned to the refrigerator and stored at
4ºC.
The validation of the test results was done by calculating the percentage of inhibition (%I) following the equation:
%I=100- [(sample O.D×100) ÷ (mean negative control O.D)] (3.3)
The mean of the negative controls should produce an optical density >
0.300 and < 2.000. The mean of the positive controls should produce the inhibition of ≥ 40%. Results were interpreted and: if the % I ≥ 40% the sample was positive and if the % I ≤ 40% the sample was negative.
The test procedure presented was adapted from the literature provided with the kits for Babesia equi (Theileria equi) antibody test kit, cELISA and
Babesia caballi antibody test kit, cELISA from (VMRD, Inc., Pullman, and
WA99163 USA). The instrument and chemicals used in this assay are mentioned in (Appendix- B).
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3.2.6.3 Polymerase chain reaction techniques
In this study, two types of reaction were achieved: firstly, to identify the
positive equids for all possible Theileria spp. and Babesia spp. employing the
universal „catch-all‟ primers (TB-F and TB-R) by conventional PCR assay.
Secondly, to differentiate between T. equi and B. caballi in all positive and
negative samples in the first reaction, by using a single forward primer (TBM-F)
and two reverse primers, one for identifying T. equi and another for B. caballi,
which were Equi-R and BC-R respectively by multiplex PCR assay (Table 3.1).
3.2.6.3.1 DNA extraction from equids blood
Piroplasms DNA was extracted from 306 equids blood samples with
® EDTA anticoagulant employing the QIAamp DNA Mini Kit (QIAgen GmbH,
Hilden, Germany). The instrument and chemicals used in this extraction kit are
descxribed in (Appendix- B). The procedure mentioned below was adapted from
the literature that came with the extraction kit.
Before starting the procedure, the waterbath was set up at 56 ºC and
Equilibrate buffer AE placed at room temperature. In addition, working solutions
were prepared as follows:
a. QIAGEN Protease solution was prepared with the addition of 5.5 ml protease ® solvent to the vial that contained lyophilized QIAGEN Protease. Dissolved
® QIAGEN Proteasem is stable for up to two months when stored at 2 - 8 ºC.
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Storage at -20 ºC is acceptable if the solution is not uses up to a maximum of two months. b. Buffer AL was prepared by mixing buffer AL thoroughly and shaking before use. The stability of Buffer AL can be maintained for up to one yeat if stored at room temperature (15-25 ºC). c. AW1 buffer was prepared by adding 125ml of ethanol (96–100%) to Buffer
AW1 concentrate as instructed on the bottle. Buffer AW1 stability can be maintained up to one year if stored closed at room temperature. d. AW2 buffer was prepared by adding 152ml of ethanol (96–100%) to Buffer
AW2 concentrate as instruction on the bottle. Buffer AW2 stability can be maintained for up to one year if stored closed at room temperature. The procedure of the extraction kit was carried out as follows:
Before starting, the water bath was set at 56ºC. Nuffer AE was equilibrated to room temperature.
& Blood samples were lysed by pipetting 20 µl QIAGEN Protease (Proteinase K) and up to 200 µl blood into a 1.5ml microcentrifuge tube, followed by the addition of 200 µl AL buffer to the sample and then vortexing the mixture vigorously for
15s, then incubated at 56ºC for 10 min. Long incubation times do not affect yield or quality of the purified DNA. This was followed by briefly centrifuging the tube to eliminate drips from the interior of the lid.
& An amount of 200μl ethanol (96–100%) was added to the sample and vortexed again for 15s. Following mixing, the tube was briefly centrifuged to eliminate drips from the interior of the lid.
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& The mixture from step 2 (in a 2ml collection tube) was carefully applied to the
QIAamp Mini spin column, taking care not to wet the rim, then the cap was closed and centrifuged at 8000 rpm for 1 min. The QIAamp Mini spin columns were placed in a clean 2 ml collection tube (provided) and the tube with the filtrate was discarded. If the lysate had not all passed through the column following centrifugation, it was centrifuged again at higher speed to completely empty the
QIAamp Mini spin column.
& The QIAamp Mini spin columns were carefully opened and 500 μl Buffer AW1 was added, taking care not to wet the rim. The cap was close and centrifuged at
8000 rpm for 1 min. The QIAamp Mini spin column was placed in a clean 2 ml collection tube (provided) and the collection tube with the filtrate was discarded.
If the lysate had not totally passed through the column following centrifugation, it was centrifuged again at higher speed to empty the QIAamp Mini spin column.
& The QIAamp Mini spin column was carefully opened and 500 μl Buffer AW2 was added, taking care not to wet the rim. The cap was closed and centrifuged at full speed 14,000 rpm for 3 min.
& The QIAamp Mini spin column was placed in a clean 1.5 ml microcentrifuge tube (not provided), and the collection tube containing the filtrate was discarded.
The QIAamp Mini spin column was carefully opened and 100 μl buffer AE or distilled water was added It was then incubated at room temperature of 15–25°C for 1 min, and then centrifuged at 8000 rpm for 1 min. Incubating for 5 min at room temperature before centrifugation generally increases DNA yield.
& The genomic DNA was stored at –25°C until needed.
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3.2.6.3.2 Determination of DNA concentration and purity
The concentration and purity of the DNA extracted from the blood TM samples were determined by using Nanophotometer P- Class (IMPLEN,
Germany). The concentration of DNA yield was determined by absorbance (A) at wavelength 260 nm. The concentration ranged between 24 and 88 ng, while the
purity was obtained by computing the ratio of A260 nm to A280 nm, the pure
DNA was an A260/A280 nm ratio of 1.7-1.9.
The concentration and purity of DNA were determined by using agarose
gel electrophoresis, and a comparison was made between the intensity of Midori
green stained bands and the bands of DNA marker that contained known amounts
of DNA. Briefly, the appropriate volume of agarose gel 1.5% (1.5 g of agarose
was dissolved in 1X Tris-borate-EDTA (TBE) buffer by microwaving) was
submerged in a suitable volume of 1x TBE electrophoresis buffer. The gel
contained 1.5% of Midori green stained. The DNA samples were mixed with the
6x DNA loading buffer dye. Then, the DNA samples were added to the wells of
the agarose gel. The gel was run at 85 volts for 45 min. Finally, the DNA was
TM visualized under the Gel Doc EZ imager (BIO RAD/ USA). This procedure
has also been mentioned by Sambrook et al. (1989).
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3.2.6.3.3 PCR amplification of piroplasms DNA from equids blood samples
Amplify the hypervariable V4 region of 18S rRNA gene of Theileria and
Babesia from equids blood (n=306), as a target in conventional PCR and
multiplex PCR techniques. DNA positive controls for T. equi and B. caballi were
prepared from the blood of clinically infected mare with both parasites (Al-Obaidi
et al., 2015). The gene bank accession numbers for DNA positive controls were
KU879042 and KU879026 for T. equi and B. caballi respectively. Further, DNA
extracted from piroplasms-free horse were used as negative control for each PCR
amplification.
In this study, the oligonucleotide primers were designed by Sloboda et al.
(2011) and provided by First BASE Laboratories Sdn. Bhd. Malaysia (Table 3.1).
To prepare these primers for PCR amplification, the primers tubes were
centrifuged at 1200 rpm for 3min and to make stock concentraton of 100μM of
primer solutions, move only one decimal point backwards from the „nMoles‟
volume in the datasheet of each primer.
For example, the: amount of oligo primer was 14.9 nMoles. The
resuspension was calculated thus: 14.9 = 149.0 μl, this amount of free nucleotides
water (dh2o) was added to the primer tube, vortexed for 5-15 min (non-stop),
followed by a short spin to remove the drops from the lids. The 100μM stock
primer solution obtained was stored at -20ºC until use. The preparation and
dilution of primers were recommended by the manufacturer.
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Table 3.1: The Oligonucleotide primers used to amplify the parasites 18S rRNA genes.
Amount of Expected Primers Sequences 5’-3’ oligo Target gene size (bp) (nMoles) CTT CAG CAC CTT GAG Universal TB_F 25.9 AGA AAT “catch-all”
496 (Babesia spp. & TCD ATC CCC RWC TB_R 25.8 Theileria spp.) ACG ATG CRB AC
CTT CAG CAC CTT GAG TBM_F 22.6 ----- AGA AAT Specific primers
TGC CTT AAA CTT CCT for T. equi Equi_R 26.5 & 360 TGC GAT
B. caballi GAT TCG TCG GTT TTG BC_R 21.3 650 CCT TGG
bp) base pair
The program was used in this work presented in the table below:
Table 3.2: PCR program for samples subject to conventional PCR and multiplex PCR.
Step Function of each step Temperature Time
1 Preheating the lid 100 ºC 5-6 min 2 Pre-denaturation of DNA 95 ºC 5 min 3 Denaturation of DNA 94 ºC 45 s 4 Annealing of primers 60.5 ºC 45 s 5 Extension 72 ºC 30 s 6 Cycling Repeat steps 3-5 36X 7 Final extension 72 ºC 10 min 8 Storage until removal 4 ºC variable
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The procedure of DNA amplification was as follows:
4. The chemicals and DNA samples were removed from the freezer and allowed to thaw at room temperature or in the refrigerator temperature.
5. The lid of the thermocycler was preheated by initiating the program as presented in Table 3.2 above.
6. 0.2 ml thin wall PCR tubes were labelled on a set corresponding to the number of samples to be analyzed.
7. This study used a master mix ready-to-use solution containing: PCR reaction buffer, 0.06U/μl of Taq DNA polymerase, 3mM of MgCl2 and 400μM of each dNTPs.
5. Table 3.3 presents what has been calculated for one reaction in a total volume of 25 μl. The components in the master mix were thoroughly mixed by repeated aspiration with pipetting tip or vortexed for 15s, to ensure that the master mix was homogenous. For this reaction volume, 12.5 μl of master mix was transferred for one reaction according to manufacturer's instructions.
6. The amount of master mix transferred was equivalent to the number of samples
(reactions) to be studied plus three more for negative and positive controls, tubes and pipetting. They were placed in sterile 1.5 ml microcentrifuge tubes.
7. 1μl of each primer was added (equivalent to the number of DNA samples), then were mixed well by repeated aspiration with pipetting tip.
8. Then 8.5μl of dh2o was added (equivalent to the number of DNA samples) in microcentrifuge tube in the first reaction (conventional PCR), while 7.5μl of dh2o
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was added (equivalent to the number of samples) in the second reaction (multiplex
PCR), which was also mixed very well by repeated aspiration with pipetting tip.
9. 23μl from the mixture was transferred to the labelled thin wall PCR tubes. Then
2μl of each DNA sample was added in corresponding PCR tube. It was mixed by vortexing for 15s.
10. The reaction tubes were taken to thermocycler and the lid was closed. The program was installed (Table 3.1) by pressing the “pause” button. The samples were removed at the end of the program when the temperature on the screen showed 4ºC.
Table 3.3: PCR procedure for DNA extracted samples subject to multiplex PCR and conventional PCR assay (reaction volume 25 μl).
Type of PCR assay Chemicals Concentration One reaction
2x PCR Master Mix 2X 12.5μl 100μM TB_F primer 20 μM 1μl Conventional PCR 100μM TB_R primer 20 μM 1μl
DNA samples ------2μl dH2o ------8.5 Total ------25μl 2x PCR Master Mix 2X 12.5 μl 100μM TBM_F primer 20 μM 1 μl 100 μM Equi_R primer 20 μM 1 μl Multiplex PCR 100 μM BC_R primer 20 μM 1 μl DNA samples ------2 μl dH2o ------7.5 Total ------25 μl
11. During the time of the thermocycler run, the gel electrophoresis was prepared by weighting 1.05g of agarose and placing it into the 500ml flask, then 70ml of
1X TBE buffer was added and microwaved for 2-3 min, until the solution was
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boiling. Checks were made to ensure that the agarose was completely dissolved otherwise, it was microwaved again. Gloves were worn when bringing out the flask from the microwave and 3μl of Midori green stained was added to the flask.
12. The agarose was allowed to cool until the flask coukd be touched with bare hands and the agarose was poured into a gel tray (16-20 wells) which was taped on the side. The gel comb was inserted into the dedicated notches on the side of the gel tray, which was then set aside for 45 min for the agarose to solidify.
13. The comb and the tape were removed from the gel tray and the gel was placed in an agarose electrophoresis apparatus. The gel was oriented so that the wells were adjacent to the negative (black) electrode. Then the gel chamber was filled with 1X TBE buffer until it covered the gel by approximately 1cm.
14. Prepared PCR samples for loading by pipetting a 3μl spot of 6X gel loading dye for each sample to be loaded onto a piece of parafilm spread on the benchtop.
Then 7μl of each PCR sample was pipetted into each spot of dye; the pipette tip was changed between samples. The sampels were well mixed with the pipettor and all were directly loaded into a well of the agarose gel. 7μl of DNA ladder was loaded into a first an empty well.
15. The electrodes covers were attached to the gel electrophoresis, then plugged into the power supply, set at 85 volts, 400 Ampere electrical current and run for
45 min.
16. After stopping the gel electrophoresis, the gel tray was removed from the apparatus for documentation. The gel was carefully taken and placed into the Gel TM Doc , EZ Imager.
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17. Results analysis, for the first reaction, visualized the DNA bands in
amplifying size 496 bp, meaning the samples were positive for Theileria spp. and
Babesia spp. without distinguishing between them. The second reaction,
visualized the DNA bands in amplifying size 360 bp, meaning the samples were
positive for T. equi, while, the samples were positive for B. caballi when the
bands iwere n amplifying size 650 bp.
The PCR program and procedural steps mentioned above were also
adapted from Nagore et al. (2004); Kouam et al. (2010b), and Qablan et al.
(2013), with few modifications. The instrument and chemicals used in PCR
amplification are provided in (Appendix- B).
3.2.7 Evaluation the efficiency of different methods to diagnosis the disease
Compative ELISA test and multiplex PCR assays were evaluated against
the microscopic examination of blood smears (Gold standard test) (OIE, 2014b;
Sumbria & Singla, 2015). The following table and formulas were used to
determine the specificity, sensitivity, accuracy, positive predictive value (PPV)
and negative predictive value (NPV), of cELISA and multiplex PCR (Table 3.4).
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Table 3.4: The epidemiological table and formulas were used to compare between tests (Thrusfield, 1995).
Gold standard test Non- Total No. Infected infected Test to be Positive TP FP compared Negative FN TN
Sensitivity Specificity Accuracy PPV NPV
TP/TP+FN TN/TN+FP TP+TN/TP+FN+TN TP/TP+FP TN/TN+FN X 100 (3.4) X 100 (3.5) +FP X 100 (3.6) X 100 (3.7) X100 (3.8)
TP) True positive: Number of samples positive to both the test to be compared and the gold standard; FP) False positive: Number of samples positive to the test to be compared but negative to the gold standard; FN) False negative: Number of samples negative to the test to be compared but positive to the gold standard; TN) True negative: Number of samples negative to both the test to be compared and the gold standard; PPV.) Positive predictive value; NPV) Negative predictive value. Sensitivity: is the capacity of the test to detect diseased animals when compared with the gold standard test (TP/TP+FN X100), Specificity: is the capacity of the test to detect non-diseased animals when compared with the gold standard test (TP/TP+FN X100). Accuracy: is the closeness between tests results (TP+TN/TP+FP+FN+TN X100). Positive predictive value: is the probability that animals with a positive screening test truly have the disease. Negative predictive value: is the probability that subjects with a negative screening test truly do not have the disease.
In addition, comparison with conventional PCR and multiplex PCR
techniques for detecting the piroplasms DNA in equids blood was also made.
3.2.8 Statistical analysis
The association between various risk factors including the type of
protozoa, equids and stables factors was determined by calculating the relative
risk (RR) and it is significant 95% confidence interval.
The difference in the prevalence between the various risk factors was
assessed by using two-sided Chi-square and Fischer‟s exact test in IBM-SPSS
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statistics version19 program. If an expected cell value in Chi-square test is less
than 5, Fisher exact test P value results recommend using factors with P values
<0.05, which are considered significant.
The relative risk (RR) for the association between risk factors for EP and TM it is 95% confidence interval was computed using 2 by 2 tables in Epi-Info 7
software (version 7).
3.3 Results
3.3.1 Morphological and biometerical finding with parasitemia
The primary identification of infectious agents of the EP was based on
morphological features and biometerical data. Microscopic examination of thin
and thick blood smears in this study demonstrated that T. equi was seen either
singly, in pairs or tetrad. Various shapes such as pyriform (a pair of joints), single
pear, arod shape, Maltese cross, and anaplasmoid shape were seen in the RBCs.
Further, T. equi appears as a small parasite measuring from 0.5µm to 3.2µm with
the mean 2.4µm in length and 0.1µm to 2µm with the mean 1.5µm in diameter
(Fig. 3.4).
The parasitemia of T. equi ranged between 0.7% and 14.2% with the
mean of 4.65% (Table 3.5). The schizonte stages: microschizontes and
macroschizontes (Khoch‟s blue bodies) of T. equi were observed within
lymphocytes in the examined blood smears of acute cases (Fig. 3.4).
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Babesia caballi was found in the erythrocytes in a single organism or in pairs of organisms with different shapes such as pyriform (double pear shape), round, signet ring, and amoeboid shape. Further, B. caballi appears as a larger parasite measuring from 2µm to 6.3µm with the mean 4.8µm in length and 1.5µm to 3.2 µm with the mean 2.5µm in diameter (Fig. 3.5).
The parasitemia of B. caballi ranged between 0.1% and 7.6% with the mean of 1.6%. Moreover, both protozoa were seen in some blood smears with a parasitemia ranging between 1% and 18.2% with the mean of 5.48% (Table 3.5).
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Figure 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 joints) 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.
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Figure 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 an obtuse angle, single pear and round shape; B) Signet ring shape; C) Amoeboid shape.
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Table 3.5: Morphological features, biometrical data and parasitemia of T. equi, B. caballi and both protozoa based on microscopic examination of blood smears.
Measuring Parasitemia Type of Morphological range µm ranges% protozoa features (mean ± SE) (mean ± SE) Pyriform (a pair of 0.2 – 3. 2 Len. joints), comma shape, ( 2.4 ± 0.21) 0.7 – 14.2 T. equi rod shape, Maltese 0.1 – 2.8 Dia. (4.65 ± 0.810) cross, and anaplasmoid (1.5 ± 0.121) shape.
Pyriform (double pear 2 – 6.2 Len. shape acute and obtuse (4.8 ± 0.231) 0.1 – 7.6 B. caballi angle), single pear, 1.5 – 3.2 Dia (1.6 ± 0.289) round, signet ring, and (2.5 ± 0.112). amoeboid shape.
------1 – 18.2 Both protozoa ------(5.48 ± 0.883)
SE) Stander error; Len.) Length; Dia) Diameter.
3.3.2 Prevalence of EP by different tests
A total of 306 equids were tested by microscopic examination, cELISA,
and multiplex PCR. The overall prevalence of EP in Kelantan was 32.02% (98 out
of 306), 80.06% (245 out of 306) and 35.62% (109 out of 306) by microscopic
examination, cELISA, and multiplex PCR respectively (Table 3.6).
The prevalence of T. equi, B. caballi and both protozoa was 16.99% (52
out of 306 equids), 22.22% (68 out of 306 equids) and 7.18% (22 out of 306
equids) respectively, by microscopic examination of thick and thin blood smears
(Table 3.7).
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By cELISA the seroprevalence of T. equi, B. caballi and both protozoa was 51.30% (157 out of 306 equids), 63.07% (193 out of 306 equids) and 34.31%
(105 out of 306 equids) respectively (Table 3.7).
By multiplex PCR the prevalence of T. equi, B. caballi and both protozoa was 18.95% (58 out of 306 equids), 22.87% (70 out of 306 equids) and 6.20% (19 out of 306 equids) respectively (Table 3.7).
The prevalence of B.caballi was significantly higher than T. equi and both protozoa for all tests used in this study (P<0.05) (Table 3.7).
Table 3.6: Overall prevalence of equine piroplasmosis (singly T. equi, B. caballi and both protozoa) in equids in Kelantan by microscopic examination, cELISA and multiplex PCR.
No. of Microscopic cELISA Multiplex PCR Type of equids examination protozoa tested No. Positive No. Positive No. Positive positive % positive % positive % c a b Singly 30 9.80 52 16.99 39 12.74 T. equi
b b c Singly 46 15.03 88 28.75 51 16.66 B. caballi 306
a c a Both 22 7.18 105 34.31 19 6.20 protozoa
Overall 98 32.02 245 80.06 109 35.62
Values significantly different (P<0.05) between type of infection are labelled with the different letters (a, b or c).
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Table 3.7: Prevalence of T. equi, B. caballi and both protozoa in equids in Kelantan by microscopic examination, cELISA and multiplex PCR.
No. of Microscopic cELISA Multiplex PCR Type of equids examination protozoa tested No. Positive No. Positive No. Positive positive % positive % positive %
b b b T. equi 52 16.99 157 51.30 58 18.95
c c c B. caballi 306 68 22.22 193 63.07 70 22.87
a a a Both 22 7.18 105 34.31 19 6.20 protozoa
Values significantly different (P<0.05) between the type of piroplasms are labelled with the different letters (a, b or c).
3.3.3 Evaluation of cELISA and multiplex PCR for detecting T. equi and B. caballi infections
The sensitivity, specificity, accuracy, positive predictive value and
negative predictive value of cELISA against microscopic examination for
detecting T. equi infection were 96.15%, 57.87%, 64.37%, 31.84% and 98.65%
respectively (Table 3.8).
The sensitivity, specificity, accuracy, positive predictive value and
negative predictive value of multiplex PCR against microscopic examination for
detected T. equi infection were 100%, 97.63%, 98.03%, 89.65% and 100%
respectively (Table 3.9).
The sensitivity, specificity, accuracy, positive predictive value and
negative predictive value of cELISA against microscopic examination for
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detected B. caballi infection were 91.17%, 44.95%, 55.22%, 32.12% and 94.69% respectively (Tables 3.10).
The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of multiplex PCR against microscopic examination for detected B. caballi infection were 97.05%, 98.31%, 98.03%, 94.28% and 99.15% respectively (Table 3.11).
These results showed that multiplex PCR and cELISA were more efficient than the microscopic examination for detecting T. equi and B. caballi infection. Further, multiplex PCR was more efficient than cELISA for detecting T. equi and B. caballi infections.
In the current study, results based on conventional PCR technique for amplified DNA fragments of Theileria spp. and Babesia spp., using „catch all‟ primers revealed a positive band approximately at 496 bp (Fig. 3.6), with detection rate of 31.69% (97 out of 306 equids) (Table 3.12).
Results based on multiplex PCR technique using specific primers showed that the overall detection of piroplasms DNA was 35.62% (109 out of 306 equids)
(Table 3.12). For T. equi, the positive bands were at approximately 360 bp (Fig.
3.7), for B. caballi the positive bands were at approximately 650 bp (Fig. 3.8), and for both protozoa the positive bands were at approximately 360 bp and 650 bp respectively (Fig. 3.9). In addition, there was no no significant difference between conventional and multiplex PCR techniques for detection of T.equi DNA and B. caballi DNA (Table 3.12).
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Table 3.8: The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of cELISA for detecting T. equi antibodies in comparison with microscopic examination (Gold standard). Microscopic examination (Gold standard) Infected Non-infected Total No. Positive 50 107 157 cELISA Negative 2 147 149 52 254 306 Positive Negative Sensitivity Specificity Accuracy predictive value predictive value 96.15% 57.87% 64.37% 31.84% 98.65%
Table 3.9: The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of multiplex PCR for detecting T. equi DNAs in comparison with microscopic examination (Gold standard).
Microscopic examination (Gold standard) Infected Non-infected Total No. Multiplex Positive 52 6 58
PCR Negative 0 248 248
52 254 306 Positive Negative Sensitivity Specificity Accuracy predictive value predictive value 100% 97.63% 98.03% 89.65% 100%
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Table 3.10: The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of cELISA for detecting B. caballi antibodies in comparison with microscopic examination (Gold standard).
Microscopic examination (Gold standard) Infected Non-infected Total No. Positive 62 131 193 cELISA Negative 6 107 113 68 238 306 Positive Negative Sensitivity Specificity Accuracy predictive value predictive value 91.17% 44.95% 55.22% 32.12 94.69%
Table 3.11: The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of multiplex PCR for detecting B. caballi DNAs in comparison with microscopic examination (Gold standard).
Microscopic examination (Gold standard) Infected Non-infected Total No. Multiplex Positive 66 4 70 PCR Negative 2 234 236 68 238 306 Positive Negative Sensitivity Specificity Accuracy predictive value predictive value 97.05% 98.31% 98.03% 94.28% 99.15%
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Table 3.12: Comparison between conventional PCR and multiplex PCR techniques for detection of piroplasms DNA in equids blood (n= 306).
Type of PCR Type of Type of Product No. positive technique primer protozoa size (bp) (%) detected a Conventional General Theileria spp. & 496 97 (31.69) PCR „catch all‟ Babesia spp. Singly T. equi 360 39 (12.74) Singly B. caballi 650 51(16.66) Multiplex PCR Specific Both protozoa 360 & 650 19 (6.20) a Overall % 109 (35.62)
Values significantly different (P<0.05) between the Type of protozoa detected are labelled with the different letters (a, b or c).
M 1 2 3 4 5 6 7 8 N M
1500 bp 500 bp 496 bp
100 bp
Figure 3.6: Gel electrophoresis image showing: lanes M) Exact Mark 100- 1500bp DNA ladder; Lane 1-8) 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.
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M P 1 2 3 4 5 6 N
1500 bp 650 bp 500 bp 360 bp
100 bp
Figure 3.7: Gel electrophoresis image showing: lane M) Exact Mark 100-1500bp DNA ladder; Lane P) DNA extracted from clinically infected case used as positive control 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.
M P 1 2 3 4 5 6 N
1500 bp 650 bp 500 bp 360 bp
100 bp
Figure 3.8: Gel electrophoresis image showing: lane M) Exact Mark 100-1500bp DNA ladder; Lane P) DNA extracted from clinically infected case used as positive control 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.
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M P 1 2 3 4 5 6 N
1500 bp 650 bp 500 bp 360 bp
100 bp
Figure 3.9: Gel electrophoresis image showing: lane M) Exact Mark 100-1500bp DNA ladder; Lane P) DNA extracted from clinically-infected case used as positive control 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.
3.3.4 Prevalence of T. equi, B. caballi and both protozoa infections by regions
The present study reported a higher prevalence of T. equi in Pasir Puteh
stables, which was 92.15%, and ranged from the lowest prervalence in stable at
sub-region Gong Jernih-3, which was 50% to highest prevalence in stables at sub-
regions Padang Pak Amat and Gong Jernih-2, which was 100%. This was
followed by Kota Bharu stables, which was 68.96%, and ranged from the lowest
in a stable at sub-region Kota-2, which was 33.33% to the highest in stables at
sub-regions Kemumin-1and Kubang Kerian-1, which was 100%. (Table 3.13)
(Fig. 3.10).
Lower prevalence of T. equi was reported in Machang stables, which was
16.66%, and ranged from free 0.00% to 33.33% in stables at sub-regions Hulu
Sat-1 and Hulu Sat-2 respectively (Table 3.12) (Fig. 3.10).
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The result of this study showed higher prevalence of B. caballi reported in Bachok stables was 93.22%, which ranged from the lowest in stables at sub- region Sering, which was 66.66% to the highest in stables at sub-regions Tawang and Perupok, which was 100%. This was followed by Pasir Mas stables, which was 88.46%, and ranged from the lowest in a stable at sub-region Tunjong, which was 66.66% to the highest in stables at sub-regions Pasir Mas and Kubang
Gadong, which was 100% (Table 3.13) (Fig. 3.11).
The lower prevalence of B. caballi reported in Machang stables was
16.66%, and ranged from 16.66% to 83.33% in stables at sub-regions Hulu Sat-1 and Hulu Sat-2. Stables at sub-regions in Gua Musang reported free from B. caballi infection (Table 3.13) (Fig. 3.11).
In addition, a higher prevalence of both protozoa reported in Pasir Puteh stables was 67.62%, and ranged from the lowest in a stable at sub-region Gong
Jernih-3, which was 50% to the highest in stables at sub-regions Padang Pakamat-
3 and Padang Pak Amat-4, which was 100%. This was followed by Kota Bharu stables, which was 42.52%, and ranged from free stables in sub-regions Kota-1,
Panji-2 and Kota-2 to the highest in stables at sub-region Sering, which was
85.71% (Table 3.13) (Fig 3.12).
Lower prevalence of both protozoa in Machang stables was 16.66%, and ranged from 16.66% to 83.33% in stables in sub-regions of Hulu Sat-1 and Hulu
Sat-2. Furthermore, the stables in sub-regions of Tanah Merah and Gua Musang reported free from both protozoa infections (Table 3.13) (Fig. 3.12).
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Table 3.13: Prevalence of T. equi, B. caballi and both protozoa in sampling stables in different regions and sub-regions in Kelantan state.
No. of Both T. equi B. caballi stables Sub-regions protozoa No. of No. No. Regions (Equids No. equids positive positive sampling stables) positive (%) (%) (%) 1 Kemumin-1 19 19(100) 12(63.15) 12(63.15) 2 Sering 7 6(85.71) 6(85.71) 6(85.71) 3 Kubang kerian-1 3 3(100) 1(33.33) 1(33.33) 4 Panji-1 12 9(75) 9(75) 7(58.33) 5 Kota-1 5 3(60) 0(0.00) 0(0.00) Kota 6 Panji-2 1 0(0.00) 1(100) 0(0.00) Bharu 7 Panchor 10 5(50) 10(100) 5(50) 8 Kubang kerian-2 4 2(50) 3(75) 1(25) 9 Kota-2 3 1(33.33) 1(33.33) 0(0.00) 10 Kemumin-2 8 3(37.5) 6(75) 2(25) 11 Wakaf Che Yeh 3 3(100) 2(66.66) 2(66.66) 12 Kubang kerian-3 12 6(50) 3(25) 1(8.33) Total 87 60 (68.96) 54 (62.06) 37 (42.52) 13 Batu Karang 8 7(87.5) 7(87.5) 6(75) 14 Tunjong 3 0(0.00) 2(66.66) 0(0.00) Pasir Mas 15 Pasir Pekan 4 1(25) 3(75) 1(25) 16 Pasir Mas 3 1(33.33) 3(100) 1(33.33) 17 Kubang Gadong 8 3(37.5) 8(100) 3(37.5) Total 26 12 (46.15) 23 (88.46) 11 (42.30) 18 Hulu Sat-1 6 2(33.33) 5(83.33) 1(16.66) Machang 19 Hulu Sat-2 6 0(0.00) 1(16.66) 0(0.00) Total 12 2 (16.66) 2 (16.66) 1 (8.33) 20 Sering 3 1(33.33) 2(66.66) 1(33.33) 21 Tawang-1 6 0(0.00) 5(83.33) 0(0.00) 22 Tawang-2 7 3(42.85) 7(100) 3(42.85) 23 Tawang-3 9 2(22.22) 9(100) 2(22.22) 24 Tawang-4 5 0(0.00) 5(100) 0(0.00) Bachok 25 Perupok-1 3 1(33.33) 3(100) 1(33.33) 26 Perupok-2 7 1(14.28) 7(100) 1(14.28) 27 Perupok-3 3 1(33.33) 3(100) 1(33.33) 28 Perupok-4 3 0(0.00) 3(100) 0(0.00) 29 Melawi 13 9(69.23) 11(84.61) 7(53.84) Total 59 18 (30.5) 55 (93.22) 17 (28.81)
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Both T. equi B. caballi Sub-regions No. of protozoa No. of No. No. Regions (Equids sampling equids No. stables positive positive stables) tested positive (%) (%) (%) 30 Padang Pak Amat-1 7 7(100) 6(85.71) 6(85.71) 31 Padang Pak Amat-2 2 2(100) 1(50) 1(50) 32 Padang Pak Amat-3 1 1(100) 1(100) 1(100) 33 Padang Pak Amat-4 3 3(100) 3(100) 3(100) Pasir 34 Gong Jernih-1 7 6(85.71) 5(71.42) 5(71.42) Puteh 35 Gong Jernih-2 3 3(100) 2(66.66) 2(66.66) 36 Gong Jernih-3 2 1(50) 1(50) 1(50) 37 Gong Datok-1 10 9(90) 5(50) 4(40) 38 Gong Datok-2 5 4(80) 4(80) 3(60) 39 Semerak 11 11(100) 9(81.81) 9(81.81) Total 51 47 (92.15) 37 (72.54) 35 (68.62) 40 Seria 4 0(0.00) 4(100) 0(0.00) 41 Belukar-1 2 2(100) 1(50) 1(50) 42 Belukar-1 10 6(60) 2(20) 1(20) 43 Kebakat 5 1(20) 1(20) 1(20) 44 Kok Seraya 5 1(20) 3(60) 0(0.00) Tumpat 45 Gelang Mas 5 0(0.00) 0(0.00) 0(0.00) 46 Belukar Perbak 5 1(20) 2(40) 1(20) 47 Chabang Empat 4 1(25) 1(25) 0(0.00) 48 Getting Tumpat 1 0(0.00) 1(100) 0(0.00) 49 Jubakar Rantai 3 0(0.00) 0(0.00) 0(0.00) Total 44 12 (27.27) 15 (34.09) 4 (9.09) 50 Kusial-1 2 1(50) 0(0.00) 0(0.00) Tanah 51 Kusial-2 9 1(11.11) 2(22.22) 0(0.00) Merah 52 Kusial-3 9 2(22.22) 1(11.11) 0(0.00) Total 20 4 (20) 7 (35) 0 (0.00) Gua 53 Galas Telaga 7 2(28.57) 0(0.00) 0(0.00) Musang Total 306 157(51.30) 193(63.07) 105(34.31)
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Figure 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.
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Figure 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.
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Figure 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.
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3.3.5 Risk factors associated with seroprevalence of EP causative agents
Based on cELISA, the results of this study demonstrate significant
(P<0.0001) differences between all types of protozoa. The seroprevalence of B.
caballi was significantly higher (RR: 1.83 times, CI: 1.53-2.19) than T. equi and
both protozoa (Table 3.14).
In the present study, the prevelance of T. equi, B. caballi and both
protozoa did not differ significantly between ponies and horses (Table 3.15).
The prevelance of EP causative agents was significant between genders
of equids (P<0.05). The seroprevalence of T. equi, B. caballi and both protozoa
was significantly higher among gelding equids (RR: 2.61, 1.92 and 4.50 times
respectively) compared to males and females. The females had a significantly
higher prevalence than males for all types of protozoa (RR: 1.42, 1.27 and 1.65
times) respectively (Table 3.15).
The seroprevalence of T. equi, B. caballi and both protozoa infections
was significantly higher among equids >5 years old (RR: 2.78, 1.96 and 2.93
times respectively) and among >2-5 years old (RR: 2.17, 1.87 and 2.74 times
respectively) compared to <1 year old. In general, the prevelance was significantly
higher among older equids than younger ones for all type of protozoa (P<0.05)
(Table 3.15).
The seroprevalence of T. equi and both protozoa did not differ
significantly among breeds of horses, whereas, the prevalence of B. caballi
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infection was significantly higher among thoroughbred horses (RR: 2.58 times,
CI: 0.79-8.45) compared to Arab breed (P<0.05) (Table 3.15).
The seroprevalence of T. equi and both protozoa was significantly higher among pony A breeds (RR: 1.57 and 2. 39 times respectively) compared to pony
C and pony B for (P<0.05), while the prevalence of B. caballi infection did not differ significantly among breeds of ponies (Table 3.15).
The seroprevalence of T. equi, B. caballi and both protozoa infections was significantly higher among imported equids (RR: 1.46, 1.24 and 1.89 times respectively) compared to local equids (P<0.05) (Table 3.15).
This study also demonstarated that there was no significant difference in the seroprevelance of T. equi, B. caballi and both protozoa among exporting countries of equids (Table 3.15).
The prevalence of all type of protozoa was significantly higher among sports equids (RR: 3.67, 3.20 and 6.43 times respectively) and among equids used for recreation (RR: 2.50, 2.23 and 4.04 times respectively) compared to those used for breeding (P<0.0001) (Table 3.15).
The present study showed that the seroprevalence of T. equi, B. caballi and both protozoa infections was significantly higher among pregnant mares (RR:
1.38, 1.40 and 1.73 times respectively) compared to non-pregnant (P<0.01).
Besides, the seroprevalence of T. equi infection was significantly higher in mares in the third stage of pregnancy (RR: 2.59 times, CI: 1.01-6.62) compared to mares in the first stage of pregnancy (P<0.0001) (Table 3.15).
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The prevalence of all type of protozoa was significantly higher among equids infested with ticks (4.09, 6.42 and 3.56 times respectively) compared to equids not infested with ticks (P<0.0001) (Table 3.15).
In general, the seroprevalence of of T. equi, B. caballi and both protozoa infections was significantly affected by geographical regions in Kelantan
(P<0.05). The prevalence of T. equi and both protozoa infections was significantly higher in Pasir Puteh region with 92.15% and 68.62 respectively (RR: 5.52 and
8.23 times respectively) compared to Machang region, which was 16.66% and
8.33 respectively, whereas, the seroprevalence of B. caballi infection was significantly higher in Bachok region which was 93.22% (RR: 5.59 times, CI:
1.57-19.85) compared to Machang region, which was 16.66% (Table 4.16).
The seroprevelance of T. equi, B. caballi and both protozoa infections was also significantly affected by sampling months (P<0.05). The seroprevalence of T. equi and both protozoa was significantly higher in September, which was
90.9% and 72.72% respectively (RR: 4.45 and 9.69 times respectively) compared to November, which was 20% and 7.5% respectively, whereas, the seroprevalence of B. caballi was significantly higher in December, which was 91.48% (RR: 5.22 times, CI: 2.65-10.30) compared to November, which was 17.5% (Table 3.17)
(Fig. 3.13).
The current study has demonstrated that the seroprevalence of T. equi, B. caballi and both protozoa infections was significantly higher among equids mixed with other animals in the stable (RR: 1.50, 1.44 and 1.75 times respectively) compared to equids isolated from other animals in the stable (P<0.0001). The
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seroprevelance of all type of protozoa was significantly higher among equids in grazing (RR: 1.64, 1.58 and 1.82 times respectively) than equids kept in the stable
(P<0.0001) (Table 3.18).
The seroprevalence of T. equi, B. caballi and both protozoa infections was significantly higher when ticks were found in the stable (RR: 1.92, 1.35 and
1.93 times respectively) compared to stables with no ticks (P<0.0001). The seroprevalence of all types of protozoa was significantly higher when ticks infested equids and nearby animals in the stable (RR: 1.36, 1.39 and 2.47 times respectively) compared to only nearby animals in the stable beign infested by ticks (P<0.05). (Table 3.18).
Regarding the climatic factors, this study noted that the seroprevalence of
T. equi, B. caballi and both protozoa was significantly higher when the means of monthly temperature ranged from 26.4ºC to 28.5ºC (RR: 2.16, 1.33 and 4.08 times respectively) compared to the the means of monthly temperature ranging from
25.6ºC to 25.8ºC (P<0.05) (Table 3.19) (Fig. 3.14). The seroprevalence of all types of protozoa was significantly higher when the mean of rainfall amount ranged from 0.6mm to 95mm (Low) and when it reanged from 220.2mm to
289.4mm (Moderate), compared to the mean of rainfall amount ranging from
500.2mm to > 500.2 (Very high) (Table 3.19) (Fig. 3.15). In addition, the seroprevalence of T. equi, B. caballi and both protozoa was significantly higher when the mean of monthly relative humidity ranged from 80.9% to 86.6% (RR:
1.65, 1.97 and 3.33 times respectively) compared to the mean of monthly relative humidity ranging from 77.1% to 77.7% (P<0.05) (Table 3.19) (Fig. 3.16).
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Table 3.14: Relative risk of EP associated with the type of protozoa in equids.
No. No. positive Type of protozoa equids RR 95% CI P (%) tested a Both protozoa 105 (32.31) 1
b T. equi 306 157 (51.30) 1.49 1.23-1.80 0.00
c B. caballi 193 (63.07) 1.83 1.53-2.19 0.00
RR) Relative risk; 95% CI) 95% confidence interval; P) P value; Values significantly different (P a, b or c < 0.05) between stables factors are labelled with the different letters ( ).
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Table 3.15: Relative risk of equids factors associated with seropositivity of T. equi, B. caballi and both protozoa.
No. T. equi infection B. caballi infection Both protozoa infections Factors equids n. (%) RR (95% CI) P n. (%) RR (95% CI) P n. (%) RR (95% CI) P tested
Type of equids a a a Horse 148 75 (50.67) 1 92 (62.16) 1 49 (33.10) 1 a a a Pony 158 82 (51.89) 0.97 (0.78-1.21) 0.92 101 (63.92) 1.02 (0.86-1.22) 56 (35.44) 1.07 (0.78-1.46) 0.75 Gender a a a Male 81 31 (38.27) 1 42 (51.85) 1 18 (22.22) 1 Female 218 119 (54.58)b 1.42 (1.05-1.92) 0.01 144 (66.05)b 1.27 (1.01-1.60) 0.03 80 (36.69)b 1.65 (1.06-2.57) 0.02 Gelding 7 7 (100)c 2.61 (1.98-3.44) 0.00 7 (100)c 1.92 (1.56-2.37) 0.01 7 (100)c 4.50 (2.99-6.76) 0.00 Age a a a <1 year 23 5 (21.73) 1 8 (34.78) 1 3 (13.04) 1 >1-2 years 26 9 (34.61)a,b 1.59 (0.62-4.06) 0.36 12 (46.15)a,b 1.32 (0.66-2.66) 0.60 6 (23.07)a,b 1.76 (0.49-6.28) 0.47 >2-5 years 95 45 (47.36)b 2.17 (0.97-4.87) 0.03 62 (65.26)b,c 1.87 (1.05-3.34) 0.01 34 (35.78)b 2.74 (0.92-8.15) 0.04 >5 y ears 162 98 (60.49)c 2.78 (1.26-6.10) 0.00 111 (68.51)c 1.96 (1.11-3.48) 0.00 62 (38.27)b 2.93 (1.00-8.58) 0.01 Breed of horse a a a Arab breed 7 3 (42.85) 1 2 (28.57) 1 1 (14.28) 1 Crossbred 95 45 (47.36)a 1.10 (0.45-2.66) 0.87 56 (58.94)a,b 0.48 (014-1.58) 0.24 28 (29.47)a 0.48 (0.07-3.05) 0.66 Thoroughbred 46 27 (58.69)a 1.36 (0.56-3.33) 0.70 34 (73.91)b 2.58 (0.79-8.45) 0.02 20 (43.47)a 0.32 (0.05-2.07) 0.22 Breed of pony b a b Pony C 31 13 (41.93) 1 17 (54.83) 1 7 (22.58) 1 Pony B 56 25 (44.64)b 1.06 (0.64-1.76) 0.98 36 (64.28)a 1.17 (0.8-1.7) 0.52 13 (23.21)b 1.02 (0.45-2.30) 0.84 Pony A 50 33 (66)a 1.57 (0.99-2.49) 0.05 37 (74)a 1.34 (0.94-1.93) 0.12 27 (54)a 2.39 (1.18-4.81) 0.01 Crossbred 21 11 (52.38)a,b 1.24 (0.69-2.23) 0.64 11(52.38)a 0.95 (0.56-1.60) 0.91 9 (42.85)a,b 1.89 (0.83-4.29) 0.21
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No. T. equi infection B. caballi infection Both protozoa infections Factors equids n. (%) RR (95% CI) P n. (%) RR (95% CI) P n. (%) RR (95% CI) P tested Origin of equids a a a Local 232 107 (46.12) 1 138 (59.48) 1 66(28.44) 1 Imported 74 50 (67.56)b 1.46 (1.18-1.80) 0.00 55 (74.32)b 1.24 (1.05-1.48) 0.03 39(52.70)b 1.85 (1.37-2.49) 0.00 Exporting countries a a a New Zealand 5 2 (40) 1 2(40) 1 2(40) 1 a a a France 4 2 (50) 0.80 (0.18-3.42) 1.00 3 (75) 0.53 (0.15-1.79) 2(50) 0.80 (0.18-3.42) 1.00 India 5 3 (60)a 0.66 (0.18-2.42) 1.00 3 (60)a 0.66 (0.18-2.42) 1.00 2(51.61)a 1.00 (0.21-4.56) 1.00 Australia 6 4 (66.66)a 0.60 (0.17-2.01) 0.56 4 (66.66)a 0.60 (0.17-2.01) 0.56 3(42.85)a 0.80 (0.20-3.05) 1.00 Thailand 30 20 (66.66)a 0.60 (0.19-1.80) 0.33 24 (80)a 0.50 (0.16-1.48) 0.17 16(40)a 0.75 (0.24-2.30) 0.65 a a a Singapore 4 3 (75) 0.53 (0.15-1.79) 0.52 3 (75) 0.53 (0.15-1.79) 0.52 2(50) 0.80 (0.18-3.42) 1.00 Argentina 20 16 (80)a 0.50 (0.16-1.49) 0.11 16(80)a 0.50 (0.16-1.49) 0.11 12(60)a 0.66 (0.21-2.06) 0.62 Purpose of keeping a a a Breeding 87 19 (21.83) 1 26 (29.88) 1 8 (9.19) 1 b b b Recreation 148 81 (54.72) 2.50 (1.64- 3.82) 0.00 99 (66.89) 2.23 (1.59-3.14) 0.00 55 (37.16) 4.04 (2.02-8.07) 0.00 c c c Sports 71 57 (80.28) 3.67 (2.43-5.56) 0.00 68 (95.77) 3.20 (2.31-4.43) 0.00 42 (59.15) 6.43 (3.23-12.8) 0.00 Pregnancy a a a No 148 72 (48.64) 1 89 (60.13) 1 45 (30.40) 1 b b b Yes 70 47 (67.14) 1.38 (1.09-1.74) 0.01 59 (84.28) 1.40 (1.18-1.65) 0.00 37 (52.85) 1.73 (1.25-2.41) 0.00 Stage of pregnancy a a a First stage < 3 9 3 (33.33) 1 7 (77.77) 1 3 (33.33) 1 Second stage 3-7 39 25 (64.10)a,b 1.92 (0.74-4.98) 0.13 31 (79.48)a 1.02 (0.69-1.50) 0.73 20 (51.28)a 0.65 (0.24-1.72) 0.54 Third stage > 7 22 19 (86.36)b 2.59 (1.01-6.62) 0.00 21 (95.45)a 1.22 (0.85-1.76) 0.39 14 (63.63)a 0.52 (0.19-1.39) 0.25 Tick found on equids a a a No 272 127 (46.69) 1 160 (58.82) 1 75 (27.57) 1 b b b Yes 34 31(91.17) 4.09 (2.86-5.86) 0.00 34 (100) 6.42 (4.38-9.40) 0.00 31 (91.17) 3.56 (2.30-5.50) 0.00
n.) Number of positive; %) Prevalence; RR) Relative risk; 95% CI) 95% confidence interval; P) P value; Values significantly different (P < 0.05) between stables factors are labelled with the different letters (a, b or c).
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Table 3.16: Relative risk of regional factors associated with seropositivity of T. equi, B. caballi and both protozoa.
No. T. equi infection B. caballi infection Both protozoa infections Regions risk No. Equids factors Stables n. (%) RR (95% CI) P n. (%) RR (95% CI) P n. (%) RR (95% CI) P tested a a,b a Machang 2 12 2 (16.66) 1 2 (16.66) 1 1 (8.33) 1
a b a Tanah Merah 3 20 4 (20) 1.20 (0.25-5.59) 0.34 7 (35) 2.10 (0.51-8.50) 0.42 0 (0.00) ------
a b a Tumpat 10 44 12 (27.27) 1.63 (0.42-6.33) 0.70 15 (34.09) 2.04 (0.54-7.73) 0.30 4 (9.09) 1.09 (0.13-8.87) 1.00
a a a Gua Musang 1 7 2 (28.5) 1.71 (0.30-9.61) 0.60 0 (0.00) ------0 (0.00) ------
a d a,b Bachok 10 59 18 (30.5) 1.83 (0.48-6.86) 0.48 55 (93.22) 5.59 (1.57-19.85) 0.00 17 (28.81) 3.45 (0.50-23.55) 0.27
a,b c,d b,c Pasir Mas 5 26 12 (46.15) 1.99 (0.49-7.30) 0.07 23 (88.46) 5.30 (1.48-18.95) 0.00 11 (42.30) 5.07 (0.73-34.95) 0.05
b,c c b,c Kota Bharu 12 87 60 (68.96) 4.13 (1.15-14.77) 0.00 54 (62.06) 3.72 (1.03-13.33) 0.00 37 (42.52) 5.10 (0.76-33.86) 0.02
c c,d c Pasir Puteh 10 51 47 (92.15) 5.52 (1.55-19.64) 0.00 37 (72.54) 4.35 (1.21-15.59) 0.00 35 (68.62) 8.23 (1.24-54.27) 0.00
n.) Number of positive; %) Prevalence; RR) Relative risk; 95% CI) 95% confidence interval; P) P value; Values significantly different (P < 0.05) between stables factors are labelled with the different letters (a, b or c).
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Table 3.17: Relative risk of monthly factors associated with seropositivity of T. equi, B. caballi and both protozoa.
Months No. of T. equi infection B. caballi infection Both protozoa infections No. of risk Stables Equids n. (%) RR (95% CI) P n. (%) RR (95% CI) P n. (%) RR (95% CI) P factors tested a a a November 8 40 8 (20) 1 7 (17.5) 1 3 (7.5) 1
a,b c,b a February 7 52 18 (34.61) 1.73 (0.83-3.56) 0.19 40 (76.92) 4.39 (2.20-8.75) 0.00 7 (13.46) 1.79 (0.49-6.50) 0.28
b,c b a,b January 13 60 29 (48.33) 2.41 (1.23-4.73) 0.00 24 (40) 2.28 (1.08-4.79) 0.00 14 (23.33) 3.11 (0.95-10.13) 0.05
c,d d b,c December 11 47 27 (57.44) 2.87 (1.47-5.59) 0.00 43 (91.48) 5.22 (2.65-10.30) 0.03 24 (51.06) 6.80 (2.21-20.94) 0.00
c,d c c October 6 46 27 (58.69) 2.93 (1.50-5.70) 0.00 29 (63.04) 3.60 (1.77-7.31) 0.00 18 (39.13) 5.21 (1.65-16.41) 0.00
d,e d c,d March 5 39 28 (71.79) 3.58 (1.87-6.87) 0.00 33 (84.61) 4.38 (2.43-9.60) 0.00 23 (58.97) 7.86 (2.56-24.08) 0.00
e c,d d September 3 22 20 (90.9) 4.54 (2.41-8.56) 0.00 17 (77.27) 4.41 (2.17-8.98) 0.00 16 (72.72) 9.69 (3.17-29.66) 0.00
n.) Number of positive; %) Prevalence; RR) Relative risk; 95% CI) 95% confidence interval; P) P value; Values significantly different (P < 0.05) between stables factors are labelled with the different letters (a, b or c).
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Figure 3.13: Seroprevalence of T. equi, B. caballi and both protozoa by the sampling months. * * * * * * * * * * * * * * *
Figure 3.13: Seroprevalence of T. equi, B. caballi and both protozoa by months; *) Values significantly different (P < 0.05) compared to November month.
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Table 3.18: Relative risk of management and ticks factors associated with seropositivity of T. equi, B. caballi and both protozoa.
No. T. equi infection B. caballi infection Both protozoa infections No. Factors Equids RR RR RR Stables n. (%) P n. (%) P n. (%) P tested (95% CI) (95% CI) (95% CI) Animals in stables a a Only equids 26 133 53 (39.84) 1 67 (50.37) 1 1 b b b Mixed with other 27 173 104 (60.11) 1.50 0.00 126 (72.83) 1.44 0.00 73 (42.19) 1.75 0.00 animals (1.18-1.92) (1.19-1.75) (1.23-2.48) Management a a In stable 47 273 131 (47.98) 1 162 (59.34) 1 1 b b b In grazing 6 33 26 (78.78) 1.64 0.00 31 (93.93) 1.58 0.00 19 (57.57) 1.82 0.00 (1.32-2.03) (1.38-1.80) (1.29-2.57) Ticks found in stables a a No 36 195 75 (38.46) 1 109 (55.89) 1 1 b b b Yes 17 111 82 (73.87) 1.92 0.00 84 (75.67) 1.35 0.00 55 (49.54) 1.93 0.00 (1.55-2.36) (1.14-1.59) (1.42-2.61) Presence of ticks a a On nearby animals in 9 54 36 (66.66) 1 37 (68.51) 1 1 the stable a,b a a At least one equid in 5 34 25 (73.52) 1.10 0.65 25 (73.52) 1.07 0.79 16 (47.05) 1.33 037 the stable (0.83-1.45) (0.81-1.40) (0.80-2.22) b b b On equids and nearby 3 23 21 (91.30) 1.36 0.02 22 (95.65) 1.39 0.00 20 (86.95) 2.47 0.00 animals in the stable (1.09-1.71) (1.14-1.70) (1.66-3.66)
n.) Number of positive; %) Prevalence; RR) Relative risk; 95% CI) 95% confidence interval; P) P value; Values significantly different (P < 0.05) between stables factors are labelled with the different letters (a, b or c).
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Table 3.19: Relative risk of climatic factors associated with seropositivity of T. equi, B. caballi and both protozoa.
No. of T. equi infection B. caballi infection Both protozoa infections Factors equids n. (%) RR (95% CI) P n. (%) RR (95% CI) P n. (%) RR (95% CI) P tested
Means of monthly temperature a a 25.6 – 25.8 ºC 92 26 (28.26) 1 47 (51.08) 1 10 (10.86) 1 b b 26.4 – 28.5 ºC 214 131 (61.21) 2.16(1.53-3.05) 0.00 146 (68.22) 1.33 (1.07-1.66) 0.00 95 (44.39) 4.08 (2.23-7.47) 0.00 Means of monthly rainfall amount 500.2 – > 500.2 40 8 (20) 1 7 (17.5) 1 3 (7.5) 1 (Very high) 220.2 – 289.4 mm 153 83 (54.24) 2.71 (1.43-5.12) 0.00 96 (62.74) 3.58 (1.80-7.10) 0.00 56 (36.60) 4.88 (1.61-14.78) 0.00 (Moderate) 0.6 – 95.0 (Low) 113 66 (58.40) 2.92 (1.45-5.53) 0.00 90 (79.64) 4.55 (2.30-8.97) 0.00 46 (40.70) 5.42 (1.78-16.48) 0.00 Means of monthly relative humidity 77.1 – 77.7 % 152 61 (40.13) 1 71 (46.71) 1 24 (15.78) 1 88.9 – 86.9 % 154 102 (66.23) 1.65 (1.31-2.06) 0.00 122 (79.22) 1.97 (1.59-2.43) 0.00 81 (52.59) 3.33 (2.24-4.95) 0.00
n.) Number of positive; %) Prevalence; RR) Relative risk; 95% CI) 95% confidence interval; P) P value; Values significantly different (P < 0.05) between stables factors are labelled with the different letters (a, b or c).
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* * * * * * * * * * * * *
* *
Figure 3.14: Seroprevalence of T. equi, B. caballi and both protozoa by means of monthly temperature (ºC); *) Values significantly different (P < 0.05) compared to November month.
Figure 3.15: Seroprevalence of T. equi, B. caballi and both protozoa by means of monthly rainfall amount (mm); *) Values significantly different (P < 0.05) compared to November month.
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Figure 3.16: Seroprevalence of T. equi, B. caballi and both protozoa by means of monthly relative humidity (%); *) Values significantly different (P < 0.05) compared to November month.
3.3.6 Ixodid ticks: identification and infestation rate
Out of 53 stables in Kelantan visited during the study, 17 (32.07%)
were infested with Ixodid ticks. The stables infested rate with Ixodid ticks was
higher in Machang (100%) and lower in Pasir Puteh (10%), whereas, stables in
Pasir Mas and Gua Musang were absent from Ixodid ticks (Table 3.20).
A total of 533 ticks were collected from equids and nearby animals such
as cattle, sheep, goats, and dogs. After concluding microscopic examination,
comparison was made of photographs with the morphotoxonomic features in the
identification standard keys available for this purpose (Walker et al., 2000;
Bouattour, 2002; Pavlidou et al., 2008). Eight species of Ixodid tick
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belonging to three genera were recorded during this study. The tick species were Rhipicephalus (Boophilus) microplus, Rh. (Boophilus) annulatus, Rh. appendeculataus, Rh. bursa, Rh. sanguineus, Haemaphysalis punctata, H. longicornis, and Dermacentor marginatus. These species were reported for the first time in Kelantan, Malaysia, except Rhipicephalus (Boophilus) microplus and Rh. Sanguineus, which had been identified previously. The identified
Ixodid ticks were in various stages (nymph, larvae and adult) and sex (male and female engorged or non-engorged) (Table 3.21) (Fig. 3.17).
In this study, most types of stables with Ixodid tick presence showed highly seropositive equids with piroplasms infections reaching 100% (Table
3.21).
Fifty-three Ixodid ticks, including all species identified in this study were seen on 34 of 306 equids examined. The predilection sites of attachment of these species on equids were inside and outside the ears, face, neck, shoulder, flank and genital area (Table 3.22).
Five Ixodid tick species were identified from 54 of 78 cattle (calves and cows) near and in equids stables including Rh. (Boophilus) annulatus, Rh.
(Boophilus) microplus, Rh. bursa, Rh. sanguineus, and H. punctata. Five species of Ixodid ticks were identified from 24 of 92 sheep near and in equids stables, including Rh. (Boophilus) annulatus, Rh. (Boophilus) microplus, Rh. appendeculataus, Rh. bursa, D. marginatus. four species from 44 of 136 goats in different equids stables, including Rh. (Boophilus) annulatus, Rh.
(Boophilus) microplus, H. punctata, D. marginatus (Table 3.22).
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Three species of Ixodid ticks were identified from 12 of 24 dogs in equids stables, including Rh. (Boophilus) microplus, Rh. bursa and Rh. sanguineus (Table 3.22).
The present study revealed that the infested rate of Ixodid ticks on equids, cattle, sheep, goats, and dogs was 11.11%, 69.23%, 26.08%, 32.35% and 50% respectively (Fig. 3.18). Rhipicephalus bursa was the most common tick species infesting equids with 33.96%. Rhipicephalus (Boophilus) microplus was the most common tick species infesting cattle, sheep and goats, with 52.15%, 34.75% and 62.50% respectively. Furthermore, Rh. sanguineus was the most common tick species infesting dogs, with 54.54%, (Table 3.23).
Rh. (Boophilus) microplus and Rh. (Boophilus) annulatus were the predominant ticks reported in Kelantan with 45.4%, 27.39% respectively, while, Haemaphysalis longicornis was reported in a low percentage in
Kelantan at 0.18% (Fig. 3.19).
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Table 3.20: The stables infested rate with Ixodid ticks in various regions in Kelantan.
No. of No. of infested Infestation % Region stables field Kota Bharu 11 6 54.54
Machang 2 2 100.0
Bachok 10 5 50.0
Pasir Puteh 10 1 10.0
Pasir Mas 5 0 0
Tanah Merah 3 1 33.3
Tumpat 11 2 18.18
Gua Musang 1 0 0
Total 53 17 32.07
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Table 3.21: Details and number of identified Ixodid tick species infesting equids and nearby animals related to the prevalence of EP in each field.
Presence of Type Type of animal Total No. No. of No. of No. & species of No. of ticks in of (No. of collected ticks) No. of of engorged Normal identification ticks seropositive each stable stable ticks Male female female equids for EP (%) Mixed Equids (5), Cattle (0) 5 3 1 1 1RBM, 1RBA, 3RB 9/12 (75%) At least one Mixed Equids (10), Goats (0) 10 4 3 3 3RBM, 6RB, 1HP 3/3 (100%) equid Mixed Equids (10), Cattle (0) 10 3 3 4 1RBM, 2RBA, 4RB, 3DM 6/6 (100%) infested Non Equids (8) 8 6 1 1 2RBA ,5RB, 1RA 1/6 (16.66%) Non Equids (2) 2 0 2 0 1HL, 1DM 3/9 (33.33%) Mixed Horse (0), Dogs (5), Cattle (54) 59 21 10 28 24RBM, 16RBA, 14RS, 5RB 19/19 (100%) Mixed Horse (0), Sheep (18), Goats (29) 47 15 12 20 34RBA, 5RA, 8DM 6/7 (85.71%) Mixed Horse (0), Sheep (15) 15 3 3 9 12RBM, 3RB 1/2 (50%) Only on Mixed Horse (0), Dogs (5), Sheep (20) 25 3 16 6 16RBA, 5RS, 4DM 3/3 (100%) nearby Mixed Horse (0), Sheep (10), Goats (56) 66 27 15 24 56RBM, 4RA, 6HP 3/3 (100%) animals Mixed Horse (0), Cattle (38), Goats (18) 56 17 32 7 36RBM, 17RBA, 3DM 3/3 (100%) Mixed Horse (0), Dogs (21) 21 10 3 8 7RBM, 8RB, 6RS 4/4 (100%) Mixed Horse (0), Sheep (6), Goats (9) 15 6 7 2 11RBM, 3HP, 1DM 2/2 (100%) Mixed Horse (0), Cattle (62) 62 28 18 16 32RBM, 23RBA, 7HP 6/10 (60%) On equids Mixed Equids (7), Dogs (11), Cattle (52) 70 22 18 30 31RBM, 18RBA, 11RB, 10RS 5/6 (83.33%) and nearby Mixed Equids (8), Cattle (37) 45 9 8 28 26RBM, 9RBA, 6HP, 4RA 7/7 (100%)
animals Mixed Equids (3), Dogs (2), Cattle (12) 17 8 3 6 5RBM, 7RBA, 2RB, 3RS 10/10 (100%)
No.) Number of samples; RBM) Rhipicephalus (Boophilus) microplus; RBA) Rh. (Boophilus) annulatus; RB) Rh. bursa; RA) Rh. appendeculataus; RS) Rh. sanguineus; HP) Haemaphysalis punctata; HL) H. longicornis; and DM) Dermacentor marginatus.
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A C E G I
B D F H J
K M O Q T
L N P S U
Figure 3.17: A&B) Rhipicephalus (Boophilus) microplus dorsal & ventral view male; C&D); Rhipicephalus Boophilus annulatus female; E&F) Rhipicephalus sanguineous dorsal & ventral view female; G&H) Rhipicephalus appendeculataus dorsal & ventral view male; I&J) Rhipicephalus bursa dorsal & ventral view female; K&L) Dermacentor marginatus dorsal & ventral view male; M&N) Haemaphysalis punctata dorsal & ventral view engorged female; O&P) Haemaphysalis Longicornis dorsal & ventral view engorged female; Q&S) Larval stage of H. punctata dorsal & ventral view normal female; T&U) Larval stage of H. punctata dorsal & ventral view engorged female.
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Table 3.22: Distribution of identified Ixodid tick species and their predilection sites on equids and nearby animals.
Type of No. of No. of No. of Species of Site of collection animal observed infested ticks tick Ears, Face, , neck, RBM, RBA, Equids 306 34 53 shoulder, flank, RB, RS, RA, genital area HP, HL, DM Face, ears, neck, RBM, RBA, Cattle 78 54 255 dewlap, shoulder, RB, RS, HP belly, legs, udder RBM, RBA, Ear, axilla, Sheep 92 24 69 RB, RA, perineum, udder DM Ears, neck, axilla, RBM, RBA, Goats 136 44 112 perineum HP, DM Face, ears, neck, RBM, RB, Dogs 24 12 44 axilla, perineum, RS
RBM) Rhipicephalus (Boophilus) microplus; RBA) Rh. (Boophilus) annulatus; RB) Rh. bursa; RA) Rh. appendeculataus; RS) Rh. sanguineus; HP) Haemaphysalis punctata; HL) H. longicornis; and DM) Dermacentor marginatus.
Figure 3.18: Pie chart showing the infested rate of Ixodid tick on equids and nearby animals.
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Table 3.23: The abundance and infested rate of Ixodid tick species on equids and nearby animals.
Ticks species No. of ticks Host Equids (%) Cattle (%) Sheep (%) Goats (%) Dogs (%)
RBM 244 10 (18.86) 133 (52.15) 24 (34.78) 70 (62.50) 7 (15.90)
RBA 146 6 (11.32) 90 (35.29) 21 (30.43) 29 (25.89) 0 (0.00)
RB 47 18 (33.96) 13 (5.09) 3 (4.34) 0 (0.00) 13 (29.54)
RS 38 4 (7.54) 10 (3.92) 0 (0.00) 0 (0.00) 24 (54.54)
RA 14 5 (9.43) 0 (0.00) 9 (13.04) 0 (0.00) 0 (0.00)
DM 20 4 (7.54) 0 (0.00) 12 (17.39) 4 (3.57) 0 (0.00)
HP 23 5 (9.43) 9 (3.52) 0 (0.00) 9 (8.03) 0 (0.00)
HL 1 1 (1.88) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) Total 533 53 255 69 112 44
RBM) Rhipicephalus (Boophilus) microplus; RBA) Rh. (Boophilus) annulatus; RB) Rh. bursa; RA) Rh. appendeculataus; RS) Rh. sanguineus; HP) Haemaphysalis punctata; HL) H. longicornis; and DM) Dermacentor marginatus.
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Ixodid tick species in Kelantan 50
45
40 35 30 25
20 Percentage % Percentage 15 10 5 0 RBM RBA RB RS RA DM HP HL
Figure 3.19: The infested rate of Ixodid tick species in Kelantan.
3.4 Discussion
EP is a global problem due to limitations in meeting international
regulations with regard to exportation or involvement in equestrian sporting
events (Friedhoff et al., 1990). This is the first epidemiological study of T. equi
and B. caballi infections in Kelantan using different methods to detect the disease
which are microscopic examination method, cELISA and multiplex PCR.
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3.4.1 Morphological and biometerical findings with parasitemia
The microscopic detection of piroplasms by visual examination of blood
smears stained with Giemsa is the simplest, most rapid and accessible diagnostic
method for confirming the clinical cases of piroplasms (Irwin, 2010). The present
study primarily identified the equine piroplasms based on morphological features
and biometrical data, which indicated that T. equi was smaller than B. caballi,
which is in agreement with Soulsby, (1982) and Malekifard et al. (2014), who
described T. equi as a small Theileria spp. being 2 – < 2.5μm and 1.14–1.6μm in
length, and 1–2 μm and 1.4–1.88μm in diameter respectively, according to the
parasite shape. On the other hand, they described B. caballi as a larger Babesia
spp. measuring 2.5–4 (> 2.5)μm and 2.6–3.53μm in the length, 1.3–3.0μm and
2.88–3.91μm in diameter respectively, was according to the parasite shape. Ueti et
al., (2008) added that B. caballi sometime measuring 6μm in the length and 2μm
in diameter.
In examining the thin and thick blood smears , it was observed that the
various stages and shapes of T. equi and B. caballi were in agreement with
Soulsby, (1982); Ueti et al. (2005); Alsaad et al. (2010) and Malekifard et al.
(2014). Microschizonts and macroschizonts stages of T. equi were also detected in
lymphocytes of examining blood smears thus indicating an acute form of the
disease (Mehlhorn & Schein, 1998).
The result of this study confirmed the simultaneous infection of equids
with both T. equi and B. caballi with a parasitemia ranging between 1% and
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18.2%, which was consistent with that observed by Alsaad and Al-Mola, (2006)
and Alsaad et al. (2010), who found the percentage of hemoparasitism
(parasitemia) for both protozoa to be 17.63% and 18.72% respectively.
3.4.2 Determing the prevalence of EP infections using different methods
The present study , showed that the prevalence of T. equi, B. caballi and
both protozoa in equids was 16.99%, 22.22% and 7.18% respectively. This result
is in contrast to a previous study conducted in the same state which
microscopically observed that the prevalence of the aforementioned parasites was
0% out of 91 equids samples (Chandrawathani et al., 1998). The negative results
by microscopic examination may have be due to low parasitemia (<0.01-0.001%)
in carrier state, in acute onset of the disease, in chronic infection of B. caballi and
inexperienced and unskilful examiner inspecting the blood smears (Knowles,
1988; Ali et al., 1996; Krause, 2003).
The positive result for EP from microscopic examination of blood smears
from equids in the current study, was confirmed by cELISA and this study
showed that the seroprevalence of EP in Kelantan by cELISA was 80.06% for all
types of protozoa; 51.30% for T. equi, 63.07% for B. caballi and 34.31% for both
protozoa. This rate was higher compared to previous studies in Malaysia and other
neighboring countries. Zawida et al. (2010) showed that the seroprevalence for T.
equi was 20% and B. caballi was 1% in 12 states in Malaysia using cELISA.
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Studies undertaken in other countries reported a higher seroprevalence for EP in equids. In the Philippines, the prevalence was 10% for T. equi, 71% for
B. caballi and 19% for both protozoa using immunochromatographic (ICT) assay
(Cruz-Flores et al., 2010). The seroprevelance for T. equi was 11.51%, B. caballi was 51.16% and both parasites were 7.64% by using indirect ELISA test in China
(Wang et al., 2014). In Pakistan, by using cELISA, the overall seroprevalence of
EP was 52.6%: for T. equi it was 41.2%, B. caballi was 21.6% and both protozoa were 10.2% (Hussain et al., 2014). In Thailand, the infection rates ranged from
5% to 49.83% for T. equi and 2% to 5% for B. caballi by using cELISA and IFAT respectively (Chungvipat & Viseshakul 2005; Kamyingkird et al., 2014). Lower seroprevalence of EP has been reported in other countries such as Korea, 1.1% for
T. equi and 0% for B. caballi using cELISA (Seo et al., 2011). Furthermore, in
Japan, 5.4% was reported for T. equi and 2.2% for B. caballi using an indirect
ELISA test (Ikadai et al., 2002).
Another confirmed method used in this study was multiplex PCR technique, which used for the first time in Kelantan to diagnose the EP infections.
The results illustrated that the overall prevalence of EP in Kelantan was 35.62%, (
T. equi 18.95%; B. caballi 22.87%; and both protozoa 6.20%), which was higher compared to other countries that used PCR technique to diagnose this disease. The prevalence was 1.25% for T. equi and 0.0% for B. caballi in equids by using multiplex PCR in Thailand (Kamyingkird et al., 2014). In India, the prevalence of
T. equi was 14.14% and for B. caballi it was 0.0% in equids using the same PCR technique (Sumbria et al., 2016a). In Tunisia, the prevalence by quantitative
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real time PCR (qPCR) was 12.5% for T. equi and 1.92% for B. caballi (Ros-
Garcia et al., 2013). In Italy, the infection rate was 17.4% for T. equi, 3.6% for B. caballi and 0.0% for both protozoa using quantitative PCR (Laus et al., 2015).
The results of the current study using multiplex PCR, showed that the prevalence of EP was lower compared to other countries that also used PCR technique. In Turkey, the prevalence of EP by using the conventional PCR technique was 66.7% for T. equi and 25% for B. caballi (Kizilarslan et al., 2015).
The infection rate of T. equi was 66.5% and that of B. caballi was 19.1% in horses using multiplex PCR technique in Mongolia (Rüegg et al., 2007). In Venezuela, the overall prevalence was 66.2% for all piroplasms; 61.4% for T. equi and 0.0% for B. caballi in horses using multiplex PCR technique (Rosales et al., 2013). In addition, the results of the present study are almost similar to those reported in
Jordan, where the overall prevalence was 27.1% comprising 18.8% for T. equi,
7.3% for B. caballi in equids, using the same PCR technique (Qablan et al., 2013).
Both protozoa species (T. equi and B. caballi) were reported in this study using single reaction multiplex PCR technique. Additionally, this PCR assay was suitable dor the detection of both protozoa simultaneously. This finding corresponds with Munkhjargal et al. (2013) and Gallusová et al. (2014). In other studies, there was an absence of mixed infections when using multiplex PCR technique (Qablan et al., 2013; Sumbria & Singla, 2015) as they only reported only single infection with T. equi or B. caballi.
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The prevalence of EP differs from country to country and the reasons could be the following: different management practices; how sensitive the diagnostics methods used are; incidence of competent tick vectors, equids activity; presence and efficacy of ticks control programs, and the climatic variations (Kouam et al., 2010a; Grandi et al., 2011).
This study showed that the prevalence of B. caballi was significantly higher than T. equi, by all diagnostic methods (microscopic examination, cELISA, mPCR). The results indicate continuing shifts in the nature of the epidemiology of
EP in peninsular Malaysia, which is in agreement with reports from Brazil, Italy,
Venezuela, and Mongolia (Kerber et al., 2009; Moretti et al., 2010; Mujica et al.,
2011; Munkhjargal et al., 2013). They observed that B. caballi was more commonly detected than T. equi. This result may be due to more spreading of the tick species transmitting B. caballi than the species transmitting T. equi in the areas and because of variations in the B. caballi and T. equi life cycles, they have different forms of transmission: B. caballi can be transovarial and transstadial transmission, conversely, T. equi is a transstadial transmission (de Waal, 1992;
Ueti et al., 2008). This may give more chance for spreading of B. caballi than T. equi. However, further reports suggested that there was higher prevalence of T. equi than B. caballi in Spain, Portugal and Romania (Garcia-Bocanegra et al.,
2013; Ribeiro et al., 2013; Gallusová et al., 2014).
Chauvin et al. (2009) explained that the high prevalence of T. equi was due to longer persistence after infection. Furthermore, it may be due to transplacental transmission, which has been reported in the case of T. equi, while
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not documented in B. caballi, and this phenomenon contributes to the high rate in
the spreading of T. equi infection (Georges et al., 2011). It may also be due to the
higher parasitemia of T. equi in the infected animals which heightens the risk of
the tick vectors (de Waal & van Heerden, 1994).
3.4.3 Evaluating of cELISA and multiplex PCR for detecting T. equi and B. caballi infections.
In this current study, cELISA and multiplex PCR were observed to be
more sensitive for the detection of T. equi and B. caballi infections compared to
microscopic examination of blood smears. This finding agrees with the results of
Ibrahim et al. (2011) who found higher sensitivity, specificity and accuracy for
ELISA tests which were 100%, 85% and 88%, while for multiplex PCR the rates
were 100%, 90% and 88% compared to blood film examination method. In
addition, this result was consistent with that of many researchers (Heim et al.,
2007; Alanazi et al., 2012; Gizachew et al., 2013; Ribeiro et al., 2013; Malekifard
et al., 2014).
A visual detection of piroplasms in the RBCs by microscopic examination
of stained blood smears is possible during an acute form of EP. During the
persistent or subclinical infection, the piroplasms are detected rearly due to very
low parasitemia in the animals (Knowles, 1988; Zweygarth et al., 2002). Salim et
al. (2008) and Servinc et al. (2008) proposed that c-ELISA test was an alternative
method for detecting acute and latent infections of EP. Moretti et al. (2010) added
that PCR techniques had been recommended for detection of latent
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infection with T. equi and B. caballi which cannot be detected microscopically.
Despite, the fact that microscopic method has low sensitivity, it remains the common method used in many laboratories, but its usage should be supplemented with other more accurate and sensitive methods such as serological tests and molecular techniques.
This present study also showed that multiplex PCR was more sensitive and specific than cELISA for the diagnosis of T. equi and B. caballi. This finding agrees with Sibeko et al. (2008) and Ibrahim et al. (2011). Multiplex PCR assay is a simpler and more rapid technique which can detecte T. equi and B. caballi in one reaction with higher sensitivity and specificity (Alhassan et al., 2005).
Moreover, this PCR assay has sufficient sensitivity to detect the parasites DNA in
2.5 μl of blood with an estimated parasitaemia of 0.000001% (Xuan et al., 2001a;
Alhassan et al., 2007a). Despite multiplex PCR being more efficient than ELISA, both tests were recommended as simple tools to diagnose EP in routine diagnostic laboratory and epidemiological studies (Alsaad et al., 2010; Alhassan et al., 2005;
OIE, 2013; Rosales et al., 2013).
Different targeting genes have been used for detecting T. equi and B. caballi. Thus study used 18S rRNA gene, which is commonly used in epidemiological and genetic diversity studies (Hunfeld et al., 2008; Ros-Garcaia et al., 2013; Qablan et al., 2013). The 18S rRNA gene is used for detection of piroplasms because the DNA sequences of this gene at the species level are mostly conserved. This gene contains highly frequented sequences, existing in
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diverse copies in the genome and the sequences of this gene are available in
molecular databases (Alhassan et al., 2005; Bhoora et al., 2009).
In the current study, no significant differences between conventional and
multiplex PCR techniques for detection piroplasms DNA in equids blood and
multiplex PCR was particularly suitable for detection of both protozoa in a single
reaction. This result was consistent with Alhassan et al. (2005) and Qablan et al.
(2013), who found almost similar sensitivity between conventional PCR and
multiplex PCR.
3.4.4 Equids factors associated with T. equi, B. caballi and both protozoa
This study showed that there was no significant difference in the
seroprevalence of T. equi, B. caballi and both protozoa between horses and ponies
in Kelantan. This may be due to the rearing together of horses and ponies in the
same environment in Kelantan, resulting in a similar exposure to protozoa. A
similar result was recorded by Kouam et al. (2010a).
The result revealed that the seroprevalence of T. equi, B. caballi and both
protozoa was significantly higher among gelding equids than in males and
females. This finding was reported by Guidi et al. (2015). This may be due to
accidental transmission of the protozoa through contaminated surgical instruments
during castration and because of the small sampling size of gelding equids in the
study (Barros, 2008; Kouam et al., 2010a). The seroprevalence was significantly
higher in female than male, which was consistent with Munkhjargal et al. (2013)
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and Davitkov et al. (2016). The immune-suppression caused by stress during the third stage of pregnancy and parturition might be the result of higher protozoan infections in female equids, especially if persistently infected (Lewis et al., 1999).
In addition, mares and geldings were found to be more significantly affected than stallions. The reason could be that the males were maintained under strict care for breeding, resulting in less exposure to ticks infestation (Shkap et al., 1998; Javed et al., 2014). Other reports showed no significant difference between the equids genders for all types of infections (Kouam et al., 2010a; Bahrami et al., 2014;
Malekifard et al., 2014).
The current study showed that the seroprevalence of T. equi, B. caballi and both protozoa among equids older than five years was significantly higher than that of newborn and younger equids. This finding corresponds with the findings of other researchers (Garcia-Bocanegra et al., 2013; Sumbria et al.,
2016b). The higher seroprevalence of infections in older age equids may be due to more exposure of older equids to the protozoa and persistence level of antibodies from a previous infection (de Wall & Van Heerden, 1994). Moreover, the reason for the increase in the prevalence of the causative agents in older age animals was the complete elimination of B. caballi infection from the blood within four years, on the contrary, T. equi infection may remain as lifelong carriers (Holman et al.,
1993; de Waal & van Heerden, 1994).
The lower seroprevalence of the piroplasms in newborn and young growing equids may be due to passive immunity through the colostrum and activity of hemopoietc system and thymus (Soulsby, 1982; de Waal & van
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Heerden, 1994). These results differed from those of Farkas et al. (2013);
Malekifard et al., (2014) and Prochno et al., (2014) who concluded that there wa no difference between T. equi and B. caballi prevalence in all ages of equids.
This study showed that the seroprevalence of B. caballi infection was significantly higher among thoroughbred horses than Arab breed. The seroprevalence of T. equi and both protozoa infections was significantly higher among pony A breed than pony C, due possibly to the fact that most of the thoroughbred horses and pony A were used for sport in Kelantan that makes them more susceptible to EP infection. The low prevalence of infections in Arab breed and pony C may be due to most of the Arab breed being used for breeding and the pony C for recreation in Kelantan. These breeds were less exposed to ticks and they were better managed.
The result revealed that the seroprevalence of T. equi, B. caballi and both protozoa was significantly higher among imported equids than local equids. This may be due to the fact that these equids were imported from countries where the disease had been documented,, such as Thailand (Kamyingkird et al., 2014), India
(Sumbria et al., 2016a), France (Fritz, 2010) and Argentina (Asenzo et al., 2008).
It should also be noted that imported equids were often brought into Kelantan without border control and quarantine (as learned from stables owners).
Other reports have shown that the prevalence of seropositive equids was significantly higher in native equids compared to the imported ones (Kouam et al.,
2010a; Dos Santos et al., 2011).
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The present study showed that the seroprevalence of all types of protozoa
was much higher among sports equids than equids used for breeding and
recreation. This finding can be the result of stress that the sports equids are being
moved frequently for training or competition. The spleen contracts during stress
and may result in the release of the protozoa from the spleen (Hailat et al., 1997).
On the other hand, there was no significantly increase or decrease in the
antibodies of T. equi in sports horses at rest and after strenuous exercise (Baldani
et al., 2008).
This study also revealed that the seroprevalence of T. equi, B. caballi and
both protozoa was significantly higher among pregnant animals than non-pregnant
animals. This may be a surprise to people as pregnant mare receive better
management, but the higher seropositive may be related to the presence of tick
vectors in their surrounding areas or they had subclinical infections. The
seroprevalence of T. equi infection in this study was much higher among mares at
the third stage of pregnancy than at the first stage. This observation is consistent
with Lewis et al. (1999).
3.4.5 Stables factors associated with T. equi, B. caballi and both protozoa
The results revealed that the seroprevalence of T. equi and B. caballi
infections was much higher in Pasir Puteh and Bachok respectively compared to
other regions. The seroprevalence of all types of protozoa was significantly lower
in Machang. The difference in the seroprevalence may be related to management
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practices, host activity, sampling size, presence of competent tick vectors and various climatic factors such as temperature, humidity and rainfall, which have an effect on the habitat for ticks (Kouam et al., 2010a; Garcia-Bocanegra et al.,
2013).
The results showed that the seroprevalence of T. equi and both protozoa infections was significantly higher in September, while the seroprevalence of B. caballi was higher in December and significantly lower in November. In fact, these may be related to climatic factors in Kelantan during these sampling months which were in September and December, when the means of temperature were
26.9°C and 28.5ºC respectively, the means of relative humidity were 88.9% and
80.6% respectively, and the means of rainfall were 95mm and 40.6mm (low) respectively. These factors play a role in the tick development, resulting in higher prevelance of protozoa infections. Conversely, in November in Kelantan the climatic factors reduce tick population, which results in low prevalence of EP.
These finding were consistent with Golynski et al. (2008) who confirmed that these seasonal factors affect the prevelance of EP. Nevertheless, these findings contradict those of Moretti et al., (2010) who found that season does not have an influence on the incidence of EP infection.
The results showed that the seroprevalence of T. equi, B. caballi and both protozoa infections was significantly higher among equids kept with other animals compared to equids isolated in the stable and away from other animals. The same result was obtained by Guidi et al. (2014) when they found the horses with cows were associated with the positive serological results of EP. The reason for the
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higher prevalence of EP in equids kept with other animals (camel, cattle, sheep, goats, dogs) may be due to these animals acting as a reservoirs for T. equi and B. caballi, as well as tick vectors (Qablan et al., 2012a, 2012b; Zhang et al., 2015).
This work demonstrated that the seroprevalence of all types of protozoa infections was significantly higher among equids that grazed than those kept in stables. The equids that grazed are more exposed to external environmental conditions that increase the chances for direct contact with tick vectors. This result was consistent with Abutarbush et al. (2012); Garcia-Bocanegra et al.
(2013), and Ribeiro et al. (2013) who found that equids in grazing lands was a risk factor for seropositivity for T. equi, B. caballi and both protozoa.
Our results showed that the seroprevalence of T. equi, B. caballi and both
protozoa infections was considerably higher among equids in stable when ticks
were found on equids and nearby animals than among equids in stables when no
ticks were found or ticks were found on nearby animals only. These higher
seropositivity of the protozoa because the ticks are known to be their main
vectors. These results agree with Garcia-Bocanegra et al. (2013) and Sumbria et
al. (2016b), who found that the presence of ticks was the risk factor associated
with both T. equi and B. caballi. According to Kouam et al. (2010a), two
competent tick vectors, Rh. bursa, Rh. sanguineus were recovered from horses
and dogs respectively. The nearby animals act as a reservoirs for T. equi and B.
caballi, as well as tick vectors (Qablan et al., 2012a, 2012b; Zhang et al., 2015).
The cattle are the primary host for Rh. (B.) microplus and infestation of horses
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with this type of tick depends on the presence of cattle in the same area (Labruna
et al., 2001).
The current research identified eight species of Ixodidae ticks that most
of them play important role in spreading of EP in Kelantan. This is in agreement
with Mariana et al., (2005) when that found highly distribution of ticks in
Kelantan.
3.4.6 Climatic factors associated with T. equi, B. caballi and both protozoa
In this study, the seroprevalence of T. equi, B. caballi and both protozoa
was considerably higher when the mean monthly temperature ranged from 24.9ºC
to 28.5ºC, relative humidity ranged from 80.9% to 86.6% and rainfall amount
ranged from 0.6mm to 289.4mm. These results agreed with Kouam et al. (2010c);
Garcia-Bocanegra et al. (2013) and Salib et al. (2013). Peninsular Malaysia has a
tropical climate characterized by moderate temperature, higher mean of relative
humidity, and rainfall amount. These climatic factors influence the habitat of ticks
(Chilton & bull, 1994; MMD, 2009; Garcia-Bocanegra et al., 2013). It has been
found that ticks thrive and survive for longer periods in forested areas, with
predictable temperature and higher humidity (Semtner et al., 1971). Tick
oviposition rate and hatching success are influenced by climatic conditions.
However, lower temperatures and humidity inhibit oviposition, which reduces the
tick population (Chilton & bull, 1994; Estrada-Peña et al., 2011). Temperature,
therefore, has been shown to facilitate tick development and Theileria
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development rate in the ticks (Young & Leitch, 1981; Sumbria et al., 2016b).
Similarly, humidity also affects tick development (Chilton & bull, 1994). Kouam
et al. (2010c) mentioned that humidity rather than temperature plays a smaller role
in EP expansion. Rainfall has been observed to have an adverse effect on tick
infestations (Lim, 1972). On the contrary, Singh et al. (1995) observed no
relationship between rainfall and ectoparasites indices. However, Rothschild and
Knowles, (2007) reported that maximum increase of the protozoa in the rainy
seasons causes the disease.
3.4.7 Ixodid ticks: identification and infestation rate
To the best knowledge of this researcher, this is the first study conducted
on the percentage of Ixodid tick infested equids and nearby animals in equids
stables in various regions of the state of Kelantan in Peninsular Malaysia. The
study showed that 32.07% (17 of 53 stables) was infested with Ixodid ticks in
Kelantan. Stables infested rate with Ixodid ticks was higher in Machang (100%)
and lower in Pasir Puteh (10%), whereas, stables in Pasir Mas and Gua Musang
were free from Ixodid ticks. This disparity in the results may be due to climatic
determinants of the study areas and some stables with poor management practices
such as allowing the animals to graze, lack of routine preventive therapy against
ticks in stables, and different animal species in stables etc. These are important
factors that contribute to the higher prevalence of ticks in stables. In addition, the
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owners in the study regions should be educated about the significant effects of tick-borne diseases in animals as well as humans.
The current study identified eight species belonging to three genera of
Ixodid tick. It reported for the first time the presence of RBA, RB, RA, DM, HL and HP, whereas, RBM and RS were previously reported in Malaysia by Tay et al. (2014) and Koh et al. (2016). Other studies have been conducted on identification of Ixodidae ticks infested rats, rodents, bats, reptiles, birds, and small mammals in Peninsular Malaysia, such as Amblyomma spp., Dermacentor spp., Haemaphysalis spp., Ixodes granulatus, and Ixodes spp. (Mariana et al.,
2005, 2008, 2011; Che Lah et al., 2015; Mohd Zain et al., 2015). This identification of new species is new useful knowledge as to the best knowledge of this researcher, there has not been any survey done of Ixodid ticks on domestic animals in Kelantan. Tick species can be imported from other states or countries, carried on their hosts. Another reason could be that these species has infests wild mammals such as rats, rodents, birds, reptiles, etc. and although it has not been reported, it is suggested that these ticks may have transferred to domesticated animals due to ecological changes.
The current study demonstrated that the presence of Ixodid ticks in stables, resulted in highly seropositive equids with piroplasms infections in these stables reaching as high as 100%. This finding corresponds with that of García-
Bocanegra et al. (2013); Sumbria & Singla, (2015) and Sumbria et al. (2016b), who found that the presence of Ixodid tick in equids stables was a risk factor significantly associated with high prevalence of both T. equi and B. caballi.
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Moreover, various stages (nymph, larvae and adult) and sex (male and female engorged or non-engorged) were collected from different predilection sites of attachment on equids and other nearby animals. The result agreed with those of other studies (Shemshad et al., 2012; Chhillar et al., 2014; Sofizadeh et al., 2014).
The present study revealed that the percentage of Ixodid ticks infesting equids and cattle, sheep, goats, and dogs was 11.11%, 69.23%, 26.08%, 32.35%, and 50% respectively. These results indicate significant differences of tick abundance on different hosts. This is simialr to Sofizadeh et al. (2014), who reported a different infestation rate of ticks on horses, camels, cattle, sheep, and goats as 50%, 69.3%, 75.8%, 72.1% and 77.3% respectively.
The results of this study show a low of Ixodid ticks infestation rate on equids, in agreement with the findings by Ros-García et al. (2013) and Abedi et al. (2014a) who reported low tick infestation rate on horses which were 10.8% and 15% respectively. The reasons for lower ticks infestation rate on equids might be due to higher level of cleanliness, application of control measures, regular use of chemical acaricides, and recently, periodic vaccination of horses for control of ticks and tick-borne disease.
This study demonstrated that the Rh. bursa tick species was the dominant detected tick on equids. This finding agrees with those of Kouam et al. (2010a) and Scoles and Ueti, (2015) who found that the Rh. bursa was dominant on horses and has been associated with transmitting T. equi and B. caballi.
Moreover, the results demonstrated that the Rh.(Boophilus) microplus tick species was dominant tick species detected in Kelantan state. This finding
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grees with Nadchatram, (2006) and Tay et al. (2014) who reported this tick
species as being predominant in Malaysian farms.
3.5 Conclusions
Base on morphological and biometrical findings, various shapes of T.
equi and B. caballi were seen in the erythrocytes. T. equi appears as a small
parasite with mean measurements of 2.4µm in length - 1.5µm in diameter and
parasitemia ranging from 0.7% - 14.2%. Babesia caballi appears as a larger
parasite with mean measuring 4.8µm in length - 2.5µm in diameter and
parasitemia ranging from 0.1% - 7.6 %. In addition, both protozoa infections were
reported in equids.
This study revealed that EP is endemic in Kelantan with overall
prevalence of 32.02%, 80.06% and 35.62 using microscopic examination of blood
smears, cELISA and multiplex PCR. The disease was caused by T. equi, B.
caballi and both protozoa. The seroprevalence of T. equi and both protozoa was
higher in Pasir Puteh region with 92.15% and 68.62% and the seroprevalence of
B. caballi was higher in Bachok region with 93.22%.
The cELISA and multiplex PCR were the more suitable and reliable
methods for detecting T. equi and B. caballi infections in equids than microscopic
method. Furthermore, multiplex PCR is more efficient than cELISA.
The significant equids risk factors associated with high seroprevalence of
T. equi, B. caballi and both protozoa in Kelantan were: gelding equids, over five
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years old, thoroughbred horses, Pony A breed, imported equids, sports equids, pregnant mares in the third stage, and ticks found on equids.
The significant regional risk factors associated with high seroprevalence of T. equi, B. caballi and both protozoa were reported in Pasir Puteh and Bachok regions, which were 5.52 times, 5.59 times and 8.23 times respectively compared to Machang region.
The significant monthly risk factors associated with high seroprevalence of T. equi, B. caballi and both protozoa were in the months pf September and
December, which were 4.54 times, 5.22 times and 9.69 times respectively compared to the month of November.
The significant management risk factors associated with high seroprevalence of T. equi, B. caballi and both protozoa in Kelantan were: equids kept with other animals in stable, equids in grazing land, ticks found in stable, and presence of ticks on equids and nearby animals.
The significant climatic risk factors associated with high seroprevalence of T. equi, B. caballi and both protozoa in Kelantan were observed when the mean of monthly temperature ranged from 26.4ºC - 28.5ºC, rainfall amount ranged from
0.6mm - 289.4mm, and relative humidity ranged from 80.9% - 86.6%.
Eight Ixodid tick species were identified from equids and nearby animals.
The animals near equids were cattle, sheep, goats, and dogs and they may act as reservoirs for the protozoa and sources of ticks to the equids.
The author recommends that cELISA should be used in the study of the epidemiology of EP. To reduce the risk of EP in Kelantan, good management
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practices should be performed, including keeping the equids in stables without mixing with other animals, conducting tick control programs, testing newly purchased equids before introducing into the stable and implementing practicable vaccination programs. In addition, it is suggested that animal border checkpoints should be intoduced (as centers for quarantine of animals ) in Kelantan.
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CHAPTER 4
EVALUATION OF HEMATOLOGY, BIOCHEMISTRY AND ANTIBODY TITER BETWEEN EQUIDS CLINICALLY AND SUBCLINICALLY INFECTED WITH EQUINE PIROPLASMOSIS
4.1 Introduction
The incubation period of EP caused by T. equi is 12-19 days and 10-30
days when it is caused by B. caballi (Friedhoff & Soulé, 1996). The acute form of
EP is characterized by fever of more than 40°C, loss of appetite and weight,
severe sweating, congested mucous membranes with petechial hemorrhage, signs
of nervousness (e.g., incoordination), pale and/ or icteric mucous membranes,
peripheral oedema, panting, depression, colic with signs of diarrhea and/or
constipation, hemoglobin urea, muscles tremor, coughing, dehydration with rough
coat, and presence of ticks on various parts of the body. There is increased
respiratory and heart rates, and capillary refilling time in the infected equids
(Ibrahim et al., 2011; Salama, 2016). Abortion or neonatal infections can occur in
pregnant mares (Lewis et al., 1999). Other clinico-pathological signs include
hepatomegaly, splenomegaly, intravascular hemolysis, hemoglobinemia,
hemoglobinuria, and bilirubinuria (Radostitis et al., 2008).
In the subclinical form, the equids appear healthy, unapparent or no
clinical signs and low parasitemia. These equids can act as carriers and source of
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dissemination of the parasites to the ticks and susceptible horses (Bahrami et al.,
2014). Tick vectors feeding on subclinically infected horses with low T. equi
3 6 -1 parasitemia (2 ×10 - 10 ml of blood) will have the chance to successfully transmit the parasites to the non-infected horses (Ueti et al., 2008).
A suspected case of EP can be based on clinical signs. However, the clinical signs may be non-specific and the disease may be confused with a variety of other illnesses (Rothschild & Knowles, 2007).
A hematological and serum biochemistry finding varies according to the virulence of the protozoa, the equid’s health status, the presence of concurrent infections, and previous exposure to the infections (Boozer & Macintire, 2003).
Equine piroplasmosis has a severe and complicated pathogenic effect on the tissue and organs of infected equids such as mechanical effect exhibited by the destruction and the hemolysis of the red blood cells (RBCs).
Studies of EP in the state of Kelantan, Malaysia are very scarce and to the best knowledge of this researcher, no information on hematology, biochemistry and antibody titer has been reported. Therefore, this study has been designed to:
15. Determine the seroprevalence of the clinical and subclinical forms of EP in equids and their relative risk in Kelantan.
16. Determine the antibody titers against T. equi and B. caballi in equids with the clinical and subclinical forms of EP.
17. Determine the clinical, hematological and serum biochemistry parameters in equids with the clinical and subclinical forms of EP.
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4.2 Materials and Methods
4.2.1 Animals and study area
From September 2013 to March 2014, 306 equids, including horses and
ponies were investigated. The equids were from 53 stables in eight regions in
Kelantan: Kota Bharu, Pasir Mas, Machang, Bachok, Pasir Puteh, Tumpat, Tanah
Merah, and Gua Musang.
Based on cELISA, 61 equids were healthy, 30 equids had a clinical form
of EP - based on clinical examination - and 215 equids had a subclinical form of
the disease. Twenty-five equids from the healthy group served as control.
4.2.2 Recording clinical signs
Clinical examination was conducted on all equids and findings were
recorded in the clinical examination card (Fig. 3.2).
4.2.3 Fecal samples collection
Fecal samples were collected from all equids and screened for internal
parasitic infestation employing standard techniques such as direct saline or iodine
smears, floatation and sedimentation (Zajac & Conboy, 2012). In this study, the
equids positive for internal helminths were excluded.
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4.2.4 Determination of antibody titers against T. equi and B. caballi
A method was developed to standardize the titration of antibodies for T.
equi and B. caballi, using the positive control serum of the cELISA kits. A serial
diluent log10 for the positive control serum in the commercial c-ELISA kits
competitive-inhibition ELISA for T. equi and B. caballi provide by (VMRD, Inc,
Pullman, and WA99163 USA), was conducted (as mentioned in Chapter 3) with
the serum samples separated from the blood for screened equids to obtain a
standardized optical density for each diluent of the positive control serum and
optical density reading for each serum sample.
4.2.5 Hematological analysis
The blood in tubes with anticoagulant was utilized to verify the total
erythrocytes counts (TECs), hemoglobin concentration (Hb), packed cell volume
(PCV), the mean corpuscular volume (MCV), mean corpuscular hemoglobin
concentration (MCHC), thrombocytes count, total leukocytes counts (TLCs), and
differential leukocytes counts (DLCs) by using a Hematology analyzer (Mythic
18VET/France). Erythrocyte sedimentation rate (ESR) was determined using
Westergren pipette. Reticulocytes count was performed by mixing 2 drops of
blood with 2 drops of new methylene blue 0.5%, 20 minutes later a thin blood
smear prepared and was examined under a microscope. The percentage of
reticulocytes was calculated following the equation of Jain (2000):
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Number of reticulocytes Reticulocyte % = X 100 (4.1) Number of calculated reticulocytes
4.2.6 Serum biochemistry analysis
Serum was used for biochemical analysis, including aspartate amino
transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP),
blood urea nitrogen (BUN), total bilirubin, total protein, albumins, globulins,
calcium, phosphorous, glucose, and creatine using special cassettes for each in a
Chemistry analyzer (IDEXX - Vet Test, Arachem/USA). A total of 40μl of serum
sample was needed to conduct the test. Briefly, the procedure was as follows:
8. A serum sample was transferred to a vet test analyser sample cup.
9. Sample cup was entered in the vet test analyser and the equine information was
entered by following the screen prompts.
10. The cassettes of aforementioned tests were inserted when prompted.
11. The tip was carefully placed on the vet test analyser pipettor, which was then
kept in a vertical position.
12. The tip was placed in the center of the sample then the pipettor button pressed.
13. When 1 BEEP was heard, it indicated that the sample was being aspirated into
the pipettor tip.
14. When 2 BEEPS were heard, the pipettor was removed from the sample cup
15. When 3 BEEPS were heard, the pipette tip was wiped down in a twisting
motion with a lint free wipe.
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12. The pipettor was replaced in the vet test analyser holder and the rest of the
testing process proceeded automatically.
4.2.7 Statistical analysis
The differences in the prevalence between the various equids health
status factors were evaluated using two-sided Chi-square and Fischer’s exact test
in IBM-SPSS statistics version 19 program.
The relative risk (RR) for the association between the equids healthy
status for EP was 95% confidence interval, which was computed using 2 by 2 TM tables in Epi-Info 7 software (version 7).
One way analysis of variance (ANOVA) was followed by post hoc test
(Duncan) in IBM-SPSS statistics (version19) program, which was used to make a
comparison between equids with hematological and serum biochemistry
parameters. All the significant differences were determined at (P<0.05).clinical
form, subclinical form and healthy equids (control group), in hematological and
serum biochemistry parameters. All the significant differences were determined at
(P<0.05).
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4.3 Results
4.3.1 Seroprevalence of clinical and subclinical forms in equids with the relative risk.
The current study demonstrated that the overall seroprevalence of the
clinical form of EP in equids was 9.80%; for T. equi 8.82%, B. caballi 8.49%,
and 7.51% for both protozoa. The overall seroprevalence of the subclinical form
was 70.26%; for T. equi 42.48%, B. caballi 54.57% and for both protozoa it was
26.79%. The seroprevalence of T. equi, B. caballi and both protozoa was
significantly higher in the subclinical form in equids (RR: 4.81, 6.42 and 3.56
times respectively) compared to the clinical form in equids (P<0.05) (Tables 4.1,
4.2).
In this study, examination of the faecal samples showed that some of
examined equids were positive for internal helminths and helminths eggs. These
equids were excluded from this study.
4.3.2 Level of antibodies titration against piroplasms in equids with clinical and subclinical forms of equine piroplasmosis
The results demonstrated that the antibody titers against T. equi in the
equids with clinical form were 1/160 (n=3), 1/320 (n=8), 1/640 (n=10), and
1/1280 (n=6), whereas, in the equids with subclinical form, they were 1/5 (n=6),
1/10 (n=18), 1/20 (n=23), 1/40 (n=51), and 1/80 (n=32), (Fig. 4. 1).
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Moreover, the antibody titers against B. caballi in the equids with clinical
form were 1/160 (n=8), 1/320 (n=16), and 1/640 (n=2), whereas in the equids with
subclinical form they were 1/5 (n=62), 1/10 (n=27), 1/20 (n=44), 1/40 (n=22), and
1/80 (n=12) (Fig. 4.2). These indicated higher antibody titers against T. equi and
B. caballi in the equids with clinical form.
4.3.3 Hematological and serum biochemistry parameters in equids with clinical and subclinical form of equine piroplasmosis
The result of this study revealed that the equids with clinical form of EP
were suffering from an acute form of the disease and showed depression, loss of
appetite, emaciation, congestion of mucous membranes with petechial
rd hemorrhage on the 3 eye lid and conjunctivae, pale and/ icterus mucous
membrane, severe sweating, difficulty in movement and incoordination with
oedema of fetlock joint, hemoglobin urea, muscles tremor, coughing, colicky
signs, diarrhea and/or constipation, signs of nervousness, dehydration with rough
coat and presence of ticks on various parts of the body (Fig. 4.3). The various
percentages of each clinical sign were recorded for the clinical form of the disease
(Fig. 4.4). In contrast, equids with a subclinical form of EP appeared healthy with
no clinical signs.
The present study found that the equids with clinical form had º significantly increased body temperature (40.2C ), respiratory rate (44.33/min),
heart rate (76.25/min) and capillary refilling time (3.9/Sec) compared to the
healthy group (P<0.01) (Table 4.3). The equids with subclinical form showed no
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statistically significant differences in these clinical symptom parameters compared to clinical form and healthy group (control group) (Table 4.3).
The haemogram of the equids with clinical form was found to be significantly decreased in TEC, Hb, PCV, thrombocytes, MCHC and significantly increased in MCV, reflecting a macrocytic hypochromic type of anemia (P<0.05), along with significant increase in the ESR, reticulocytosis and the TLCs
(neutrophilia and lymphocytosis), compared to the healthy group (P<0.05) (Table
4.4).
The equids with subclinical form showed no statistically significant differences in these hematological parameters in the subclinical cases compared to clinical form and healthy group (Table 4.4).
Serum biochemistry analysis of the equids with clinical from revealed significant increase in AST, ALT, ALKP, BUN, and total bilirubin, along with significantly decrease in the total protein (albumins and globulins), calcium, phosphorous, glucose, and creatinine, compared to the healthy group (P<0.05)
(Table 4.5). The equids with subclinical form showed no statistically significant difference in these biochemistry parameters in the subclinical cases compared to clinical cases and healthy group (Table 4.5).
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Table 4.1: Health status factor of equids associated with seroprevalence of T. equi, B. caballi and both protozoa.
cELISA Health status of Total No. of No. of Overall T. equi B. caballi Both protozoa equids tested equids equids % n (%) n (%) n (%) Healthy 61 19.93 0 0 0
a a a Clinical form 306 30 9.80 27 (8.82) 26 (8.49) 23 (7.51)
b b b Subclinical form 215 70.26 130 (42.48) 167 (54.57) 82 (26.79)
n) Number of positive; Values significantly different (P < 0.05) between equids status are labelled with the vertically different letters (a,b).
Table 4.2: Relative risk of equids health status factors associated with the T. equi, B. caballi and both protozoa.
Factors/ Health T. equi infection B. caballi infection Both protozoa infection
status n (%) RR 95% CI P n (%) RR 95% CI P n (%) RR 95% CI P
Clinical form 27 (8.82) 1 26 (8.49) 1 23 (7.51) 1
Subclinical form 130 (42.48) 4.81 3.28-7.06 0.00 167 (54.57) 6.42 4.38-9.40 0.00 82 (26.79) 3.56 2.30-5.50 0.00
n) Number of positive; %) Prevalence; RR) Relative risk; 95% CI) 95% confidence interval; P) P value.
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Figure 4.1: A) Determination the optical density of serial dilution log10 positive control serum for T. equi in cELISA kit, used as standerd; B) The antibody titers against T. equi in the clinical and subclinical forms of EP.
Figure 4.2: A) Determination the optical density of serial dilution log10 positive control serum for B. caballi in cELISA kit, used as standard; B) The antibody titers against B. caballi in the clinical and subclinical forms of EP.
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A C
B D
Figure 4.3: Some of the clinical signs shown in equids with clinical form of EP: 14. Loss of body weight (Emaciation) and depression; B) Pale mucous membrane in rd rd the 3 eye lid; C) Petechial hemorrhage of the mucous membrane in the 3 eye lid; D) Presence of ticks under the ear.
Figure 4.4: Percentage of clinical signs on equids with clinical form (n=30).
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Table 4.3: The clinical symptom parameters in the equids with clinical form compared to the equids with subclinical form and healthy group.
Healthy group Clinical form Subclinical Parameters (Control) form
Mean ±S.E. Mean ±S.E. Mean ±S.E.
Body temperature a b a 37.26 ± 0.11 40.20 ± 0.16 37.50 ± 0.18 / ºC
Respiratory rate a b a 17.90 ± 0.40 44.33 ± 0.87 18.03 ± 0.51 /min
a b a Heart rate /min 46.76± 1.55 76.25± 3.59 46.67± 1.03
a b a Capillary refilling 1.11 ± 0.12 3.9 ± 0.19 1.22 ± 0.13 time /Sec
Mean values ± Standard error (S.E.) significantly different at (P<0.05) between equids status are labelled with the horizontal different letters (a, b).
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Table 4.4: Hematological changes in the equids with clinical form compared to the equids with subclinical form and healthy group.
Healthy group Clinical form Subclinical form (Control) Parameters Mean ±S.E. Mean ±S.E. Mean ± S.E.
6 a b a TECs ×10 µl 8.85 ± 0.40 5.22 ± 0.24 8.98 ± 0.21 a b a Hb mg/100 ml 12.10 ± 0.14 6.92 ± 0.22 11.90 ± 0.21 a b a PCV % 36.98 ± 0.85 24.16 ± 0.51 36.75 ± 0.92 3 a b a MCV / µm 34.49 ± 0.59 38.23±0.94 34.71±0.60 a b a MCHC mg/dl 36.17 ± 0.97 30.82 ± 0.31 35.94 ± 0.94 a b a ESR mm/20 min 24.48 ± 1.69 78.27 ± 2.70 25.82 ± 2.28 3 a b a Thrombocytes x10 µl 478.96 ± 18.28 277.40 ±12.06 475.23 ± 16.19 a b a Reticulocytes % 0.00±0.0 3.10 ± 0.13 0.04 ± 0.01 3 a b a TLCs x10 µl 8.87 ± 0.25 12.45 ± 0.32 9.03 ± 0.24 3 a b a Lymphocyte x10 µl 4.65 ± 0.25 7.37±0.38 4.99 ± 0.28 (%) (45.66 ± 1.52) (87.94 ± 0.67) (44.93±0.72) 3 a b a Neutrophil x10 µl 4.82 ± 0.20 6.29 ± 0.25 4.84 ± 0.20 (%) (47.62 ± 0.43) (64.53 ± 0.72 ) (46.24 ± 0.63) a a a 3 0.66 ± 0.06 0.74 ± 0.07 0.68 ± 0.03 Monocyte x10 µl (%) (5.63 ± 0.1) (5.71 ± 0.2) (5.20 ±0.2)
a a a 3 0.67 ± 0.47 0.72 ± 0.64 0.71 ± 0.05 Basophile x10 µl (%) (7.84 ± 0.2) (7.58 ± 0.22) (6.77± 0.1)
3 a a a Eosinophil x10 µl 0.40 ± 0.05 0.43 ± 0.04 0.40± 0.24 (%) (3.27 ± 0.2) (3.17± 0.1) (3.26 ± 0.1)
S.E.) Mean values ± standard error; TECs) Erythrocytes counts; Hb) Hemoglobin concentration; PCV) Packed cell volume, MCV) Mean corpuscular volume; MCHC) Mean corpuscular hemoglobin concentration; (TLCs) Total leukocytes counts; DLCs) Differential leukocytes counts; ESR) Erythrocyte sedimentation rate. Significantly different at (P<0.05) between equids status are labeled with the horizontal different letters (a, b).
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Table 4.5: Serum biochemistry changes in the equids with clinical form compared to the equids with subclinical form and healthy group.
Healthy group Clinical form Subclinical Parameters (Control) form Mean ±S.E Mean ±S.E Mean ±S.E a b a AST U/L 264.6 ± 6.18 370.43 ±13.19 264.26± 6.38 a b a ALT U/L 20.92 ± 0.59 38.93 ± 0.97 20.70 ± 0.57 a b a ALP U/L 145.51 ± 5.02 227.83±10.34 146.83 ± 4.53 a b a BUN mg/dl 21.24 ± 0.54 55.96 ± 1.79 21.53 ±0.50 a b a Total bilirubin mg/dl 1.70 ± 0.55 2.85 ± 0.16 1.62 ± 0.46 a b a Total protein g /dl 7.58 ± 0.09 3.22 ± 0.11 7.63 ± 0.09 a b a Albumins g/dl 3.22 ± 0.17 1.57 ± 0.10 3.11 ± 0.15 a b a Globulins g /dl 4.63 ± 0.16 1.85 ± 0.10 4.40 ± 0.16 a b a Calcium mg/dl 11.24 ± 0.28 6.61 ± 0.39 10.92 ± 0.25 a b a Glucose mg/dl 131.88 ± 6.17 68.16 ± 2.87 129.26 ± 5.87 a b a Phosphorus mg/dl 6.86 ± 0.23 3.52 ± 0.17 6.59 ± 0.28 a b a Creatinine mg/dl 1.62 ± 0.10 0.63 ± 0.07 1.67± 0.08
S.E.) Mean values ± standard error; AST) Aspartate amino transferase; ALT) Alanine amino transferase; ALP) Alkaline phosphatase; BUN) Blood urea nitrogen. Significantly different at (P<0.05) between equids status are labelled with the horizontal different letters (a, b).
4.4 Discussion
In the context of Malaysia, and to the best knowledge of this study, this is
the first report of equids with an acute clinical form of EP. Previous studies
conducted on clinically healthy equids in Peninsular Malaysia, showed that the
prevalence of T. equi and B. caballi was 0% in Kelantan based on microscopic
examination method of stained blood smears (Chandrawathani et al., 1998). Based
on cELISA, the seroprevalence of T. equi was 20% and B. caballi was 1% in
Malaysia (Zawida et al., 2010).
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This study revealed that the overall seroprevalence of T. equi, B. caballi and both protozoa was significantly higher in equids with subclinical form
(70.29%) than clinical form (9.80%) (P<0.05). This result approximates the prevalence reported by Alsaad et al. (2012) in Iraqi equids.
Equids with acute clinical form may finally recovered, but remain in subclinically form with low parasitemia (Kappmeyer et al., 2012). Ticks feed on
3 6 -1 equids with a subclinical form with low parasitemia (2 x 10 - 10 ml of blood) can successfully transmit T. equi to non-infected susceptible horses (Ueti et al.,
2008). Alanazi et al., (2014) noted that horses with subclinical form act as an important reservoir for the protozoa.
The results showed that equids with acute clinical form have higher antibody titers against T. equi (1/160 – 1/1280) and B. caballi (1/160 – 1/640), whereas equids with subclinical form have low titers against T. equi and B. caballi
(1/5–1/80). This observation is in agreement with Holbrook et al., (1972) and
Friedhoff and Soule, (1996), who stated that the antibody titer in acute clinical cases is 1/160 or above , while remaining at low levels (1/80 or less) in horses in carrier’s stage or subclinical cases.
Clinical examination of equids with clinical form highlight the acute signs of EP, and these signs are the same as those reported by Garba et al. (2011);
Salib et al. (2013); Alsaad, (2014) and Salama, (2016).
rd The result of this study showed hemorrhages on the 3 eye lid and conjunctivae in some equids with clinical form. This may be due to thrombocytopenia (Schalm et al., 2003). Other equids showed paleness of mucous
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membranes. This may indicate anemia, and the excessive destruction and elimination of infected RBCs by reticulo-endothelial macrophages change the color of mucus membranes from pale to become icteric and increase the level of bilirubin in the urine of infected equids, thus leading to a progressive type of anemia (Alsaad, 2009).
Equids with clinical form in this study showed difficulty in movement, incoordination and muscle tremors. This was due to generalized weakness and hypocalcaemia, which is in agreement with Radostitis et al. (2008).
The fetlock joint oedema which appeared in some equids in this study could be due to the differences between arterial hydrostatic pressure and different osmotic pressure causing fluids to escape from the vessels and accumulate in the distal parts of the body. Furthermore, hypoprotenemia may also have a significant part in the dialysis and accumulation of edematous fluids (Romerrio & Dyson.,
1997).
The results showed that hemoglobinurea was seen in equids with clinical form of EP. This was due to the intravascular destruction of RBCs and the release of hemoglobin, which then passes through the kidney and discolors the urine to brownish or dark coffee-like color and this is in agreement with Niven, (2002).
The damage to RBCs could be due to various reasons such as the result of infections, including the increased intracellular pressure of infected cells during multiplication of the protozoa, toxic mechanism by haemolytic factor produced by parasite and increase in erythrocyte fragility due to protozoal consumption of the
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phosphorus component or disturbance in the accumulation of proteins and fats of cell wall leading to more destruction (de Gopegui et al., 2007; Zobba et al., 2008).
Some equids in the current research showed colic signs. This could have been caused by disturbances of intestinal movements from diarrhea or constipation; as was also stated by Hailat et al. (1997) and Alsaad, (2009).
Hepatic insufficiency lead to a deficit of bile salts secretion and subsequent digestive disturbances, while frequent hemoglobinurea might cause glomerulo- nephrosis and renal damage and the microthrombosis in intestinal capillaries, all of which are a reflection of colicky signs (Radostitis et al., 2008).
In the current study, signs of nervousness were recorded in equids with clinical form, manifested by walking in a circle, ataxia, mild tonic-clonic spasms and paralysis of hind limbs, in agreement with the findings of Soulsby, (1982).
There was evidence of dehydration in diseased equids, which could be due to the lack of body fluids resulting in anuria or oliguria, increase thirst and rough hair coat, which were also observed by Lewis et al. (1999).
The present study noted the presence of ticks on different body parts of infected equids. This may refer to the fact that ticks are important vectors of piroplasms, as indicated by Kouam et al. (2010a).
This study also showed that higher body temperatures coincide with the presence of protozoa in the circulating blood of equids with clinical form. This result is consistent with Soulsby, (1982). The severity of fever depends on the virulence of protozoa, disease stage, type of lesions and generalized infection
(Krause, 2002). The body temperature rises in the infected equids due to release of
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the endogenous pyrogens after cellular lysis which stimulates the thermoregulatory centers of the hypothalamus (Radostitis et al., 2008).
The result also showed increase in the heart and respiratory rates in equids with clinical form. This may be due to hypoxia (hypoxic anaemia), which occurs as a compensatory mechanism.
Results of hemogram revealed a significant decrease in RBCs, Hb, PCV, and thrombocytes, which reflected anemia in equids with clinical form of EP in this study. These findings were consistent with Zobba et al. (2008); Alsaad et al.
(2010) and Sumbria et al. (2016b). The type of anemia may differ according to the severity and stage of the disease. In this study, macrocytic hypochromic type of anemia was observed in the equids with clinical form and this was due to a significant increase in the MCV and MCHC values. This was also reported by
Ibrahim et al. (2011) and Sumbria et al. (2016b). Anemia has been reported in naturally infected foals with EP (Alsaad, 2009). The advancement of anemia in equids is a clear clinical sign of T. equi infection (de Waal & van Heerden, 2004).
The results of hemogram in this study confirm the anaemia. This was also
reported by other researchers (Zobba et al., 2008; Alsaad et al., 2010).
There are three mechanisms for hemolytic anemia: mechanical hemolysis by parasites intra erythrocyte binary fission, auto-immunity of the anti-erythrocytic autoantibodies enhancing more erythrophagocytosis (infected and uninfected erythrocytes) and the toxic effect by the production of the hemolytic factor from the parasites or may also inhibit the hemopoitic system (Zygner et al., 2007; Zobba et al., 2008). In addition, the direct effect of the parasites on the infected
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erythrocytes causes lipid peroxidase and oxidative stress, which may lead to incriminate the life span of infected RBCs and erythrolysis (Sellon, 1997; Kumar et al., 2009).
The results showed increase in the ESR values in the equids with clinical form. This was in agreement with Alsaad et al. (2010). There is a correlation between the ESR value and intensity of anemia due to infections, and the increase in the sedimentation of RBCs will occur with increased intensity of anemia (Jain,
2000).
The appearance of reticulocytes in the blood of equids with clinical form in this study revealed that reticulocytosis synchronizes with the presence of a regenerative form of anemia and recorded the highest numerical value (5.6 -
5.7%) on days 23-25 post-infection (Maxis, 2011).
This study showed significantly increased TLCs. This was due to neutrophilia and lymphocytosis observed in infected equids. This was also recorded by Ibrahim et al. (2011) and Javed et al. (2014).
Results of serum biochemistry showed considerable increase in AST,
ALT, ALKP, BUN, total bilirubin and decrease of total protein level in equids with clinical form. This may be indicative of the damage to the skeletal and heart muscles, hepatocytes, renocytes and erythrocytes. These enzymes may be released and detected during the pathological situation. These findings concurred with the evidence in the literature (Zobba et al., 2008; Alsaad et al., 2010; Sumbria et al.,
2016b).
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The results showed that there was significant hypocalcaemia,
hypophosphatemia, and hypoglycemia in the equids with clinical form of EP.
These may be due to starvation, malabsorption, and hepatic depletion, which were
consistent with Alsaad et al. (2010) and Sumbria et al. (2016b)
This study proved and recorded the existence of a subclinical form of EP
in equids in Kelantan. In these equids no clinical signs appeared and no significant
hematological and serum biochemistry changes were observed when compared to
equids with clinical form. This result was consistent with Kappmeyer et al.
(2012); Alanazi et al. (2014) and Bahrami et al. (2014), but inconsistent with Laus
et al. (2015) who observed high prevalence of piroplasms associated with the
clinical and hematochemical alterations in subclinical donkeys.
Chemoprophylaxis using imidocarb dipropionate at a dose of 2 mg/kg body
weight IM conferred protection for 4 to 6 weeks in equids with subclinical form of
EP (Maslin et al., 2004).
4.5 Conclusion
In conclusion, this study showed that the overall seroprevalence of T.
equi, B. caballi and both protozoa was significantly higher and more risky in
equids with subclinical form (70.29%) (RR: 4.81, 6.42 and 3.56 times
respectively) compared to equids with clinical form (9.80%) (P<0.05).
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Equids with acute clinical form had higher antibody titers against T. equi
(1/160 – 1/1280) and B. caballi (1/160 – 1/640), whereas equids with subclinical form had low titers against T. equi and B. caballi (1/5–1/80).
This study recorded the wide existence of a subclinical form of EP among equids in Kelantan. In these equids, there were silent clinical signs with no significant changes in hematological and serum biochemistry parameters.
These equids with subclinical form should be monitored because it act as reservoirs of piroplasms, can transmitted to susceptible equids via tick vectors and treatment was recommended such as imidocarb dipropionate.
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CHAPTER 5
GENETIC DIVERSITY AND PHYLOGENIC ANALYSES OF THEILERIA EQUI AND BABESIA CABALLI DETECTED IN EQUIDS AND IXODID TICKS IN KELANTAN
5.1 Introduction
Rapid developments in molecular biological techniques have enabled
more efficient ways to detect, identify and characterize many hemoprotozoan
(Nagore et al., 2004; Ros-García et al., 2013; Kashyap et al., 2014). The PCR
assays have been used to detect numerous species of Theileria and Babesia and
have been shown to be more highly sensitive and more accurate in serological
tests (Buling et al., 2007; Jefferies et al., 2007).
Multiplex PCR can be used to detect Theileria and Babesia parasites in
one reaction, identify the species and genotype the protozoa. Multiplex PCR had
been proposed as a simple tool in routine diagnostic laboratory and in
epidemiological studies (Alhassan et al., 2005; Rosales et al., 2013; Qablan et al.,
2013). This assay has been recommended to detect latent infection of T. equi and
B. caballi. It can also be used to determine the genetic diversity of parasites and
the morphologically identical species that cannot be detected microscopically
(Moretti et al., 2010). In addition, the assay had provided important data on the
biogeographical distribution of piroplasms species (Kouam et al., 2010b).
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Various targeting genes have been used for detecting T. equi such as 18S rRNA gene, 16S rRNA gene and equi merozoites antigen -1, 2 and 3 (EMA-1,
EMA-2, EMA-3) genes (Bashiruddin et al., 1999; Kappmeyer et al., 2012; Qablan et al., 2013; Vianna et al., 2014). For B. caballi the target genes are 18S rRNA gene, 16S rRNA gene, Babesia caballi 48 (BC48) gene, and rhoptry associated protein-1 (RAP-1) gene (Suarez et al., 2003; Vargas et al., 2004; Alhassan et al.,
2005; Zhang et al., 2015).
The 18s ribosomal ribonucleic acid (rRNA) gene is the most common gene used to identify piroplasms in epidemiology and genetic diversity studies
(Bhoora et al., 2009; Ros-Garcaia et al., 2013). Based on PCR amplicons sequencing five genotypes of T. equi 18s rRNA gene have been distinguished; genotype A, genotype B, genotype C, genotype D, and genotype E. Whereas, three genotypes of B. caballi 18s rRNA gene have been distinguished; genotype
A, genotype B and genotype C (Qablan et al., 2013; Gallusová et al., 2014).
Many researches have been conducted on genetic diversity of T. equi and
B. caballi in equids and ticks in different countries, In China, T. equi genotype A was detected in horses (Tian et al., 2013). Theileria equi genotype E was detected in horses in South Korea (Seo et al., 2013). In Turkey, T. equi genotype E and B. caballi genotype A were detected in horses (Kizilarslan et al., 2015). Theileria equi genotypes (B and D) and B. caballi genotypes (A, B and C) were detected in equids in Jordan (Qablan et al., 2013). In Tunisia, of 104 horses examined for detection of piroplasms DNA using RLB PCR technique, T. equi genotype A
(8.7%), genotype C (1.0%) and genotype D (2.9%) were detected, besides B.
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caballi genotypes A and B (1.0%) for each, whereas, of 120 Ixodid ticks, T. equi
genotypes A and B (9.2%) and B. caballi genotype B (1.6%) were detected (Ros-
Garcaia et al., 2013). In Romania, T. equi genotypes (A, C, D and E) and B.
caballi genotype B were detected in horses. Furthermore, T. equi genotype D was
detected in dogs (Gallusová et al., 2014).
Variables in distribution of piroplasms genotypes due to insertion or
deletion in the 18s rRNA gene sequences, can be valuable to the parasite through
modifications in the gene expression, supporting their adaptation to a new
environment such as a different host (equids and ticks), climatic changes, etc.
(Kouam et al., 2010b; Bhoora et al., 2009).
The genotypes of T. equi and B. caballi that cause EP in Kelantan are
unknown and there is no gene bank database on the etiology of EP in Kelantan.
Therefore, the aims of this work were to:
1. Determine the detection rate of T. equi and B. caballi and both protozoa in
Ixodid ticks collected from equids.
2. Determine the similarity rates for detection of T. equi, B. caballi and both protozoa DNAs between equids and Ixodid ticks.
Determine the genotypes of T. equi and B. caballi detected in equids and
Ixodid ticks collected from equids.
iv) Investigate genetic diversity and phylogenetic analysis of T. equi and B.
caballi in equids and Ixodid ticks.
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5.2 Materials and Methods
5.2.1 Collection of ticks from equids
At the time of sampling, equids were inspected on the face, ears, neck,
pectoral, armpit, inguinal and under tail areas, in order to determine whether the
equids were infested with ticks or not. A total of 53 Ixodid ticks comprising 31
adult engorged females were collected from 34 equids.
5.2.2 DNA extraction from Ixodid ticks
Piroplasms DNA were extracted from 31 engorged female Ixodid ticks
that included Rhipicephalus bursa (n=8), Rh. Sanguineous (n=3), Rh. (Boophilus)
microplus (n=4), Rh. (Boophilus) annulatus (n=4), Rh. appendeculataus (n=3),
Dermacentor marginatus (n=4), Haemaphysalis longicornis (n=1), and H.
TM punctata (n=4) using ReliaPrep gDNA Tissue Miniprep system kit (Promega,
USA).
The procedure of extraction was as follows:
1. Each Ixodid tick was cut into small pieces under stereo microscope. The salivary glands, mid gut and ovaries were put into 1.5 ml eppendrof tube.
2. A total of 160μl PBS was added to each tube and mixed by vortexing. The samples were homogenized with a plastic tissue grinder.
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3. A total of 20μl proteinase K (PK) solution was added into each sample, then
200μl of cell lysis buffer (CLD) was added to the tubes, mixed by vortexing for
10 sec.
4. The samples were incubated at 56ºC for 2 hours.
5. A total of 20μl RNase A solution was added to each sample, mixed by vortexing for 10 sec, then incubated at 56ºC for 10 min.
6. A total of 250μl binding buffer (BBA) was added to each sample, mixed by vortexing for 10 sec.
7. The liquid portion of samples was transferred to the binding column, then centrifuged for 1min at full speed of 14,000 rpm (1,568 G- force).
8. The collection tubes that contained flowthrough were removed and discard.
The binding columns were placed into fresh collection tubes.
9. An amount of 500μl of column wash solution (CWD) was added to each
column and centrifuged at full speed for 2 min, and then the flowthrough
discarded.
10. Step 9 was repeated twice for a total of three times washing.
11. The columns were placed in clean 1.5ml eppendrof tubes.
12. An amount of 50μl of nuclease-free water was added to each column, and centrifuge at full speed for 1min.
13. The binding columns were discarded and the collection tubes containing the eluate genomic DNA placed in freezer at -20ºC until needed.
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The instrument and chemicals used in this extraction kit are mentioned in
(Appendix- B). The procedure mentioned above was adapted from the literature
on the extraction kit.
Determination of DNA concentration and purity were the same as
mentioned in DNA extraction from equids blood (Section 3.2). The concentration
ranged between 18 and 93 ng. Further, the purity was an A260/A280 nm ratio of
1.7-1.9.
5.2.3 PCR amplification of piroplasms DNA extracted from Ixodid ticks
The hypervariable V4 region of 18S rRNA gene of Theileria and Babesia
from 31 engorged females of Ixodid ticks were amplified to serve as a target in
multiplex PCR. The PCR amplification procedure, program, primers used in
multiplex PCR and DNA positive and negative controls were the same as
mentioned in PCR amplification from equids blood (Chapter 3.2).
Amplified DNA of T. equi (58 sequences) and B. caballi (70 sequences)
from equids blood were used in this study (Chapter 3.2).
5.2.4 DNA sequencing and phylogenetic analyses
A total of 140 PCR amplicons from equids blood and Ixodid ticks that
were positive for T. equi and B. caballi by multiplex PCR, comprised 58
amplicons for T. equi and 70 amplicons for B. caballi from equids blood and 5
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amplicons for T. equi and 7 amplicons for B. caballi from ticks. These amplicons were sent to the commercial company for purification and sequencing (My TACG
Bioscience Enterprise, Kuala Lumpur, Malaysia).
Sequences of the DNA were analyzed by using Bioedit program version
7.2.5 (Hall, 1999) and blasted against other published T. equi and B. caballi sequences from the Genbank using NCBI BLAST (BLASTn) from NCBI
(available at http://www.ncbi.nlm.nih.gov). Sequences similarity analyses
(between and within genotypes) of T. equi and B. caballi were performed using online multiple sequences alignment-CLUSTALW (GenomeNet) (available at http://www.gen ome.Jp/tools/clustalw/).
Multiple sequence alignment was done employing ClustalX (NCBI) program and phylogenetic tree was generated using the same ClustalX (NCBI) and Neighbor-joining (NJ) programs (Saitou & Nei, 1987). The 18S rRNA gene sequences of the Theileria parva (L02366) and Babesia bigemina (EF458206) were used as outgroup respectively in the construction of phylogenetic trees (100 replicates).
Finally, 20 variable sequences of T. equi (15 sequences from equids blood and 5 sequences from ticks) and 32 variable sequences of B. caballi (27 from equids blood and 5 Ixodid ticks), were deposited into GeneBank and to get the accession numbers, BankIt submission tool (available at http://www
.Ncbi.nlm.nih.gov/WebSub/?tool= GeneBank) was used.
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5.2.5 Statistical analyses
Statistical analysis conducted by using a two-sided Chi-square in IBM-
SPSS statistics version19 program, to analyze the detection rate of T. equi and B.
caballi in ticks and their genotypes. P values of <0.05 were considered
statistically significant.
5.3 Results
5.3.1 Detection rate of T. equi, B. caballi and both protozoa in Ixodid ticks using multiplex PCR
Out of 31 engorged female ticks analyzed by multiplex PCR, piroplasms
detected in 10 ticks 32.25%. Theileria equi was detected in 5 ticks (16.12%) of
the species Rhipicephalus bursa (n=2), Rh. sanguineous (n=1), Rh. (Boophilus)
microplus (n=1) and Rh. (Boophilus) annulatus (n=1). Babesia caballi was
detected in 7 ticks (22.58%) of the species Rhipicephalus bursa (n=2), Rh.
sanguineous (n=2), Dermacentor marginatus (n=2) and Haemaphysalis
longicornis (n=1). Both protozoa were detected in 2 ticks (6.45%) of the species
Rh. bursa (n=1) and Rh. sanguineous (n=1). No piroplasms were detected in Rh.
appendeculataus and H. punctata (Table 5.1). These findings indicated that B.
caballi was commonly detected in ticks compared to T. equi and both protozoa
(P<0.05).
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Table 5.1: Detection of T. equi and B. caballi in Ixodid ticks (n= 31) using multiplex PCR.
Tick No. of No. of Type of piroplasms DNA No. of species tested positive sequences Both ticks ticks T. equi B. caballi protozoa (%) 1 0 0 1 RB 8 3 1 1 1 2 0 1 0 1 1 1 1 2 RS 3 2 0 1 0 1 RBM 4 1 1 0 0 1 RBA 4 1 1 0 0 1 RA 3 0 0 0 0 0 0 1 0 1 DM 4 2 0 1 0 1 HL 1 1 0 1 0 1 HP 4 0 0 0 0 0 10 5 7 2 Total (%) 12 (32.25%) (16.12%) (22.58%) (6.45%)
No.) A number of samples; RB) Rhipicephalus bursa; RS) Rh. sanguineus; RBA) Rh. (Boophilus) microplus; RBM) Rh. (Boophilus) annulatus; RA) Rh. appendeculataus; DM) Dermacentor marginatus; HL) H. Longicornis; HP) Haemaphysalis punctata.
5.3.2 Similarity, rates for detection of T. equi, B. caballi and both protozoa DNAs between equids and Ixodid ticks.
From 34 equids infested with Ixodid ticks (males and female engorged or
non-engorged), 19 equids were infested with 31 engorged females Ixodid tick
species, including RB, RS, RBM, RBA, RA, DM, HL, and HP, which were tested
to study the similarity rates for the detection T. equi, B. caballi and both protozoa
between equids and Ixodid ticks based on multiplex PCR.
This study showed only one case (pony B number 303), which was
infested with Rhipicephalus bursa and was incompatible in the detection of T.
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equi, which was negative in a tick (RB) and positive in the pony B (PB-303)
(Table 5.2).
In addition, two cases (horse number 45 and pony B number 170) which were infested with Rhipicephalus sanguineous and Rhipicephalus (Boophilus) microplus respectively showed incompatibility in detection of B. caballi. The first case was positive in the tick (RS) and negative in the horse (H-45), while the second case was negative in the tick (RBM) and positive in the pony B (PB-170)
(Table 5.2).
The results in this study showed also one case (horse number 45) which was infested with Rhipicephalus sanguineous incompatible in the detection of both protozoa, which was positive in a tick (RS) and negative in the horse (H-45)
(Table 5.2).
These results indicated that the similarity rates for detection of T. equi, B. caballi and both protozoa between equids and Ixodid ticks were 94.73%, 89.47% and 94.73% respectively (Table 5.2).
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Table 5.2: Similarity on detection of T. equi, B. caballi and both protozoa in the equids and Ixodid ticks using multiplex PCR.
Piroplasms in Ixodid ticks Piroplasms in equids blood No. of No. of Equids Species of Both ticks engorged Both T. equi B. caballi No. ticks T. equi B. caballi protozoa collected females protozoa
H-28 1 0 RBM - - - H-34 1 0 RBM - - - PB-40 2 2 DM - + - - + - H-45 1 1 RS + + + + - - H-57 1 0 RBA - + - H-59 1 0 RB - - - H-62 2 2 RS, RA ------H-63 1 0 RBM + PB-65 1 0 RBM + - - PB-77 2 2 RBA ------H-78 2 2 RB ------PC-83 1 0 RA - - - RB,H H-90 2 1 P - + - - + - PC-I07 1 1 RBA + - - + - - PB-109 1 1 RBA ------
RBM(2), H-126 5 1 + + + + + + RA,RB,RS PB-169 2 2 RB ------PB-170 1 1 RBM - - - - + -
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Equids No. of No. of Species of mPCR results of ticks mPCR results of equids blood No. ticks engorged ticks T. equi B. caballi Both T. equi B. caballi Both collected females protozoa protozoa PA-175 1 0 RA - - - H-176 1 1 RA ------H-177 1 1 RB ------PA-190 2 2 RBM, DM + - - + - - PA-215 1 1 RS - + - - + - PC-230 2 1 RB, HP ------H-233 1 0 RBA - - - H-244 1 0 RB + + + H-247 2 0 RBM, RB - - - H-253 1 0 RB - - - H-267 3 3 HP ------H-269 3 2 RB + - - + - - H-270 1 1 HL - + - - + - PB-271 2 1 DM, RB - + - - + - H-279 1 0 RB - - - PB-303 2 2 RBM, RB - - - + - -
No.) A number of samples; mPCR) multiplex PCR; H) Horse; PA) Pony A; PB) Pony B; PC) Pony C; RB) Rhipicephalus bursa; RS) Rh. sanguineus; RBA) Rh. (Boophilus) microplus; RBM) Rh. (Boophilus) annulatus; RA) Rh. appendeculataus; DM) Dermacentor marginatus; HL) H. Longicornis; HP) Haemaphysalis punctata.
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5.3.3 Genotypes of T. equi and B. caballi in equids and ticks
This study varied our individual sequencing analysis (BLASTn) of 58
sequences of 18S rRNA gene for T. equi and 70 sequences of B. caballi out of 306
equids blood samples.
Three genotypes of T. equi were detected: genotype A 1.30%, genotype
D 12.09%, and genotype E 5.55% (Table 5. 3). Theileria equi genotype D was the
most commonly found in equids (P<0.05), commonly detected in equids in Pasir
Puteh (19.60%). Theileria equi E was commonly detected in Machang (16.66%).
Theileria equi genotype A was commonly detected in Tanah Merah (10.00%)
(Table 5.4).
Three genotypes of B. caballi were detected: genotype A (4.24%),
genotype B (15.03%), and genotype C (3.59%) (Table 5.3). Babesia caballi
genotype B was the most commonly detected in equids (P<0.05), and commonly
detected in equids in Machang (25%). Babesia caballi genotype C was also
commonly detected in Machang 8.33%. Babesia caballi genotype A was
commonly detected in Tanah Merah (15%) (Table 5.4).
In addition, this study carried out, individual BLASTn analyses of 5
sequences of T. equi and 7 sequences of B. caballi out of 31 Ixodid ticks. Three
genotypes of T. equi were detected in ticks: genotype A (3.22%), genotype D
(9.67%), and genotype E (3.22%). Theileria equi genotype D was more common
(P <0.05) and was detected in Rh. bursa, Rh. (Boophilus) microplus and Rh.
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(Boophilus) annulatus. Theileria equi genotypes A and E were detected in Rh. sanguineous and Rh. bursa respectively.
Two genotypes of B. caballi were detected in ticks: genotype A (6.45%) and genotype B (16.12%). Babesia caballi genotype B was more commonly detected (P <0.05) and was found in Rh. bursa, Rh. sanguineous, Dermacentor marginatus. Babesia caballi genotype A was detected in D. marginatus and H. longicornis. Babesia caballi genotype C was absent in the Ixodid tick species
(Table 5.5).
All sequences of T. equi and B. caballi obtained from equids blood and
Ixodid ticks are shown in (Appendix- C).
Table 5.3: Detection rate of T. equi and B. caballi genotypes in equids base on individual BLASTn analysis of positive samples.
No. of Type of No. of Percentage equids Genotypes protozoa sequence (%) tested a A 4 1.30 b Theileria 306 D 37 12.09
equi c E 17 5.55 Total 58 18.95
a A 13 4.24 b Babesia 306 B 46 15.03
caballi c C 11 3.59 Total 70 22.87
Values significantly different at (P<0.05) between genotypes are labelled with the different letters (a, b or c).
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Table 5.4: Detection rate of T. equi and B. caballi genotypes in Kelantan regions.
T. equi genotypes B. caballi genotypes No of Regions A D E A B C samples NP (%) NP (%) NP (%) NP (%) NP (%) NP (%) Kota Bharu 87 0 (0.0) 13 (14.94) 6 (6.89) 2 (2.29) 15(17.24) 3 (3.44)
Pasir Mas 26 0 (0.0) 2 (7.69) 1 (3.84) 2 (7.69) 4 (15.38) 1 (3.84)
Machang 12 0 (0.0) 0 (0.0) 2 (16.66) 0 (0.0) 3 (25.0) 1 (8.33)
Bachok 59 0 (0.0) 4 (6.77) 5 (8.47) 2 (3.38) 5 (8.47) 1 (1.69)
Pasir Puteh 51 0 (0.0) 10 (19.60) 2 (3.92) 2 (3.92) 6 (11.76) 3 (5.88)
Tumpat 44 2 (4.54) 4 (9.09) 1 (2.27) 2 (4.54) 10 (22.72) 2 (4.54)
Tanah Merah 20 2 (10.0) 2 (10.0) 0 (0.0) 3 (15.0) 4 (20.00) 0 (0.0)
Gua Musang 7 0 (0.0) 2 (28.57) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Total 306 4 (1.30) 37 (12.09) 17 (5.55) 13 (4.24) 46 (15.03) 11 (3.59)
NP) Number of positive equids blood samples for the genotype; %) Percentage of genotype.
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Table 5.5: Detection rate of T. equi and B. caballi genotypes in Ixodid ticks (n=31) based on individual BLASTn analysis of sequences.
No. of Percentage Genotypes Tick species sequences % a T. equi- A Rh. sanguineous 1 3.22
Rh. bursa b T. equi- D Rh. (Boophilus) microplus 3 9.67 Rh. (Boophilus) annulatus a T. equi- E Rh. bursa 1 3.22 Total 5 16.12
a B. caballi- A Dermacentor marginatus 2 6.45 Haemaphysalis longicornis Rh. bursa a B. caballi- B Rh. sanguineous 5 16.12 Dermacentor marginatus
B. caballi- C ------0 0.00 Total 7 22.58
Values significantly different at (P<0.05) between type genotypes are labelled with the different letters (a, b).
5.3.4 Similarity within and between the genotypes of T. equi and B. caballi
This study showed that the similarity within T. equi genotypes was 100%
for genotype A sequences (n=5), from 92.74% to 100% for genotype D sequences
(n=40) and from 96.69% to 100% for genotype E sequences (n=18) (Table 5.6).
The similarity between T. equi genotypes: A and D genotypes ranged
from 76.92% to 87.76%, A and E genotypes ranged from 79.72% to 84.61% and
between D and E genotypes the range was from 87.70% to 95.37% (Table 5.6).
Multiple sequence alignment for T. equi genotypes using ClustalX
(NCBI) program confirmed these similarities (Appendix D).
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The results in this study also showed that the similarity within B. caballi genotypes ranged from 98.68% to 100% for genotype A sequences (n=15), from
92.66% to 100% for genotype B sequences (n=51) and from 92.77% to 100% for genotype C sequences (n=11) (Table 5.7).
The similarity between B. caballi genotypes: A and B genotypes ranged from 80.30% to 86.37%, A and C genotypes ranged from 82.05% to 86.37% and between B and C genotypes the range was from 89.93% to 96.48% (Table 5.7).
Multiple sequence alignment B. caballi genotypes using ClustalX
(NCBI) program confirmed these similarities (Appendix- D).
Table 5.6: Similarity within and between T. equi genotypes using multiple sequence alignment- CLUSTALW (GenomeNet).
Category Genotypes Similarity (%) A 100 Within genotype D 92.74 - 100 E 96.69 - 100
A: D 76.92 - 87.76 Between genotypes A: E 79.72 - 84.61 D: E 87.70 - 95.37
Table 5.7: Similarity within and between B. caballi genotypes using multiple sequence alignment- CLUSTALW (GenomeNet).
Category Genotypes Similarity % A 98.68 - 100 Within genotype B 92.66 - 100 C 92.77 - 100
A : B 80.30 - 86.37 Between genotypes A : C 82.05 - 86.37 B : C 89.93 - 96.48
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5.3.5 Phylogenic analysis of T. equi and B. caballi sequences
In this study, a total of 140 amplicons obtained from extracted equids
blood and Ixodid ticks using multiplex PCR were sequenced (T. equi = 63 and
B. caballi = 77).
Fifteen variable sequences of T. equi obtained from equids were
deposited in the GeneBank under accession numbers (KU879042 – KU879056).
Five variable sequences of T. equi obtained from Ixodid ticks were also
deposited in the GeneBank under accession numbers (KX348233 – KX348237)
(Table 5.8).
In addition, 37 variable sequences of B. caballi obtained from equids
were deposited in the GeneBank under accession numbers (KU879042 –
KU879056). Five variable sequences of B. caballi obtained from Ixodid ticks
were deposited in the GeneBank under accession numbers (KX348233–
KX348237 (Table 5.9).
The genotypes which were obtained from Ixodid ticks represented the
same genotypes in the equids except B. caballi genotype C, which was absent in
a tick (Appendix- C).
In this study, the homology between T. equi 18S rRNA gene sequences
obtained from equids blood and ticks and GeneBank database demonstrated that
genotype D sequences (n=10) were highly related (100% identity) to those
sequences of Romania (KJ908946, KJ90894647, KJ90894674) (Table 5.10).
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Genotype E sequences (n=5) were observed (100% identity) together
with those sequences of South Korea (HM229407), China (KF559357) and
Romania (KJ908953, KJ908954) (Table 5.10).
Genotype A sequences (n=5) were shown (100% identity) to be similar
to those sequences of Trinidad (KU289090, KU289093, KU289094) and USA
(JX177672) (Table 5.10).
In addition, the homology of B. caballi 18S rRNA gene sequences
obtained from equids blood and ticks and GeneBank database observed, showed
that genotype C sequences (n=3) were closely related (99% identity) to Jordan
(JQ417253) and (98% identity) to those sequences of South Africa (EU642514)
and Jordan (JF827602) (Table 5.11).
Genotype B sequences (n=24) were observed with high similarity (98% c) 100% identity) to those sequences of Jordan (JQ417254, JQ417261, JQ417262,
JN596977, JN596978), and South Africa (EU888901-04) (Table 5.11).
Genotype A Sequences (n=5) were found to have lower relation (96%
identity) to those sequences of Jordan (JQ417260) and Romania (KJ908930) and
(95% identity) to the sequence of Jordan (JN596980) (Table 5.11).
In this study, phylogenetic trees analysis using the neighbor-joining, T.
equi and B. caballi yielded trees with almost identical topologies and high
bootstrap or nodal support values.
Kelantan T. equi 18S rRNA gene sequences exhibited five major clads
comprising the previously published genotypes of T. equi (Bhoora et al., 2009;
Salim et al., 2010; Qablan et al., 2012a; Hall et al., 2013; Qablan et al., 2013;
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Seo et al., 2013; Gallusová et al., 2014). Except genotypes B and C, other genotypes A, D and E, were present in the current study (Fig 5.1).
T. equi tree demonstrated close relation between genotypes C and D, and between genotypes B and E. The clustering of T. equi sequences exposed the presence of three genetically distinguishing genotypes represented by cladding (genotype A), clades (genotypes C and D) and clades (genotypes B and
E). The tree was rooted with Theileria parva (Accession number EF458206) as an outgroup (Fig. 5.1).
Kelantan B. caballi 18S rRNA gene sequences showed tree major clads comprising the previously published genotypes of B. caballi (Bhoora et al.,
2009; Qablan et al., 2012a, b; Qablan et al., 2013). B. caballi tree revealed the existence of three genetically distinct genotypes comprising clad A, clad B and clad C. The tree was rooted with Babesia bigemina (Accession number
EF458206) as an outgroup (Fig. 5.2).
In the phylogenetic trees of T. equi and B. caballi, the samples were obtained from horses, ponies and Ixodid ticks from different regions in Kelantan,
Malaysia.
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Table 5.8: GeneBank accession numbers of Kelantan T. equi genotypes in equids and ticks.
Accession Accession No. Equids No. of 18S of 18S rRNA Genotypes No. rRNA gene gene from from equids Ixodid ticks H-126 KU879042 -- D PC-82 KU879043 -- E PA-172 KU879044 -- E H-80 KU879045 -- E H-269 KU879046 KX348236 E PC-140 KU879047 -- D H-45 KU879048 KX348235 D PA-74 KU879049 -- D PC-107 KU879050 KX348234 D H-281 KU879051 -- D PB-303 KU879052 KX348237 D H-187 KU879053 -- A PA-190 KU879054 KX348233 A PB-218 KU879055 -- A H-222 KU879056 -- A
H) Horse; PA) Pony A; PB) Pony B; PC) Pony C
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Table 5.9: GeneBank accession numbers of Kelantan B. caballi genotypes in the equids and ticks.
Accession Accession No. Equids No. of 18S of 18S rRNA Genotypes No. rRNA gene gene from from equids Ixodid ticks PA-305 KU879015 -- C PC-295 KU879016 -- C PA-174 KU879017 -- C PB-170 KU879018 -- B H-267 KU879019 -- B H-63 KU879020 -- B H-49 KU879021 B H-90 KU879022 KX349895 B PA-148 KU879023 -- B H-178 KU879024 -- B H-160 KU879025 -- B H-126 KU879026 -- B H-181 KU879027 -- B PA-231 KU879028 -- B H-57 KU879029 -- B PB-108 KU879030 -- B PC-82 KU879031 -- B PA-215 KU879032 KX349896 B H-214 KU879033 -- B H-257 KU879034 -- B H-261 KU879035 -- B H-223 KU879036 -- B PB-40 KU879037 KX349894 B H-15 KU879038 -- B PB-271 KU879039 KX349898 A H-278 KU879040 -- A H-270 KU879041 KX349897 A
H) Horse; PA) Pony A; PB) Pony B; PC) Pony C
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Table 5.10: Homology between obtained sequences (GeneBank accession numbers) of T. equi and GeneBank database using online sequence BLASTn.
Sequence GeneBank- Query Sequence accession Gaps Genotypes NCBI No. cover Identity No. KU879042 KJ908946 100% 298/298(100%) 0/298(0%) D KU879043 HM229407 100% 306/306(100%) 0/306(0%) E KU879044 KF559357 100% 306/306(100%) 0/306(0%) E KU879045 KJ908954 97% 298/298(100%) 0/298(0%) E KU879046 KJ908953 100% 306/306(100%) 0/306(0%) E KX348236 KJ908954 97% 298/298(100%) 0/298(0%) E KU879047 KJ908947 100% 295/295(100%) 0/298(0%) D KU879048 KJ908974 100% 306/306(100%) 0/306(0%) D KU879049 KJ908974 100% 428/428(100%) 0/428(0%) D KU879050 KJ908946 100% 317/317(100%) 0/317(0%) D KU879051 KJ908946 96% 289/289(100%) 0/289(0%) D KU879052 KJ908946 100% 288/2288(100%) 0/288(0%) D KX348235 KJ908946 100% 286/288(99%) 0/288(0%) D KX348234 KJ908946 100% 303/306(99%) 1/306(0%) D KX348237 KJ908946 100% 304/306(99%) 0/306(0%) D KU879053 KU289094 100% 286/2286(100%) 0/286(0%) A KU879054 KU289093 100% 286/2286(100%) 0/286(0%) A KU879055 KU289090 100% 286/2286(100%) 0/286(0%) A KU879056 JX177672 100% 286/2286(100%) 0/286(0%) A KX348233 KU289094 100% 286/2286(100%) 0/286(0%) A
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Table 5.11: Homology between obtained sequences (GeneBank accession numbers) of B. caballi and GeneBank database using online sequence BLASTn.
Sequence GeneBan Query Sequence accession k- NCBI Gaps Genotype cover Identity No. No. KU879015 JQ417253 100% 453/457(99%) 4/457(0%) C KU879016 EU642514 100% 450/457(98%) 4/457(0%) C KU879017 JF827602 100% 447/457(98%) 4/457(0%) C KU879018 JQ417262 100% 453/455(99%) 2/455(0%) B KU879019 JQ417262 100% 453/453(100%) 0/453(0%) B KU879020 JQ417254 99% 453/455(99%) 2/455(0%) B KU879021 JQ417254 99% 453/453(100%) 0/453(0%) B KU879022 JQ417254 100% 453/453(100%) 0/453(0%) B KU879023 JN596977 98% 447/449(99%) 0/449(0%) B KU879024 JQ417262 99% 452/456(99%) 3/456(0%) B KU879025 JQ417262 99% 452/453(99%) 0/453(0%) B KU879026 JQ417262 100% 452/453(99%) 0/453(0%) B KU879027 JQ417262 100% 452/455(99%) 2/455(0%) B KU879028 JQ417262 100% 452/455(99%) 2/455(0%) B KU879029 JQ417258 100% 452/457(99%) 4/457(0%) B KU879030 JQ417262 99% 451/454(99%) 1/454(0%) B KU879031 JQ417261 99% 446/450(99%) 0/450(0%) B KU879032 EU888901 98% 442/444(99%) 0/444(0%) B KU879033 JQ417262 100% 453/453(100%) 0/453(0%) B KU879034 EU888904 99% 449/456(98%) 3/456(0%) B KU879035 JQ417262 100% 452/455(99%) 2/455(0%) B KU879036 JQ417262 100% 52/455(99%) 2/455(0%) B KU879037 JN596978 88% 449/451(99%) 2/451(0%) B KU879038 JQ417254 100% 447/453(99%) 0/453(0%) B KX349894 JQ417262 100% 452/455(99%) 2/455(0%) B KX349895 JQ417254 100% 453/453(100% 0/453(0%) B KX349896 JQ417262 100% 452/456(99%) 3/456(0%) B KU879039 JQ417260 100% 444/461(96%) 14/461(3%) A KU879040 KJ908930 99% 426/445(96%) 12/445(2%) A KU879041 JN596980 99% 436/457(95%) 14/457(3%) A KX349897 JQ417260 99% 444/462(96%) 15/462(3%) A KX349898 JQ417260 99% 444/462(96%) 15/462(3%) A
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Figure 5.1: Phylogenetic tree of T. equi obtained with partial sequences of the 18S rRNA gene. The numbers at the branches indicate bootstrap supports (100 replications). Sequences in bold indicate to those obtained sequences in this study. Equids, ticks and locations are in between brackets. A, B, C, D and E indicate to genotypes.
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Figure 5.2: Phylogenetic tree of B. caballi obtained with partial sequences of the 18S rRNA gene. The numbers at the branches indicate bootstrap supports (100 replications). Sequences in bold indicate to those obtained sequences in this study. Equids, tick and locations are in between brackets. A, B and C indicate to genotypes.
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5.4 Discussion
Equine piroplasmosis was previously reported in Malaysia using a
microscopic examination of stained blood smears and competitive enzyme-linked
immunosorbent assay (cELISA) test (Chandrawathani et al., 1998; Zawida et al.,
2010). In the current study, multiplex PCR assay was used to diagnose equine
piroplasms by specific primers to differentiate between T. equi and B. caballi and
to detect both protozoa in a single reaction.
Regarding Ixodid ticks investigation, this study showed that the detection
rate of piroplasms in Ixodid ticks was 32.25%; for T. equi, 16.12% was detected
in RB, RS, RBM, and RBA. For B. caballi, 22.58% was detected in RB, RS, DM,
and HL. For both protozoa, 6.45% was detected in RB and RS. These findings
were consistent with Scoles and Ueti, (2015) who found that these tick species are
able to transmit T. equi and B. caballi or both protozoa. Other studies for
detection piroplasms DNA in Ixodid tick species using PCR technique have been
done in different countries such as: In Japan, T. equi was detected in
Haemaphysalis longicornis (Ikadai et al., 2007). In India, T. equi was detected in
five of 74 ticks including Rh. microplus, Hyalomma spp. and Haemaphysalis spp.
(Kashyap et al., 214). In Iran, T. equi was detected in three tick species,
Hyalomma excavatum (n=2) and Rh. bursa, while no ticks were found in B.
caballi (Abedi et al., 2014a). Theileria equi and B. caballi were detected in
Dermacentor nuttalli in Mongolia (Battsetseg et al., 2001). In Brazil, T. equi and
B. caballi were detected in eggs and larvae of Rh. (Boophilus) microplus
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(Battsetseg et al., 2002). T. equi and B. caballi were detected in Hyalomma marginatum (9.1%), Ixodes ricinus (5.1%), Dermacentor marginatus (5%),
Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%), in Italy (Lori et al.,
2010).
The results in this study revealed that the scarcity of T. equi infected D. marginatus, and H. longicornis. This may be due to the small number of tick samples tested (n=4, n=1) respectively.
In this study, Rh. appendeculataus and H. punctata were collected from equids. However, they were negative for piroplasms DNA. This finding corresponds with Taylor et al. (2007) and Scoles and Ueti, (2015) who mentioned that these tick species can infest equids but it has not been confirmed yet as vectors for equids piroplasms.
In this study, higher similarity rates were recorded in the detection of T. equi, B. caballi and both protozoa between Ixodid ticks species and equids using multiplex PCR. This result may indicate tick vector capacity of RB, RS, RBM,
RBA, DM, and HL for transmission of T. equi, B. caballi and both protozoa to the susceptible equids. This finding is in agreement with other studies (Lori et al.,
2010; Ros-García et al., 2013; Scoles & Ueti, 2015), whereas it disagrees with
Estrada-Peña et al. (2013) who mentioned that the ticks feeding on reservoir animals are likely to ingest some pathogens with the blood meal, thus pathogens in engorged ticks must always be interpreted with great caution, as positive findings might be caused by remnants of imbibed blood meals containing microorganism DNA, which does not necessarily implicate the ticks as a vector.
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In the current study regarding sequencing BLASTn of PCR amplicons of
18s rRNA gene for T. equi and B. caballi obtained from equids blood samples and
Ixodid ticks, three genotypes of T. equi were detected for the first time in
Kelantan including genotypes A, D and E. This is very similar to previously known genotypes in other parts of world such as Trinidad, USA-Florida, Brazil,
South Korea, China, and Romania (Bhoora et al., 2009; Salim et al., 2010; Qablan et al., 2012a; Hall et al., 2013; Qablan et al., 2013; Seo et al., 2013; Gallusová et al., 2014). The absence of T. equi genotypes B and C in this current study reflects the findings of other studies by Kouam et al. (2010b) and Hall et al. (2013).
In addition, three genotypes of B. caballi were detected for the first time in Kelantan, and, included genotypes A, B, C. B. caballi genotype A was 95% to
96% identical to those identified in Jordan and Romania (Qablan et al., 2012a;
Qablan et al., 2013; Gallusová et al., 2014). Babesia caballi genotypes B and C were 98% to 100% identical to those identified in Jordan and South Africa
(Bhoora et al., 2009; Qablan et al., 2012a, b; Qablan et al., 2013).
In this study, same T. equi and B. caballi genotypes detected in equids blood were found in the Ixodid ticks except B. caballi genotype C, which was absent, and could be due to the small sampling of tested ticks.
The results of this study regarding the distribution of T. equi and B. caballi genotypes were obtained from equids and ticks in Kelantan. Surprisingly,
T. equi genotype D was the more commonly detected genotype in equids and
Ixodid ticks in Kelantan, with rates of 12.09% and 9.67% respectively, and commonly found in Pasir Puteh region. This observation is consistent with the
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study by Salim et al. (2010) which had described this genotype as the most widespread in Sudan.
In contrast to other studies that found T. equi genotype A as the most prevalent in the humid and sub-humid bioclimatic zones in Tunisia (Ros-García et al., 2013), relatively well distributed in all coastal countries around the
Mediterranean Sea (Bashiruddin et al., 1999; Kouam et al., 2010b) and in Africa,
America and Asia (Mehlhorn & Schein, 1984; Bhoora et al., 2009).
In Europe, T. equi genotype B has been found to be most prevalent
(Nagore et al., 2004; Kouam et al., 2010b) but this is not reported in this current study. This is in agreement with the absence of this genotype in South Africa and
Romania (Bhoora et al., 2009; Gallusová et al., 2014).
Babesia caballi genotype B was the genotype most commonly detected in equids and Ixodid ticks in Kelantan with 15.09%, and 16.12% respectively and commonly found in the Tumpat region. This finding agrees with Ros-García et al., (2013) who observed both B. caballi genotypes A and B, in the sub-humid zone in Tunisia being highest presence of genotypes B.
Babesia caballi genotypes B has been detected in different countries like
Spain, South Africa, Romania, and Jordan (Nagore et al., 2004; Bhoora et al.,
2009; Qablan et al., 2013; Gallusová et al., 2014), while B. caballi genotypes A was observed in Greece and novel genotypes C has been detected in Jordan
(Kouam et al., 2010b; Qablan et al., 2013).
These variables in distribution of piroplasms genotypes can be interpreted based on the insertion or deletion in the 18s rRNA gene sequences,
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which can be valuable to the parasite by way of modifying in the gene expression,
which supports their adaptation to a new environment such as a different host
(equids and ticks) and climatic changes (Kouam et al., 2010b; Bhoora et al.,
2009)
In this study, phylogenetic analysis of T. equi tree showed five majer
sequence diversity of the targeting 18S rRNA gene with a closely relation
between genotypes C and D, represented in one clad and between genotypes B
and E, also represented in one clad. These findings are consistent with Qablan et
al., (2013) and Gallusová et al., (2014).
The analysis of B. caballi tree demonstrated less sequence diversity of
the targeting 18S rRNA gene represented by only three clads A, B and C. This
agrees with Qablan et al. (2013), and Gallusová et al. (2014).
5.5 Conclusions
To the best knowledge of this researcher, this is the first molecular study
in Kelantan where multiplex PCR was used to detect T. equi and B. caballi equine
in Ixodid tick species collected from equids.
Theileria equi was detected in Ixodid ticks of the species RB, RS, RBM
and RBA. Babesia caballi was detected in Ixodid ticks of the species RB, RS,
DM and HL, whereas, no piroplasms were detected in RA and HP.
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High similarity rate on detection of T. equi, B. caballi and both protozoa in the Ixodid ticks and equids blood indicate that these ticks are one of the important predisposing factors in the spread of EP in Kelantan.
For the first time, three genotypes of T. equi (A, D and E) obtained from equids blood and Ixodid ticks were reported in Kelantan, Malaysia, and were deposited in the GeneBank under accession numbers KU879042–KU879056 and
KX348233–KX348237 respectively.
In addition, for the first time three genotypes of B. caballi (A, B and C) obtained from equids and two genotypes (A and B) obtained from ticks were reported in Kelantan, Malaysia and were deposited in the GeneBank under accession numbers KU879042–KU879056 and KX348233–KX348237 respectively.
Theileria equi genotype D and B. caballi genotype B were the most detected in Kelantan and commonly found in Pasir Puteh and Machang regions respectively.
Phylogenetic analyses for T. equi and B. caballi trees indicated a high level of genetic diversity within 18S rRNA genes.
In this study, ticks play an important role as EP transmitter. Therefore, good control programs for ticks should be implemented to prevent the spread of the disease.
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CHAPTER 6
CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK
6.1 General conclusions
This study showed three types of infections that cause EP, which are T.
equi, B. caballi and both protozoa, They were determined using microscopic
examination of blood smears stained with Giemsa as a primary method of
diagnosis. In addition, various shapes, sizes and parasitemia of T. equi and B.
caballi. Theileria equi appear smaller than B. caballi inside the infected RBCs.
This study demonstrated that Kelantan is endemic for EP and the
prevalence of the disease is highest in Pasir Puteh and Bachok regions. The
cELISA test and multiplex PCR technique are more efficient for detecting T. equi
and B. caballi infections in equids than microscopic method. Further, multiplex
PCR is more efficient than cELISA. There is no significant difference between
multiplex PCR and conventional PCR.
The significance of different risk factors associated with high
seroprevalence of T. equi, B. caballi and both protozoa in Kelantan such as
gelding equids, over five years old, thoroughbred horse, pony A breed, imported
equids, sports equids, pregnant mares in the third stage, subclinically equids, ticks
found on equids, Pasir Puteh and Bachok regions, months of September and
December, equids kept with other animals in stable, equids in grazing land, ticks
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found in stable, presence of ticks on equids and nearby animals, when the mean of monthly temperature ranges from 26.4ºC - 28.5ºC, low rainfall amount ranging from 0.6mm - 289.4mm, and higher relative humidity ranging from 80.9% -
86.6%.
Eight ixodid ticks species were identified after collection from equids and nearby animals. The animals near equids: such as cattle, sheep, goats, and dogs act as reservoirs for piroplasms and sources of ticks to the equids. The RBA,
RB, RA, DM, HL and HP, were reported for the first time in Kelantan, Malaysia, whereas, RBM and RS had been reported previously. Six species of these Ixodid ticks play an important role in spreading EP in Kelantan and they include RBM,
RBA, RB, RS, HL and DM.
The seroprevalence of T. equi, B. caballi and both protozoa was significantly higher in equids with subclinical form with low antibody titers ranging from 1/5 – 1/80 compared to equids with clinical form with higher antibody titers ranging from 1/160 – 1/1280. Equids with subclinical form were silent clinical signs with no significant changes in hematological and biochemical parameters.
Molecular techniques were used for the first time in this study for detection equine piroplasms DNA in the blood of the equids and in Ixodid ticks collected from equids in Kelantan.
Theileria equi was detected in Ixodid ticks of the species RB, RS, RBM and RBA. Babesia caballi was detected in Ixodid ticks of the species RB, RS,
DM and HL, while, no piroplasms were detected in RA and HP.
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Higher similarity rate on detection of T. equi, B. caballi and both
protozoa DNAs between ixodid ticks and equids, indicate that these ticks are one
of the important predisposing factors for spreading EP in Kelantan.
For the first time three genotypes of T. equi (A, D and E) obtained from
equids blood and Ixodid ticks were reported in Kelantan, Malaysia, and were
deposited in the GeneBank under accession numbers KU879042–KU879056 and
KX348233–KX348237 respectively.
In addition, for the first time, three genotypes of B. caballi (A, B and C)
obtained from equids and two genotypes (A and B) obtained from ticks were
reported in Kelantan, Malaysia and were deposited in the GeneBank under
accession numbers KU879042–KU879056 and KX348233–KX348237
respectively. Theileria equi genotype D and B. caballi genotype B were the most
frequently detected in Kelantan and commonly found in Pasir Puteh and Machang
regions respectively. Phylogenetic analyses for T. equi and B. caballi trees
demonstrated a high level of genetic diversity within 18S rRNA gene of T. equi
and B. caballi.
6.2 Recommendations
Competitive ELISA should be used to study the epidemiology of EP and
multiplex PCR technique for molecular detection of etiological agents of EP. To
reduce the risk of EP in Kelantan, good management practices should be adopted,
including keeping the equids in stables without being mixed with other
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animals, testing newly purchased equids before entry into the stable, and
implementing practicable chemotherapy for carriers and disease equids. In
addition, Equids quarantine facilities should be available in Kelantan because high
demand of equine from Thailand. Good tick control programs should be
implemented to prevent spreading of the disease (i.e. Chemical acaricides, anti-
ticks vaccine, etc.). Equids with subclinical form of EP should be monitored and
treated for strategic control of the disease in the Kelantan state.
6.3 Future Work
Epidemiological study of EP should be conducted in the other states of
the east coast of peninsular Malaysia, that is, Terengganu, Pahang and Johor.
More investigation should be conducted to compare the specific and
more effective treatments of the disease.
Further studies should be conducted on the molecular detection of T. equi
and B. caballi in reservoir animals (sheep, goats, cattle, donkey, and dogs) and
their ticks in Kelantan.
Future study on vaccine preparation against T. equi and B. caballi base
on their detected genotypes for strategically control of disease in Malaysia.
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APPENDIX - A
Meteorology department data (Climatic factors)
289
290
291
292
APPENDIX - B
Chemicals and instruments used in this study
B.1 Microscopic examination of blood smears
B.1.1 Purpose
The prepared of Giemsa stained thin and thick blood smears with the
view to detecting the piroplasms based on the morphological features and
biometerical data with parasitemia by microscopic examination.
B.1.2 Chemicals
4. Giemsa’s solution (Azur-eosin-methylene blue solution for
microscopy), Merck Sdn. Bhd., Germany.
5. Absolute methanol
6. Immersion oil for microscopy, Mark
7. Distilled water
293
B.1.3 Instruments
4. Microscopic slides
5. Plastic Pasture pipettes
6. Toothpicks
7. Glass staining jars
8. Microscope (Olympus BX53, FL- CCO. Japan) connected to an image
analysis system (Image pro plus version 3.0.0.1.0.0 software).
B.2 Competitive-inhibition enzyme linked immunosorbent assay (cELISA)
B.2.1 Purpose
To detect the antibodies against T. equi and B. caballi antigens by
cELISA test.
B.2.2 Chemicals
Each test cELISA kit contains: Chemicals or reagents stored at 2-7ºC (35-
45 ºF):
5. Antigen-coated plates, 2 plates.
6. Positive control, 2 ml.
7. Negative control, 2ml.
294
11. 100X Primary antibody, 300 μl.
12. 100X Secondary antibody-peroxidase, 300 μl.
13. Antibody diluting buffer, 60 ml.
14. Serum diluting buffer, 9 ml.
15. 10X Wash solution concentrate, 120 ml.
16. Substrate solution, 30 ml.
17. Stop solution, 30 ml.
B.2.3 Instruments
The following instruments were needed not included in the test kits:
15. Single and multichannel adjustable-volume pipettor.
16. Disposable yellow plastic tips.
17. Test tubes or non-antigen-coated transfer plates.
18. ELISA microplate reader or spectrophotometer with 620, 630 or
650 nm filters.
19. Deionized or distilled water.
20. Paper towels.
21. Multichannel pipettor reservoirs.
22. Wash bottle.
23. Timer (clock)
295
B.3 DNA extraction from equids blood samples
B.3.1 Purpose
To extract the piroplasms DNA from equids blood samples to use
in PCR techniques.
B.3.2 Materials / Chemicals
e) QIAamp min spin column, 250 columns.
f) Collection Tubes (2 ml), 750 tubes.
g) Buffer AL, 54 ml.
h) Buffer AW1 (concentrate), 95 ml.
i) Buffer AW2 (concentrate), 66 ml.
j) Buffer AE, 60 ml.
® k) QIAGEN Protease, 1 vial.
l) Protease solvent, 5.5 ml.
m) Other chemical needed but not included in the kit: 96-100% ethanol.
296
B.3.3 Instruments
j) Clean view UV Cabinet, Cleaver scientific Ltd. UK.
k) Microcentrifuge mini spin® plus, Fisher scientific (M) Sdn. Bhd.
l) Eppendrof centrifuge 5804, Germany.
m) Vortex mixer, Belgium.
n) Sensitive balance, Germany.
o) Water bath memert, Germany.
p) Incubator BINDER, Germany.
q) Freezer -20ºC for storing crude genomic DNA samples, Fisher scientific
(M) Sdn. Bhd.
B.4 DNA extraction from ixodid tick species
B.4.1 Purpose
To extraction the piroplasms DNA from ixodid tick species to use
in PCR technique.
297
B.4.2 Materials / Chemicals
TM f) ReliaPrep binding columns (50/ pack), 2 packs.
g) Collection tubes (40/ pack), 10 packs.
h) Cell Lysis buffer (CLD), 40ml.
i) Proteinase K (PK) solution, 2 x 1.1ml.
j) Binding buffer (BBA), 50ml.
k) Column wash solution (CWD), 165ml.
l) Nuclease-free water, 25ml.
m) RNase A solution, 2.25ml.
n) Other chemical needed but not included in the kit: Phosphate-buffer
saline (PBS).
B.4.3 Instruments
& Clean view UV Cabinet (Cleaver scientific Ltd. UK)
& Plastic tissue grinder, Fisher Scientific (M) Sdn Bhd, Malaysia
& I.5 ml eppendrof tubes
& Microcentrifuge mini spin® plus (Fisher scientific (M) Sdn. Bhd.)
& Eppendrof centrifuge 5804, Germany.
& Vortex mixer, Belgium.
& Shaker/Incubator, Germany.
298
& Incubator BINDER, Germany.
& Stereo microscope (Olympus SZX16, Japan)
& Blade or scalpel and forceps
& Freezer -20 ºC for storing crude genomic DNA samples (Fisher scientific
(M) Sdn. Bhd.)
B.5 PCR amplification of piroplasms DNA from equids blood and ixodid ticks
B.5.1 Puropose
To amplify the hypervariable V4 region of 18S rRNA gene of
Theileria and /or Babesia from equids blood and Ixodid ticks to serve as target
in the conventional PCR and multiplex PCR techniques.
B.5.2 Chemicals
& Oligonucleotide primers; catch-all (TB-F and TB-R) and specific (TBM-F, st Equi-R and BC-R), 1 BASE Laboratories Sdn. Bhd. Malaysia.
st & 2X Master Mix, 1000 reactions, ready to use, 1 BASE Pte Ltd, Singapore.
& Nuclease-free water (dh2o), Vivantis. Inc. US.
& Agarose, Biotechnology grad, 100g, Axon scientific Sdn Bhd, Malaysia.
& 10X Tris-borate-EDTA (TBE) buffer, Axon scientific Sdn Bhd, Malaysia.
& Midori green stain, 1ml, Axon scientific Sdn Bhd, Malaysia.
299
8. 6X Gel loading dye, Axon scientific Sdn Bhd, Malaysia.
9. Exact Mark DNA ladder (100-1500 bp), ready to use, Axon scientific Sdn
Bhd, Malaysia.
10. 100 μl Stock genomic DNA
B.5.3 Instruments
9. 1.5 ml microcentrifuge tubes (sterile)
10. 0.2 ml PCR thin- walled tubes (sterile)
TM 11. Thermal cycler (1000 Touch , BIO-RAD, Singapore)
12. Electrophoresis (BIO-RAD, Singapore)
13. Sensitive balance (Sartorius, Germany)
14. Glass flask
15. Microwave
16. Parafile
17. Laboratory tape
18. Gel comb and tray
TM 19. Gel Doc , EZ Imager (BIO-RAD, USA)
300
APPENDIX - C
Sequences obtained in this study
C.1 Sequences of T. equi genotypes from equids blood and Ixodid ticks
Genotype A
>187-KU879053 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGG CACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTG ACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTT AATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG
>190-KU879054 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGG CACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTG ACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTT AATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG
>218-KU879055 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGG CACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTG ACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTT AATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG
>227-KU879056 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGG CACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTG ACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTT AATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG
>KX348233-Tick CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGG CACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTG ACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTT AATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG
Genotype D
>126-KU879042 GGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAA ACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAG TTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>140-KU879047 GGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAA ACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAG TTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>45-KU879048
301
GGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTG CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGG TGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>74-KU879049 GGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTG GGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG CTAAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAG GCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTGAGTGTATCCT TGGCTGAGAGGCTTGGGTAATCTTGAGTATGCATCGTG
>107-KU879050 GGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTG GGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG CTAAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAG GCAAT
>281-KU879051 GGCTCGACCTTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGA AACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCC GTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGA GTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>303-KU879051 ACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACC AGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAG TTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTT CTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>19- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGT GTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAGTTTTAAGGCAA
>37- GGTCCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGT GTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>39- GGTCGCAAGGCTAGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTG CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGG TGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>48- GGGGGAGTATGGTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTT GACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGG TGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCT AAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGC AATAAAGGTCTGTGATGCCCTTAATGTCCTGGGCTGCACGCGCGCTACACTGATGCTTCACTGAGTGTATCCTTGGCT GAGAGGCTTGGGTAATCTTGAGTCGCATCGTGACGGGGAT
302
>118- CTGGGCACAGAGGCGGATCACGGACGACAAGCAATAGAACTTAAAGGATTGACGGAAGGGCACCACCAGGCGTGGAGC CTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTT CTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGA GACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCA AGGAAGTTTAAGGCAAA
>121- GGGGGAGTAAGAGTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGG GTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGC TAAATAGGATGTTGGAGTTGTGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG CAATA
>127- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>128- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>129- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>138- GGCTCGACCTTTACAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGA AACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCC GTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGA GTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>139- ACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACC AGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTAGTGCATGGCCGTTCTTAG TTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTT CTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>152- ACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACC AGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGGGGTGCATGGCCGTTCTTAG TTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTT CTACACTGCTTCTTAGAGGGACTTTGCGGTCATTAATCGCAAGGAAGTTTAAGG
>161- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAG GTCCAGACAGAGGAAGGTTTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTT GGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCAAAATAGGGTGTTGGAGTTATGTTCT ACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>169- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAG GTCCAGACAGAGGAAGGATTGACAGATTAATGGCTCTTTCTTGATTCTTTGGGTGGAGGTGCATGGCCGTTCTTAGTT
303
GGGGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCAAAATAGGGTGTTGGAGTTATGTTCT ACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>170- ACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAGGGGGAAACTCACC AGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAG TTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGTTAAATAGGGTGTTGGAGTTATGTT CTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>179- GGGGGAGTATGGTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTT GACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGG TGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCT AAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGC AATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTGAGTGTATCCTTG GCTGAGAGGCTTGGGTAATCTTGAGTGCGCATCGTGACGGGG
>180- GGTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCA TGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTG TGGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAATAACAGGT CTGTGA
>181- GGGGGGGGGAGTATGGTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTA ATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTT TGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACC TGCTAAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTA AGGCAATAAAGGTCTGTGATGCCCTTAATGTCCTGGGCTGCACGCGCGCTACACTGATGCTTCACTGAGTGTATCCTT GGCTGAGAGGCTTGGGTAATCTTGAGTCGCATCGTGACGGGGATCGAAAAGCCTT
>215- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAG GTCCAGACAGAGGAAGGATTGACAGATTAATGGCTCTTTCTTGATTCTTTGGGTGGAGGTGCATGGCCGTTCTTAGTT GGGGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCAAAATAGGGTGTTGGAGTTATGTTCT ACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>232- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGT GTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAGTTTTAAGGCAA
>235- GGTCCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGT GTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>236- GGTCGCAAGGCTAGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTG CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGG TGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>244- GGTCGCAAGGCTAGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTG
304
CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGG TGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>251- GGGGGAGTATGGTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTT GACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGG TGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCT AAATAGGGTGTTGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGC AATAAAGGTCTGTGATGCCCTTAATGTCCTGGGCTGCACGCGCGCTACACTGATGCTTCACTGAGTGTATCCTTGGCT GAGAGGCTTGGGTAATCTTGAGTCGCATCGTGACGGGG
>256- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>272- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>275- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>281- GGCTCGACCTTTACAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGA AACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCC GTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGA GTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>293- ACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACC AGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTAGTGCATGGCCGTTCTTAG TTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTT CTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
>296- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAACCTGCGGCTTAATTTGACTCAACACGGGGAAA ATCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTGAATAGGGTGTTGGAGT TATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTCAGG
>KX348234-Tick GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCAT GGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTT GGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAGTTTTAAGGCAA
>KX348235-Tick ACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAGGGGGAAACTCACCA GGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTT GGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGTTAAATAGGGTGTTGGAGTTATGTTCTA CACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
305
>KX348237-Tick GGTCGCAAGGCTAGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGATTCTTTGGGTGGTGGTGCA TGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGT TGGAGTTATGTTCTACACTGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
Genotype E
>82-KU879043 GGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTG CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGA TGCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>172-KU879044 GTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAATAAC
>80-KU879045 GGTCGCTCCGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGT GCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGG ATGCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>269-KU879046 GGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTG CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGA TGCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>49- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAATTTTAAGGCAA
>53- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>63- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>65- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>72- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC
306
ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>81- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>87- GGCTAGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAA ACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGAT TTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>108- GGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAA CTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATT TGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>171- GTCGCAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACG GGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCAT GGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGC GAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>256- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAATTTTAAGGCAA
>264- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>268- GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>270 GGTCGCAAGGCTGAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACA CGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGAT GCGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
>KX348236-Tick GGTCGCTCCGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAAC ACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGC ATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATG CGAGATTTGGTCTCGTTATCGCTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGGCAA
307
C.2 Sequences of B. caballi genotypes from equids blood and Ixodid ticks
Genotype-A
>271-KU879039
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>278-KU879040
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>270-KU879041
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
>275-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
>239
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>288-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
308
>293-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
>296-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>225-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>233-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
>197
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG ATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>200-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
>297-
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACAACG
309
ATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
>KX349897-Tick
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
>KX349898-Tick
GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCG TTAACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTAGAGGGACTTTACCAAC GATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGCCTTAAAATGTCCTGGGCTGCACGCGCGCTACACTG ATGCATTCAGTGCGTTTTTCCTGGTCCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGTT
Genotype-B
>171-KU879018 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>267-KU879019 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>63-KU879020 AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGA CAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTTGGGTTTGCTTCTTAGAGGGACTTTA CAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTAC ACTGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>49-KU879021 AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>90-KU879022 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG
310
ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>148-KU879023 AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>178-KU879024 AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTA CACTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>160-KU879025 AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>125-KU879026 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>181-KU879027 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>231-KU879028 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>57-KU879029 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATTCGT
>108-KU879030
311
AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGA CAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTCCCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>82-KU879031 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCCAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGCACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATGGT
>215-KU879032 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>214-KU879033 AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTA CACTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>257-KU879034 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>261-KU879035 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>223-KU879036 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATT CCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>40-KU879037 AAAGAGAAAAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGA AGGGCACCAACCAGGCGTGGAGCCTGCGGCTTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAG GATTGACAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCT GGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGA
312
CTTTACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGC GCTACACTGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGTTTTTCCTGCTC CAAAGGTGTGGGTAATCTGTCCGCATCCGCATCGT
>15-KU879038 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGAGTTTTCTTCTTTTAGGAACTTTACCGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>150- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>190- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>12- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>13- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT >14- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGA CAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTTGGGTTTGCTTCTTAGAGGGACTTTA CAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTAC ACTGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>16- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
313
>44- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>68- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>69- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTA CACTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>70- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>100- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>103- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGA CAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTTGGGTTTGCTTCTTAGAGGGACTTTA CAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTAC ACTGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>106- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>115- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC
314
GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>117- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>134- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTA CACTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>135- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>136- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>167- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGA CAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTTGGGTTTGCTTCTTAGAGGGACTTTA CAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTAC ACTGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>202- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>203- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
315
>204- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAAATTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACA GCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>281- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGAC AGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTA CACTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>240- AAAGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGA CAGATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAA TTCCGTTAACGAACGAGACCTTAACCTGCTAACTCCCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>235- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCCAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGCACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATGGT
>KX349894-Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>KX349895-Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGA TTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC GTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGCG ACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGA TGCATTCACTAAGTTTTTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>KX349896-Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATT CCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC
316
CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAGC GACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACACT GATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
>Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATT CCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTACAG CGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTTGGGCTGCACGCGCGCTACAC TGATGCATTCACTAAGTTTTTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT
Gemotype-C
>305-KU879015 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC CACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>295-KU879016 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGGTTTGCTTCTTAGAGGGACTTTACA GCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCCGCATCGT
>174-KU879017 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATT CCGTTAACGAACGAGACCTTTAACCTGCTAACCTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>1- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC CACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>6- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC CACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>35- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC CACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTAC
317
AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>62- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGGTTTGCTTCTTAGAGGGACTTTACA GCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCCGCATCGT
>157- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATT CCGTTAACGAACGAGACCTTTAACCTGCTAACCTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>184- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC CACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
>185- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAG ATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTC CGTTAACGAACGAGACCTTAACCCTGCTAACTAGCTTCCCTTTTTTTGTTTGGGGTTTGCTTCTTAGAGGGACTTTACA GCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACAC TGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCCGCATCGT
>53- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAGGAATTGACGGAAGGGCACC ACCCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACA GATTGATAGCTCTTTCTTGATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATT CCGTTAACGAACGAGACCTTTAACCTGCTAACCTAGCTTCCCTTTTTTTGTTTGGGTTTGCTTCTTAGAGGGACTTTAC AGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACA CTGATGCATTCACTACGTTTTTCCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT
318
APPENDIX - D
Alignment between obtained sequences
D.1 Multiple Alignment between T. equi sequences
Genotype A
187-KU879053 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTA 190-KU879054 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTA 218-KU879055 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTA KX348233-Tick CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTA 227-KU879056 CTTGAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTA ************************************************************ 187-KU879053 AAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC 190-KU879054 AAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC 218-KU879055 AAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC KX348233-Tick AAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC 227-KU879056 AAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC ************************************************************ 187-KU879053 GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGAT 190-KU879054 GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGAT 218-KU879055 GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGAT KX348233-Tick GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGAT 227-KU879056 GGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTTGAT ************************************************************ 187-KU879053 TCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 190-KU879054 TCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 218-KU879055 TCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC KX348233-Tick TCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 227-KU879056 TCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC ************************************************************ 187-KU879053 GTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG 190-KU879054 GTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG 218-KU879055 GTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG KX348233-Tick GTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG 227-KU879056 GTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGAGACTTGG **********************************************
Genotype D
19- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 232- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA KX348234-Tick TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 169- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 215- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 161- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 152- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 296- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAACCTGCGGCTTAATTTGACTCAA 121- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 138- TTACAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 281- TTACAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 180- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 126-KU879042 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 140-KU879047 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 107-KU879050 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 48- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 179- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA
319
129- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 244- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 272- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 139- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 293- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 170- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA KX348235-Tick TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 37- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 39- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 74-KU879049 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 303-KU879051 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA KX348237-Tick TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 181- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 236- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 256- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 275- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 251- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 235- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 128- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 127- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 281-KU879051 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 45-KU879048 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 118- TTAAAGGA-TTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA *** **** **************************** **********************
19- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 232- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT KX348234-Tick CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 169- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTAATGGCTCTTTCTT 215- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTAATGGCTCTTTCTT 161- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGTTTGACAGATTGATGGCTCTTTCTT 152- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 296- CACGGGGAAAATCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 121- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 138- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 281- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 180- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 126-KU879042 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 140-KU879047 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 107-KU879050 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 48- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 179- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 129- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 244- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 272- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 139- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 293- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 170- CAGGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT KX348235-Tick CAGGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 37- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 39- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 74-KU879049 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 303-KU879051 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT KX348237-Tick CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 181- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 236- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 256- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 275- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 251- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 235- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 128- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 127- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 281-KU879051 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 45-KU879048 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT 118- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATGGCTCTTTCTT ** ******* ************************ ********** ************* 19- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 232- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT KX348234-Tick GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT
320
169- GATTCTTTGGGTGGAGGTGCATGGCCGTTCTTAGTTGGGGGAGTGATTTGTCTGGTTAAT 215- GATTCTTTGGGTGGAGGTGCATGGCCGTTCTTAGTTGGGGGAGTGATTTGTCTGGTTAAT 161- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 152- GATTCTTTGGGTGGGGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 296- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 121- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 138- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 281- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 180- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 126-KU879042 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 140-KU879047 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 107-KU879050 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 48- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 179- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 129- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 244- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 272- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 139- GATTCTTTGGGTGGTAGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 293- GATTCTTTGGGTGGTAGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 170- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT KX348235-Tick GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 37- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 39- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 74-KU879049 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 303-KU879051 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT KX348237-Tick GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 181- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 236- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 256- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 275- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 251- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 235- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 128- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 127- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 281-KU879051 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 45-KU879048 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 118- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT ************** ********************** ********************* 19- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 232- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG KX348234-Tick TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 169- TCCGTTAACGAACGAGACCTTAACCTGCAAAATAGGGTGTTGGAGTTATGTTCTACACTG 215- TCCGTTAACGAACGAGACCTTAACCTGCAAAATAGGGTGTTGGAGTTATGTTCTACACTG 161- TCCGTTAACGAACGAGACCTTAACCTGCAAAATAGGGTGTTGGAGTTATGTTCTACACTG 152- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 296- TCCGTTAACGAACGAGACCTTAACCTGCTGAATAGGGTGTTGGAGTTATGTTCTACACTG 121- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGTTGGAGTTGTGTTCTACACTG 138- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 281- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 180- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTGGGAGTTATGTTCTACACTG 126-KU879042 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 140-KU879047 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 107-KU879050 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 48- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 179- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 129- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 244- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 272- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 139- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 293- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 170- TCCGTTAACGAACGAGACCTTAACCTGTTAAATAGGGTGTTGGAGTTATGTTCTACACTG KX348235-Tick TCCGTTAACGAACGAGACCTTAACCTGTTAAATAGGGTGTTGGAGTTATGTTCTACACTG 37- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 39- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 74-KU879049 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 303-KU879051 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG KX348237-Tick TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 181- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 236- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG
321
256- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 275- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 251- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 235- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 128- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 127- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 281-KU879051 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 45-KU879048 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG 118- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGGTGTTGGAGTTATGTTCTACACTG *************************** ****** *** ****** ************ 19- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAGTTTTAAGG 232- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAGTTTTAAGG KX348234-Tick CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAGTTTTAAGG 169- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 215- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 161- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 152- CTTCTTAGAGGGACTTTGCGGTCATTAATCGCAAGGAAGTTTAAGG 296- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTCAGG 121- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 138- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 281- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 180- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 126-KU879042 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 140-KU879047 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 107-KU879050 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 48- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 179- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 129- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 244- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 272- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 139- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 293- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 170- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG KX348235-Tick CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 37- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 39- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 74-KU879049 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 303-KU879051 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG KX348237-Tick CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 181- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 236- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 256- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 275- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 251- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 235- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 128- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 127- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 281-KU879051 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 45-KU879048 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 118- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG ************************* *********** *** ***
Genotype E
268- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 270- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA KX348236-Tick TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 264- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 171- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 108- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 87- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 72- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 81- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 65- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 63- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 53- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 80-KU879045 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA
322
269-KU879046 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 172-KU879044 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 82-KU879043 TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 49- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA 256- TTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAA ************************************************************ 268- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 270- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT KX348236-Tick CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 264- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 171- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 108- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 87- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 72- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 81- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 65- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 63- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 53- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 80-KU879045 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 269-KU879046 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 172-KU879044 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 82-KU879043 CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 49- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT 256- CACGGGGAAACTCACCAGGTCCAGACAGAGGAAGGATTGACAGATTGATAGCTCTTTCTT ************************************************************ 268- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 270- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT KX348236-Tick GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 264- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 171- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 108- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 87- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 72- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 81- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 65- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 63- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 53- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 80-KU879045 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 269-KU879046 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 172-KU879044 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 82-KU879043 GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 49- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT 256- GATTCTTTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT ************************************************************ 268- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 270- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG KX348236-Tick TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 264- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 171- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 108- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 87- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 72- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 81- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 65- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 63- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 53- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 80-KU879045 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 269-KU879046 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 172-KU879044 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 82-KU879043 TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 49- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG 256- TCCGTTAACGAACGAGACCTTAACCTGCTAAATAGGATGCGAGATTTGGTCTCGTTATCG ************************************************************ 268- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 270- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG KX348236-Tick CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 264- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 171- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 108- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG
323
87- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 72- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 81- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 65- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 63- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 53- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 80-KU879045 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 269-KU879046 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 172-KU879044 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 82-KU879043 CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAAGTTTAAGG 49- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAATTTTAAGG 256- CTTCTTAGAGGGACTTTGCGGTCATAAATCGCAAGGAATTTTAAGG ************************************** *******
D.2 Multiple Alignment between B. caballi sequences
Genotype A
271-KU879039 GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 239- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 296- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 297- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 278-KU879040 GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 225- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 197 GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 288- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG KX349898_Tick GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 270-KU879041 GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 275- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 293- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 233- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 200- GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG KX349897_Tick GAGAGAAATCAAAGTCCTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG ************************************************************ 271-KU879039 GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 239- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 296- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 297- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 278-KU879040 GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 225- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 197 GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 288- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG KX349898_Tick GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 270-KU879041 GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 275- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 293- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 233- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG 200- GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG KX349897_Tick GAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACGG ************************************************************ 271-KU879039 GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 239- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 296- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 297- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 278-KU879040 GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 225- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 197 GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 288- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT KX349898_Tick GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 270-KU879041 GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 275- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 293- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT 233- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT
324
200- GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT KX349897_Tick GAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATTCT ************************************************************ 271-KU879039 TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 239- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 296- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 297- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 278-KU879040 TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 225- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 197 TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 288- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA KX349898_Tick TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 270-KU879041 TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 275- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 293- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 233- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA 200- TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA KX349897_Tick TTGGGTGGTGGTGCATGGCCGTTCTTATTGGTGGAGTGATTTGTCTGGTTAATTCCGTTA ************************************************************ 271-KU879039 ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 239- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 296- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 297- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 278-KU879040 ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 225- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 197 ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 288- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA KX349898_Tick ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 270-KU879041 ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 275- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 293- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 233- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA 200- ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA KX349897_Tick ACGAACGAGACCTTAACCTGCTGCTAACTAGCTCCCCTTTTTTTGTTTGGGGTTTGCTTA ************************************************************ 271-KU879039 GAGGGACTTTAC-AACGATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 239- GAGGGACTTTAC-AACGATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 296- GAGGGACTTTAC-AACGATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 297- GAGGGACTTTAC-AACGATAAGGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 278-KU879040 GAGGGACTTTAC-AACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 225- GAGGGACTTTAC-AACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 197 GAGGGACTTTAC-AACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 288- GAGGGACTTTAC-AACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC KX349898_Tick GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 270-KU879041 GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 275- GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 293- GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 233- GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC 200- GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC KX349897_Tick GAGGGACTTTACCAACGATAAGTTTGTAGGGAAGTTTAAGGCAATAACAGGTCTATATGC ************ ********* ************************************* 271-KU879039 CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 239- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 296- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 297- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 278-KU879040 CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 225- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 197 CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 288- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT KX349898_Tick CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 270-KU879041 CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 275- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 293- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 233- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT 200- CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT KX349897_Tick CTTAAAATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCAGTGCGTTTTTCCTGGT ************************************************************ 271-KU879039 CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 239- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT
325
296- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 297- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 278-KU879040 CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 225- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 197 CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 288- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT KX349898_Tick CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 270-KU879041 CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 275- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 293- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 233- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT 200- CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT KX349897_Tick CCAAAAGGTCTGGGTAATCTCTCTAGTCCGCATCGT ************************************
Genotype-C
6- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 35- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 305-KU879015 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 184- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 1- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 62 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 185- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 295-KU879016 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 157- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 53- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 174-KU879017 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG ************************************************************ 6- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 35- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 305-KU879015 GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 184- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 1- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 62 GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 185- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 295-KU879016 GAATTGACGGAAGGGCACC-ACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 157- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 53- GAATTGACGGAAGGGCACCCACCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 174-KU879017 GAATTGACGGAAGGGCACCACCCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG ******************* *************************************** 6- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 35- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 305-KU879015 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 184- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 1- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 62 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 185- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 295-KU879016 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 157- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 53- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 174-KU879017 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT ************************************************************ 6- CTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 35- CTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 305-KU879015 CTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 184- CTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 1- CTTTGGGTGGTGGTGCATGGCCGTTCTTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCC 62 CTTTGGGTGGTGGTGCATGGCCGTTCTT-AGTTGGTGGAGTGATTTGTCTGGTTAATTCC 185- CTTTGGGTGGTGGTGCATGGCCGTTCTT-AGTTGGTGGAGTGATTTGTCTGGTTAATTCC 295-KU879016 CTTTGGGTGGTGGTGCATGGCCGTTCTT-AGTTGGTGGAGTGATTTGTCTGGTTAATTCC 157- CTTTGGGTGGTGGTGCATGGCCGTTCTT-AGTTGGTGGAGTGATTTGTCTGGTTAATTCC 53- CTTTGGGTGGTGGTGCATGGCCGTTCTT-AGTTGGTGGAGTGATTTGTCTGGTTAATTCC 174-KU879017 CTTTGGGTGGTGGTGCATGGCCGTTCTT-AGTTGGTGGAGTGATTTGTCTGGTTAATTCC **************************** ******************************* 6- GTTAACGAACGAGACCTT-AACCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGG-TTTGC 35- GTTAACGAACGAGACCTT-AACCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGG-TTTGC 305-KU879015 GTTAACGAACGAGACCTT-AACCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGG-TTTGC
326
184- GTTAACGAACGAGACCTT-AACCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGG-TTTGC 1- GTTAACGAACGAGACCTT-AACCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGG-TTTGC 62 GTTAACGAACGAGACCTTAACCCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGGGTTTGC 185- GTTAACGAACGAGACCTTAACCCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGGGTTTGC 295-KU879016 GTTAACGAACGAGACCTTAACCCTGCTAAC-TAGCTTCCCTTTTTTTGTTTGGGGTTTGC 157- GTTAACGAACGAGACCTTTAACCTGCTAACCTAGCTTCCCTTTTTTTGTTTGGG-TTTGC 53- GTTAACGAACGAGACCTTTAACCTGCTAACCTAGCTTCCCTTTTTTTGTTTGGG-TTTGC 174-KU879017 GTTAACGAACGAGACCTTTAACCTGCTAACCTAGCTTCCCTTTTTTTGTTTGGG-TTTGC ****************** * ********* *********************** ***** 6- TTCTTAGAGGGACTTTTACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 35- TTCTTAGAGGGACTTTTACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 305-KU879015 TTCTTAGAGGGACTTTTACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 184- TTCTTAGAGGGACTTTTACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 1- TTCTTAGAGGGACTTTTACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 62 TTCTTAGAGGGACTTT-ACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 185- TTCTTAGAGGGACTTT-ACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 295-KU879016 TTCTTAGAGGGACTTT-ACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 157- TTCTTAGAGGGACTTT-ACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 53- TTCTTAGAGGGACTTT-ACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 174-KU879017 TTCTTAGAGGGACTTT-ACAGCGATAAGTTGTAGGGAAGTTTAAGGCAATAACAGGTCTG **************** ******************************************* 6- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 35- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 305-KU879015 TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 184- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 1- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 62 TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 185- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 295-KU879016 TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 157- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 53- TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT 174-KU879017 TGATGCCCTTAGATGTCCTGGGCTGCACGCGCGCTACACTGATGCATTCACTACGTTTTT ************************************************************ 6- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 35- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 305-KU879015 CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 184- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 1- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 62 CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 185- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 295-KU879016 CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 157- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 53- CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT 174-KU879017 CCTTCTCCGAAAGGTGTGGGTAATCTGGAGTCCGCATCGT ****************************************
Genotype B
171-KU879018 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 70- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 135- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 12- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 108-KU879030 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 240- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 181-KU879027 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG KX349894_Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG 257-KU879034 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG 57-Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG 261-KU879035 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG 190- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG KX349896_Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG 231-KU879028 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG 223-KU879036 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG 126-Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAATTCTGAAACTTAAAG 63-KU879020 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 14- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG
327
103- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 167- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 57-KU879029 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 40-KU879037 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 82-KU879031 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCCAGTCTGAAACTTAAAG 235- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCCAGTCTGAAACTTAAAG 203- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 90-KU879022 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 44- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 115- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 106- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 15-KU879038 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 16- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 49-KU879021 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG KX349895_Tick GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 202- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 148-KU879023 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 160-KU879025 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 204- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 126-KU879026 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 150- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 68- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 117- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 100- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 13- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 136- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 215-KU879032 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 267-KU879019 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGTCTGAAACTTAAAG 178-KU879024 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG 214-KU879033 GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG 69- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG 134- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG 281- GAGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGTCTGAAACTTAAAG **************************************** ** * ************** 171-KU879018 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 70- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 135- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 12- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 108-KU879030 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 240- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 181-KU879027 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG KX349894_Tick GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 257-KU879034 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 57-Tick GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 261-KU879035 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 190- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG KX349896_Tick GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 231-KU879028 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 223-KU879036 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 126-Tick GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 63-KU879020 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 14- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 103- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 167- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 57-KU879029 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAATTTTGACTCAACACG 40-KU879037 GAATTGACGGAAGGGCACCAACCAGGCGTGGAGCCTGCGGCTTTAATTTGACTCAACACG 82-KU879031 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 235- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 203- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 90-KU879022 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 44- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 115- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 106- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 15-KU879038 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 16- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 49-KU879021 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG KX349895_Tick GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 202- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 148-KU879023 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG
328
160-KU879025 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG 204- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG 126-KU879026 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG 150- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG 68- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG 117- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-ATTGACTCAACACG 100- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 13- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 136- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 215-KU879032 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 267-KU879019 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 178-KU879024 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 214-KU879033 GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 69- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 134- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG 281- GAATTGACGGAAGGGCACCA-CCAGGCGTGGAGCCTGCGGCTTAA-TTTGACTCAACACG ******************** ********************** * ************* 171-KU879018 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 70- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 135- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 12- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 108-KU879030 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 240- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 181-KU879027 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT KX349894_Tick GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 257-KU879034 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 57-Tick GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 261-KU879035 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 190- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT KX349896_Tick GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 231-KU879028 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 223-KU879036 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 126-Tick GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 63-KU879020 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 14- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 103- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 167- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 57-KU879029 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 40-KU879037 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 82-KU879031 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 235- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 203- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 90-KU879022 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 44- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 115- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 106- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 15-KU879038 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 16- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 49-KU879021 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT KX349895_Tick GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 202- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 148-KU879023 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 160-KU879025 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 204- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 126-KU879026 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 150- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 68- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 117- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 100- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 13- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 136- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 215-KU879032 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 267-KU879019 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 178-KU879024 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 214-KU879033 GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 69- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 134- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT 281- GGGAAACTCACCAGGTCCAGACAGAGGTAGGATTGACAGATTGATAGCTCTTTCTTGATT ************************************************************
329
171-KU879018 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 70- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 135- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 12- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 108-KU879030 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 240- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 181-KU879027 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC KX349894_Tick CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 257-KU879034 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 57-Tick CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 261-KU879035 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 190- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC KX349896_Tick CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 231-KU879028 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 223-KU879036 CTTTGGGTTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 126-Tick CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 63-KU879020 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 14- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 103- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 167- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 57-KU879029 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 40-KU879037 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 82-KU879031 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 235- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 203- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 90-KU879022 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 44- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 115- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 106- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 15-KU879038 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 16- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 49-KU879021 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC KX349895_Tick CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 202- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 148-KU879023 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 160-KU879025 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 204- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 126-KU879026 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 150- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 68- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 117- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 100- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 13- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 136- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 215-KU879032 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 267-KU879019 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT-GTCTGGTTAATTC 178-KU879024 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAATTC 214-KU879033 CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAATTC 69- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAATTC 134- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAATTC 281- CTTTGGGT-GGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTTGTCTGGTTAATTC ******** ************************************* ************* 171-KU879018 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 70- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 135- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 12- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 108-KU879030 CGTTAACGAACGAGACCTTAACCTGCTAACTCCCTTCCCTTTTTTTGTTT--GGGTTTGC 240- CGTTAACGAACGAGACCTTAACCTGCTAACTCCCTTCCCTTTTTTTGTTT--GGGTTTGC 181-KU879027 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC KX349894_Tick CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 257-KU879034 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 57-Tick CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 261-KU879035 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 190- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC KX349896_Tick CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 231-KU879028 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 223-KU879036 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 126-Tick CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 63-KU879020 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTT-GGGTTTGC
330
14- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTT-GGGTTTGC 103- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTT-GGGTTTGC 167- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTT-GGGTTTGC 57-KU879029 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTTTTGGGTTTGC 40-KU879037 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 82-KU879031 CGTTAACGCACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 235- CGTTAACGCACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 203- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 90-KU879022 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 44- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 115- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 106- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 15-KU879038 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GAGTTTTC 16- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 49-KU879021 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC KX349895_Tick CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 202- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 148-KU879023 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 160-KU879025 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 204- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 126-KU879026 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 150- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 68- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 117- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 100- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 13- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 136- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 215-KU879032 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 267-KU879019 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 178-KU879024 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 214-KU879033 CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 69- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 134- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC 281- CGTTAACGAACGAGACCTTAACCTGCTAACTAGCTTCCCTTTTTTTGTTT--GGGTTTGC ******** ********************** ***************** * **** *
171-KU879018 TTCTTAGAGGGACTTTTACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 70- TTCTTAGAGGGACTTTTACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 135- TTCTTAGAGGGACTTTTACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 12- TTCTTAGAGGGACTTTTACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 108-KU879030 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 240- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 181-KU879027 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG KX349894_Tick TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 257-KU879034 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 57-Tick TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 261-KU879035 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 190- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG KX349896_Tick TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 231-KU879028 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 223-KU879036 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 126-Tick TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 63-KU879020 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 14- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 103- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 167- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 57-KU879029 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 40-KU879037 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 82-KU879031 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 235- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 203- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 90-KU879022 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 44- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 115- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 106- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 15-KU879038 TTCTTTTAGGAACTTT-ACCGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 16- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 49-KU879021 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG KX349895_Tick TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG
331
202- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 148-KU879023 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 160-KU879025 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 204- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 126-KU879026 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 150- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 68- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 117- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 100- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 13- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 136- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 215-KU879032 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 267-KU879019 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 178-KU879024 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 214-KU879033 TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 69- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 134- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 281- TTCTTAGAGGGACTTT-ACAGCGACAAGCTGTAGGGAAGTTTAAGGCAATAACAGGTCTG 1. *** ***** ** **************************************** 171-KU879018TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 70- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 135- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 12- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 108-KU879030 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 240- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 181-KU879027 TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT KX349894_Tick TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 257-KU879034 TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 57-Tick TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 261-KU879035 TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 190- TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT KX349896_Tick TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 231-KU879028 TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 223-KU879036 TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 126-Tick TGATGCCCTTAGATGTCCTTGGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 63-KU879020 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 14- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 103- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 167- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 57-KU879029 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 40-KU879037 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 82-KU879031 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 235- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 203- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 90-KU879022 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 44- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 115- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 106- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 15-KU879038 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 16- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 49-KU879021 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT KX349895_Tick TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 202- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 148-KU879023 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 160-KU879025 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 204- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 126-KU879026 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 150- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 68- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 117- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 100- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 13- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 136- TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 215-KU879032 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 267-KU879019 TGATGCCCTTAGATGTCCT-GGGCT-GCACGCGCGCTACACTGATGCATTCACTAAGTTT 178-KU879024 TGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTACACTGATGCATTCACTAAGTTT 214-KU879033 TGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTACACTGATGCATTCACTAAGTTT 69- TGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTACACTGATGCATTCACTAAGTTT 134- TGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTACACTGATGCATTCACTAAGTTT
332
281- TGATGCCCTTAGATGTTCCTGGGCTTGCACGCGCGCTACACTGATGCATTCACTAAGTTT **************** * ***** ********************************** 171-KU879018 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 70- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 135- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 12- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 108-KU879030 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 240- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 181-KU879027 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT KX349894_Tick TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 257-KU879034 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 57-Tick TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 261-KU879035 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 190- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT KX349896_Tick TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 231-KU879028 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 223-KU879036 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT KX349896_Tick TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 63-KU879020 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 14- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 103- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 167- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 57-KU879029 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 40-KU879037 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 82-KU879031 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATGGT 235- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 203- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 90-KU879022 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 44- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 115- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 106- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 15-KU879038 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 16- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 49-KU879021 TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT KX349895_Tick TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 202- TTCCTGCTCCGAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 148-KU879023 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 160-KU879025 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 204- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 126-KU879026 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 150- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 68- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 117- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 100- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 13- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 136- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 215-KU879032 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 267-KU879019 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 178-KU879024 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 214-KU879033 TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 69- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 134- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT 281- TTCCTGCTCCAAAAGGTGTGGGTAATCTGTAGTCCGCATCGT ********** **************************** **
333