A MOLECULAR SURVEY of TICK-BORNE PATHOGENS (Anaplasma and Ehrlichia Spp.) in ANIMAL and TICK SAMPLES in MALAYSIA

A MOLECULAR SURVEY OF TICK-BORNE PATHOGENS (Anaplasma AND Ehrlichia spp.) IN ANIMAL AND TICK SAMPLES IN MALAYSIA

KOH FUI XIAN Malaya of

FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR

University 2018

A MOLECULAR SURVEY OF TICK-BORNE PATHOGENS (Anaplasma AND Ehrlichia spp.) IN ANIMAL AND TICK SAMPLES IN MALAYSIA

KOH FUI XIAN Malaya

DESSERTATION SUBMITTEDof IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF MEDICAL SCIENCE

FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR

University2018

UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION

Name of Candidate: KOH FUI XIAN Registration/Matric No: MGN130009 Name of Degree: MASTER OF MEDICAL SCIENCE Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): A MOLECULAR SURVEY OF TICK-BORNE PATHOGENS (Anaplasma AND Ehrlichia spp.) IN ANIMAL AND TICK SAMPLES IN MALAYSIA Field of Study: MOLECULAR MICROBIOLOGY

I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work hasMalaya been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledgeof nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. University Candidate’s Signature Date: Subscribed and solemnly declared before,

Witness’s Signature Date: Name: Designation:

ii ABSTRACT

Anaplasmosis and ehrlichiosis are tick-borne diseases which are caused by Gram- negative obligate intracellular bacteria in the family Anaplasmataceae. The diseases have been reported in a wide variety of wild and domestic animals from different parts of the world. Little data is available on the prevalence and transmission of the diseases in

Malaysia. In this study, the occurrences of Anaplasma spp. and Ehrlichia spp. in animal and tick samples derived from different sources were determined. A total of 304 blood samples collected from livestock farms (cattle, sheep and goats) and 393 various animal blood samples provided by researchers from Veterinary Research Institute, Malaysia and

Department of Wildlife and National Parks (PERHILITAN) Peninsular Malaysia from

2013-2014 for health screening were included in this investigation. A wide variety of ticks collected from livestock farms, aboriginal villagesMalaya and the forest areas were subjected to morphological identification and molecular analysis of tick mitochondrial 16S rRNA gene to assist tick identification.of Haemaphysalis and Dermacentor ticks were the main ticks identified in this study, besides Amblyomma and Rhipicephalus ticks.

Using specific polymerase chain reaction (PCR) assays, Anaplasma DNA was detected in 136 (60.7%) cattle and 32 (80.0%) sheep from livestock farms investigated in this study. Majority of the Anaplasma spp. detected from the cattle were Anaplasma marginale, while all goats were not infected by Anaplasma spp. Anaplasma bovis DNA was detected from both wild and domestic animals in this study. A novel Anaplasma sp. tentativelyUniversity designated as Candidatus Anaplasma pangolinii was detected from three of 15 pangolins (Manis javanica) investigated in this study. Both Ehrlichia canis and

Anaplasma phagocytophilum were detected in dog blood samples and Rhipicephalus sanguineus ticks. A. phagocytophilum was mainly detected in questing ticks from the forest areas. A total of 61.5% ticks infesting livestock animals (cattle and sheep), 28.8% ticks collected from vegetation and small animals in the forest areas and 37.0% ticks

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infesting peri-domestic animals in aboriginal villages were PCR-positive for

Anaplasmataceae DNA. Sequence analyses of the bacterial 16S rRNA gene region (238 bp) provided the identification for A. marginale, A. bovis, A. phagocytophilum,

Anaplasma platys, Anaplasma spp. and Ehrlichia spp. in ticks and animal blood samples.

New sequence variants of Anaplasma spp., Ehrlichia sp. strain EBm52, Ehrlichia mineirensis and Candidatus Ehrlichia shimanensis were identified in this study. In conclusion, the high detection rates of A. marginale in cattle blood samples may affect the livestock production in the cattle farms. The detection of Anaplasma spp. and

Ehrlichia spp. in questing ticks and ticks infesting domestic and wildlife animals may pose a risk to human who come into contact with ticks or tick-infested animals. This study reports for the first time the type and distribution of various Anaplasma spp. and Ehrlichia spp. in animals and ticks present in this region. FurtherMalaya investigation should be carried out on the potential role of various tick species and the transmission dynamics of anaplasmosis and ehrlichiosis. Appropriateof measures should be instituted for prevention and control of tick-borne diseases in Malaysia.

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ABSTRAK

“Anaplasmosis” dan “ehrlichiosis” ialah penyakit bawaan sengkenit disebabkan oleh bakteria intrasel obligat Gram-negatif dalam famili Anaplasmataceae. Penyakit-penyakit ini pernah dilaporkan dalam pelbagai jenis haiwan liar dan domestik dari lokasi yang berlainan di dunia. Sehingga kini, maklumat tentang kelaziman dan transmisi kedua-dua penyakit ini di Malaysia masih terhad. Tujuan kajian ini adalah untuk menentukan kejadian “anaplasma” dan “ehrlichiae” dalam sampel haiwan dan sengkenit yang dikumpulkan dari sumber yang berlainan. Sebanyak 304 sampel darah dari ladang-ladang haiwan ternakan (lembu, kambing dan biri-biri) dan 393 sampel darah haiwan yang dibekalkan oleh penyelidik Institut Penyelidikan Veterinar Ipoh (VRI) dan Jabatan

Perlindungan Hidupan Liar dan Taman Negara (PERHILITAN) dari tahun 2013-2014 untuk pemeriksaan kesihatan juga dirangkumi dalamMalaya kajian ini. Pelbagai jenis sengkenit yang dikumpulkan dari ladang haiwan ternakan, perkampungan orang asli dan kawasan perhutanan dikenalpasti dengan kaedah morfologiof dan analisis molecular berdasarkan gen 16S rRNA mitokondria sengkenit. Selain daripada Amblyomma dan Rhipicephalus, sengkenit Haemaphysalis dan Dermacentor ialah sengkenit utama yang dikenalpasti dalam kajian ini. Dengan menggunakan teknik tindak balas berantai polimerase (PCR),

DNA Anaplasma dikesan dari 168 (63.6%) sampel darah lembu dan kambing biri-biri dalam kajian ini. Kebanyakan Anaplasma spp. yang dikesan di dalam lembu telah dikenalpasti sebagai Anaplasma marginale. Semua sampel kambing dalam kajian ini tidakUniversity dijangkiti oleh Anaplasma spp. DNA Anaplasma bovis telah dikesan di dalam haiwan liar dan domestik. Spesis Anaplasma novel dikesan dalam tiga tenggiling (Manis javanica) dan dinamakan secara tentatif sebagai Candidatus Anaplasma pangolinii.

Kedua-dua Ehrlichia canis dan Anaplasma phagocytophilum telah dikesan dalam sampel darah anjing dan sengkenit Rhipicephalus sanguineus. Kebanyakan A. phagocytophilum dikesan dalam sengkenit yang dikutip dari tumbuh-tumbuhan di hutan. Sebanyak 61.5%

sengkenit dari ladang-ladang haiwan ternakan (lembu dan kambing biri-biri), 28.8% sengkenit dari tumbuh-tumbuhan dan haiwan kecil di kawasan perhutanan dan 37.0% sengkenit dari haiwan peri-domestik di perkampungan orang asli didapati positif untuk

DNA Anaplasmataceae. Analisis penjujukan gen 16S rRNA (238 bp) mengenalpasti A. marginale, A. bovis, A. phagocytophilum, Anaplasma platys, Anaplasma spp. dan

Ehrlichia spp. dari sengkenit dan sampel darah haiwan. Urutan baru varian Anaplasma spp., Ehrlichia sp. strain EBm52, Ehrlichia mineirensis and Candidatus Ehrlichia shimanensis dikenalpasti dalam kajian ini. Kesimpulannya, kadar jangkitan A. marginale yang tinggi dalam sampel darah lembu berkemungkinan akan menjejaskan produktiviti haiwan ternakan. Pengesanan Anaplasma spp. dan Ehrlichia spp. dalam sengkenit dari tumbuh-tumbuhan, haiwan liar dan domestik mungkin menimbulkan risiko kepada manusia yang berdekatan dengan sengkenit atau haiwanMalaya yang dijangkiti oleh sengkenit. Kajian ini melaporkan jenis dan penyebaran Anaplasma spp. dan Ehrlichia spp. dalam haiwan dan sengkenit untuk kali pertamaof di rantau ini. Kajian selanjutnya perlu dijalankan untuk memahami pelbagai jenis sengkenit dan dinamik transmisi

“anaplasmosis” dan “ehrlichiosis”. Langkah-langkah pencegahan dan pengawalan penyakit bawaan sengkenit yang sewajarnya perlu dilaksanakan di Malaysia.

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ACKNOWLEDGEMENTS

I would like to express my utmost gratitude to everyone who have supported and helped me throughout the journey of completing this study. Firstly, my deepest gratitude to my supervisors, Professor Dr. Tay Sun Tee from Department of Medical Microbiology,

Faculty of Medicine, University of Malaya and Dr. Chandrawathani Panchadcharam from the Laboratory Diagnostics Division, Department of Veterinary Services, Ministry of

Agriculture and Agro-Based Industry Malaysia for all the encouragement and guidance given to me throughout this master study.

I would like to acknowledge the financial supports from various grants [UMRG

(RP013-2012A); HIR-MOHE: E000013-20001 and PPP (PG005-2013B)] provided by

University of Malaya for me to carry out this study. Besides that, I would like to express my sincere thanks to all the staff from the DepartmentMalaya of Wildlife and National Parks Peninsular Malaysia (PERHILITAN), Department of Orang Asli Development (JAKOA), Department of Veterinary Servicesof (DVS) and Veterinary Research Institute (VRI), Malaysia for their technical assistance in the sample collection. Additionally, I would like to thank Madam Harvinder Kaur from University Malaya Medical Centre

(UMMC) for providing technical support in this study.

I would like to extend my sincere appreciation to my labmates who accompany me through the ups and downs during this journey of pursuing my master degree. Last but not least, I would like to thank my family and friends for all the loves and supports. University

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TABLE OF CONTENTS

ABSTRACT ...... iii

ABSTRAK ...... v

ACKNOWLEDGEMENTS ...... vii

TABLE OF CONTENTS ...... viii

LIST OF FIGURES ...... xiv

LIST OF TABLES ...... xvi

LIST OF APPENDICES ...... xxiii

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: LITERATURE REVIEW ...... Malaya...... 4 2.1 Anaplasma spp...... 4 2.1.1 Anaplasma marginale ...... of 4 2.1.2 Anaplasma phagocytophilum ...... 5

2.1.3 Anaplasma centrale ...... 6

2.1.4 Anaplasma ovis ...... 7

2.1.5 Anaplasma bovis ...... 8

2.1.6 Anaplasma platys ...... 8

2.2 Ehrlichia spp...... 9

University2.2.1 Ehrlichia canis ...... 9

2.2.2 Ehrlichia chaffeensis ...... 10

2.2.3 Ehrlichia ewingii ...... 11

2.2.4 Ehrlichia ruminantium ...... 11

2.2.5 Ehrlichia muris ...... 12

2.3 New Anaplasma and Ehrlichia species ...... 13

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2.4 Anaplasma, ehrlichiae and their hosts/vectors...... 14

2.4.1 Human infections ...... 14

2.4.1.1 Anaplasmosis ...... 14

2.4.1.2 Ehrlichiosis ...... 15

2.4.2 Animal infections ...... 16

2.4.3 Anaplasma and ehrlichiae in ticks ...... 17

2.5 Infections caused by anaplasma and ehrlichiae ...... 22

2.5.1 Clinical symptoms in humans ...... 22

2.5.2 Clinical signs in animals ...... 23

2.6 Anaplasma and ehrlichiae infections in Asian countries ...... 24

2.7 Current status of anaplasmosis and ehrlichiosis in Malaysia ...... 25 2.8 Laboratory diagnosis of anaplasmosis and ehrlichiosisMalaya ...... 34 2.8.1 Histochemistry and immunochemistry ...... 34 2.8.2 Serology methods ...... of 34 2.8.3 Isolation of anaplasma and ehrlichiae ...... 35

2.8.4 Molecular detection of Anaplasma spp. and Ehrlichia spp...... 36

2.8.4.1 Molecular characterisation of Anaplasma spp. and Ehrlichia

spp…… ...... 38

2.9 Treatment for anaplasmosis and ehrlichiosis ...... 39

2.10 Prevention and control of anaplasmosis and ehrlichiosis ...... 40 University CHAPTER 3: MATERIALS AND METHODS ...... 41

3.1 Sample collection...... 41

3.1.1 Samples obtained from livestock farms ...... 41

3.1.2 Samples obtained from Veterinary Research Institute (VRI), Ipoh ...... 43

3.1.3 Samples obtained from Wildlife Department (PERHILITAN)...... 43

3.1.4 Tick sampling in aboriginal villages ...... 44

3.1.5 Other samples ...... 44

3.2 Identification of tick species using morphological identification methods ...... 46

3.3 Identification of tick species using molecular identification methods ...... 46

3.4 DNA extraction ...... 46

3.4.1 Tick samples ...... 46

3.4.2 Whole blood samples ...... 47

3.4.3 Organ samples ...... 47

3.4.4 Whatman® FTA® card ...... 48

3.5 Identification of Anaplasma spp. and Ehrlichia spp. from animals and ticks using

molecular methods ...... 48 3.5.1 Integrity checking of samples for amplificationMalaya ...... 48 3.5.1.1 Amplification of the 16S rRNA or 28S rRNA gene from tick DNA samples ...... of 48 3.5.1.2 Amplification of the cytochrome B (cytB) gene of animal blood

and organ samples ...... 49

3.5.2 Polymerase chain reaction ...... 49

3.5.3 Preliminary screening of Anaplasmataceae DNA ...... 50

3.5.4 Further characterisation of Anaplasma spp. and Ehrlichia spp...... 50

3.5.4.1 Amplification of the full length 16S rRNA, gltA and groEL genes Universityof anaplasma and ehrlichiae ...... 50 3.5.4.2 PCR assays targeting specific organism ...... 52

3.5.5 Detection of amplified PCR products generated in the PCR assays ...... 55

3.6 Generation of positive control for PCR assays ...... 55

3.6.1 Setting up cloning reaction and transformation of plasmid into competent

E. coli ...... 55

3.6.2 Plasmid extraction ...... 56

3.6.3 Analysing positive clones ...... 57

3.7 Sequence determination and analysis of selected amplified products ...... 57

3.7.1 Purification of PCR products ...... 57

3.7.2 Sequence determination and analysis ...... 59

3.7.3 Sequence with ambiguous nucleotide positions ...... 59

3.8 Serological detection of selective tick-borne pathogens from dog samples ...... 60

3.8.1 SNAP 4Dx® kit ...... 60

CHAPTER 4: RESULTS ...... 61

4.1 Collection and identification of tick species using morphological identification method...... Malaya 61 4.1.1 Livestock farms ...... 61 4.1.2 Forest areas ...... of 63 4.1.3 Aboriginal villages ...... 65

4.1.4 Other samples ...... 66

4.2 Identification of tick species using molecular identification methods ...... 67

4.2.1 Livestock farms ...... 68

4.2.2 Ticks collected from the forest areas ...... 72

4.2.2.1 Vegetation ...... 72 University4.2.2.2 Small animals ...... 73 4.2.3 Ticks collected from peri-domestic animals in aboriginal villages ...... 74

4.2.4 Ticks collected from other sources ...... 75

4.3 Integrity checking of extracted samples prior to amplification for Anaplasmataceae

DNA…...... 76

4.4 Seroprevalence of selective tick-borne pathogens in dog blood samples...... 78

4.5 Identification of Anaplasma spp. and Ehrlichia spp. from ticks using molecular

methods ...... 78

4.5.1 Preliminary screening ...... 78

4.5.2 Further characterisation of Anaplasma spp. and Ehrlichia spp...... 85

4.5.2.1 Amplification of nearly full length 16S rRNA gene of Anaplasma

spp. and Ehrlichia spp. from ticks ...... 85

4.5.2.2 Amplification of gltA gene of anaplasma and ehrlichiae ...... 90

4.5.2.3 Amplification of groEL gene of anaplasma and ehrlichiae ...... 92

4.5.2.4 Molecular characterisation of A. phagocytophilum based on three

genes (msp4, 16S rRNA and groEL) ...... 94

4.5.2.5 Molecular characterisation of A. bovis in tick samples ...... 97 4.5.2.6 Molecular characterisation ofMalaya E. chaffeensis in tick samples .... 99 4.6 Identification of Anaplasmataceae from animal samples using molecular methods… ...... of 101 4.6.1 Preliminary screening ...... 101

4.6.2 Further characterisation of Anaplasmataceae ...... 107

4.6.2.1 Amplification of the nearly full length 16S rRNA gene of

Anaplasma spp. and Ehrlichia spp...... 107

4.6.2.2 Amplification of gltA gene of Anaplasma spp. and Ehrlichia

spp…...... 111 University4.6.2.3 Molecular characterisation of A. phagocytophilum from animal blood DNA samples ...... 112

4.6.2.4 Molecular characterisation of A. bovis from animal blood DNA

samples ...... 119

4.6.2.5 Molecular characterisation of E. chaffeensis from animal blood

DNA samples ...... 122

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4.7 Molecular detection of Anaplasma spp. and Ehrlichia spp. in stray dogs and dog

ticks…...... 122

CHAPTER 5: DISCUSSION ...... 125

5.1 Identification of ticks ...... 125

5.1.1 Haemaphysalis spp...... 125

5.1.2 Dermacentor spp...... 128

5.1.3 Amblyomma spp...... 130

5.1.4 Rhipicephalus spp...... 130

5.2 Detection and identification of Anaplasmataceae DNA in ticks ...... 131

5.3 Molecular detection of A. marginale in tick and animal samples ...... 132 5.4 Molecular detection of A. bovis in tick and animalMalaya samples ...... 133 5.5 Molecular detection of A. platys in tick and animal samples ...... 135 5.6 Molecular detection of A. phagocytophilumof in tick and animal samples ...... 136 5.7 Molecular detection of Candidatus A. pangolinii in pangolins ...... 138

5.8 Detection of A. phagocytophilum and E. canis in ticks and dog blood samples . 139

5.9 Molecular detection of new variants of Anaplasma spp. and Ehrlichia spp. in tick

and animal samples ...... 141

5.10 Limitations of this study and future investigation ...... 143

CHAPTERUniversity 6: CONCLUSION ...... 145 REFERENCES ...... 146

LIST OF PUBLICATIONS AND PAPERS PRESENTED ...... 174

APPENDIX ...... 175

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LIST OF FIGURES

Figure 4.1 Ticks collected from livestock farms in this study……………….. 63 Figure 4.2 Ticks collected from the forest areas in this study……………….. 64 Figure 4.3 Ticks collected from aboriginal villages in this study……………. 66 Figure 4.4 Snake ticks collected from Sepang, Selangor…………………….. 67 Figure 4.5 Rhipicephalus sp. collected from a dog in Klang Valley…………. 67 Figure 4.6 Amplification of approximately 300 bp of the 16S rRNA gene fragments from tick samples using 16S-1/16S+2 primer pair……. 77 Figure 4.7 Amplification of approximately 490 bp of the 28S rRNA gene fragments from tick samples using 28SF/28SR primer pair…….... 77 Figure 4.8 Amplification of approximately 358 bp of the cytB gene fragments from animal blood samples using cytBFor/cytBRev primer pair………………………………………………………...... 77 Figure 4.9 Amplification of approximately 345 bp of the 16S rRNA gene fragments from samples using EHR16SD/EHR16SR primer pair………………………………………………………………… 80 Figure 4.10 Phylogenetic relationships among various Anaplasma spp. and Ehrlichia spp. based on partial sequences of the 16S rRNA gene (222 bp)………………………………………………………….... 84 Figure 4.11 Amplification of approximately 1500Malaya bp of the 16S rRNA gene fragments from samples using fD1/Rp2 primer pair……………... 86 Figure 4.12 Phylogenetic relationships among various Anaplasma spp. and Ehrlichia spp. based on partialof sequences of the 16S rRNA gene (887 bp)…………………………………………………………… 89 Figure 4.13 Amplification of approximately 641 bp of the gltA gene fragments from samples using EHR-CS136F/EHR-CS778R primer pair………………………………………………………………… 91 Figure 4.14 Phylogenetic relationships among various Anaplasma spp. and Ehrlichia spp. based on partial sequences of the gltA gene (533 bp)…………………………………………………………………. 91 Figure 4.15 Amplification of approximately 1320 bp of the groEL gene fragments from samples using HS3-f/HSVR nested amplification primer pair………………………………………………………… 93 Figure 4.16 Phylogenetic relationships among various Ehrlichia spp. based on Universitypartial sequences of the groEL gene (943 bp)…………………….. 93 Figure 4.17 Amplification of approximately 343 bp of the msp4 gene fragments of A. phagocytophilum from samples using msp4f/msp4r nested amplification primer pair………………………………………….. 95 Figure 4.18 Phylogenetic relationships among various A. phagocytophilum based on partial sequences of the msp4 gene (252 bp)…………… 97 Figure 4.19 Amplification of approximately 551 bp of the 16S rRNA gene fragments of A. bovis from DNA samples using AB1f/AB1r nested amplification primer pair………………………………………….. 98 Figure 4.20 Phylogenetic relationships among various A. bovis based on partial sequences of the 16S rRNA gene (450 bp)………………...... 99 xiv

Figure 4.21 Amplification of approximately 389 bp of the 16S rRNA gene fragments of E. chaffeensis from DNA samples using HE1/HE3 nested amplification primer pair………………………………….. 100 Figure 4.22 Phylogenetic relationships among various E. chaffeensis based on partial sequences of the 16S rRNA gene (261 bp)………………... 101 Figure 4.23 Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the 16S rRNA gene (224 bp)…………….. 106 Figure 4.24 Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the 16S rRNA gene (1068 bp)…………… 111 Figure 4.25 Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the gltA gene (469 bp)…………………… 112 Figure 4.26 Amplification of approximately 641 bp of the 16S rRNA gene fragments of A. phagocytophilum from samples using SSAP2f /SSAP2r nested amplification primer pair………………………... 114 Figure 4.27 Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the 16S rRNA gene (434 bp)…………….. 116 Figure 4.28 Phylogenetic relationships among various A. phagocytophilum based on partial sequences of the msp4 gene (259 bp)…………… 117 Figure 4.29 Amplification of approximately 573 bp of the groEL gene fragments of A. phagocytophilum from DNA samples using EphplgroEL(569)F/EphgroEL(1142)RMalaya nested amplification primer pair………………………………………………………………… 118 Figure 4.30 Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the groELof gene (463 bp)…………………. 119 Figure 4.31 Phylogenetic relationships among various A. bovis based on partial sequences of the 16S rRNA gene (433 bp)………………...... 121

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LIST OF TABLES

Table 2.1 Animals and ticks associated with various species of Anaplasma spp. and Ehrlichia spp……………………………………………... 18 Table 2.2 Prevalence of anaplasma and ehrlichiae infections reported in Asian region, and the method used for detection...... 26 Table 2.3 Animal host for Anaplasmataceae, prevalence (%) and detection methods for infections reported in Malaysia………………………. 33 Table 2.4 Cell lines used for successful isolation of Anaplasma spp. and Ehrlichia spp……………………………………………………….. 36 Table 3.1 Sampling sites, date of sampling and number of samples collected from livestock farms in this study………………………………….. 42 Table 3.2 Sampling sites and samples collected from aboriginal villages in this study…………………………………………………………… 45 Table 3.3 PCR reaction mixture used for amplification of Anaplasmataceae DNA……………………………………………………………….. 50 Table 3.4 Details of the DNA fragments cloned as positive control…………. 55 Table 3.5 Reagents and the volumes required in the TOPO® cloning reaction…………………………………………………………….. 56 Table 4.1 Total number of tick samples collected from different sources in this study………………………………………………………………..Malaya 62 Table 4.2 Results of BLAST analyses of the 16S rRNA gene sequences obtained from 117 ticks collected from different sources in this study………………………………………………………………..of 69 Table 4.3 Sequence analyses of the cytB sequences from one sample of each animal type………………………………………………………… 78 Table 4.4 BLAST analyses of 53 short 16S rRNA gene sequences amplified from ticks from different sources in this study…………………….. 81 Table 4.5 BLAST analyses of 23 nearly full length 16S rRNA, gltA and groEL gene sequences amplified from ticks from different sources in this study………………………………………………………...... 87 Table 4.6 BLAST analyses of the partial 16S rRNA gene sequences of A. bovis, E. chaffeensis and msp4 gene sequences of A. phagocytophilum amplified from ticks from different sources in this study……………………………………………………………….. 96 TableUniversity 4.7 BLAST analyses of 84 short 16S rRNA gene sequences amplified from animal samples from different sources in this study………… 104 Table 4.8 BLAST analyses of nearly full length 16S rRNA and gltA gene sequences amplified from animal samples from different sources in this study………………………………………………………...... 108 Table 4.9 BLAST analyses of the partial 16S rRNA, msp4 and groEL gene sequences of A. phagocytophilum amplified from animal samples from different sources in this study………………………………... 115 Table 4.10 BLAST analyses of the partial 16S rRNA gene sequences of A. bovis amplified from animal samples from different sources in this study……………………………………………………………….. 120 xvi

Table 4.11 Summary of the identification of Anaplasma spp. and Ehrlichia spp. DNA in different animal and tick samples collected in this study……………………………………………………………….. 124

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LIST OF SYMBOLS AND ABBREVIATIONS

% : percent = : equal to < : less than ≤ : less than or equal to > : greater than - : minus/to ~ : approximately ºC : degree Celsius µg : microgram µl : microliter µm : micrometre µM : micromolar A : adenine A. bovis : Anaplasma bovis A. capra : Anaplasma capra A. centrale : Anaplasma centrale A. marginale : Anaplasma marginale A. ovis : Anaplasma ovis A. phagocytophilum : Anaplasma phagocytophilumMalaya A. platys : Anaplasma platys A. americanum : Amblyomma americanum A. astrion : Amblyommaof astrion A. cajennense : Amblyomma cajennense A. cohaerens : Amblyomma cohaerens A. gemma : Amblyomma gemma A. hebraeum : Amblyomma hebraeum A. helvolum : Amblyomma helvolum A. javanense : Amblyomma javanense A. lepidium : Amblyomma lepidium A. maculatum : Amblyomma maculatum A. marmoreum : Amblyomma marmoreum A. parvum : Amblyomma parvum A. pomposum : Amblyomma pomposum A.University testudinarium : Amblyomma testudinarium A. varanense : Amblyomma varanense A. variegatum : Amblyomma variegatum B. bovis : Bartonella bovis B. burgdorferi : Borrelia burgdorferi BLAST : Basic Local Alignment Search Tool bp : base pair C. ruminantium : Cowdria ruminantium Candidatus A. pangolinii : Candidatus Anaplasma pangolinii Candidatus E. shimanensis : Candidatus Ehrlichia shimanensis

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cytB : cytochrome B CME : canine monocytotropic ehrlichiosis D. albipictus : Dermacentor albipictus D. andersoni : Dermacentor andersoni D. atrosignatus : Dermacentor atrosignatus D. auratus : Dermacentor auratus D. everestianus : Dermacentor everestianus D. immitis : Dirofilaria immitis D. marginatus : Dermacentor marginatus D. nitens : Dermacentor nitens D. niveus : Dermacentor niveus D. nuttalli : Dermacentor nuttalli D. occidentalis : Dermacentor occidentalis D. reticulatus : Dermacentor reticulatus D. silvarum : Dermacentor silvarum D. variabilis : Dermacentor variabilis DNA : deoxyribonucleic acid dNTPs : Deoxynucleotide triphosphates E : east E. canis : Ehrlichia canis E. chaffeensis : Ehrlichia chaffeensisMalaya E. coli : Escherichia coli E. equi : Ehrlichia equi E. ewingii : Ehrlichiaof ewingii E. kageus : Eothenomys kageus E. mineirensis : Ehrlichia mineirensis E. muris : Ehrlichia muris E. phagocytophila : Ehrlichia phagocytophila E. ruminantium : Ehrlichia ruminantium EDTA : Ethylenediaminetetraacetic acid ELISA : enzyme-linked immunosorbent assay EML : Ehrlichia muris-like et al. : et alia (Latin), and others G+C : guanine+ cytosine gltA : citrate synthase gene groUniversityEL : heat shock protein H. aborensis : Haemaphysalis aborensis H. asiatica : Haemaphysalis asiatica H. bispinosa : Haemaphysalis bispinosa H. canis : Hepatozoon canis H. concinna : Haemaphysalis concinna H. douglasii : Haemaphysalis douglasii H. flava : Haemaphysalis flava H. formosensis : Haemaphysalis formosensis H. hystricis : Haemaphysalis hystricis

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H. langrangei : Haemaphysalis langrangei H. leporispalustris : Haemaphysalis leporispalustris H. longicornis : Haemaphysalis longicornis H. megaspinosa : Haemaphysalis megaspinosa H. obesa : Haemaphysalis obesa H. punctata : Haemaphysalis punctata H. qinghaiensis : Haemaphysalis qinghaiensis H. shimoga : Haemaphysalis shimoga H. sulcata : Haemaphysalis sulcata H. wellingtoni : Haemaphysalis wellingtoni H. yeni : Haemaphysalis yeni H. anatolicum : Hyalomma anatolicum H. asiaticum : Hyalomma asiaticum H. detritum : Hyalomma detritum H. marginatum : Hyalomma marginatum HEE : human ewingii ehrlichiosis HGA : human granulocytic anaplasmosis HIV : human immunodeficiency virus HME : human monocytic ehrlichiosis I. dentatus : Ixodes dentatus I. frontalis : Ixodes frontalisMalaya I. nipponensis : Ixodes nipponensis I. ovatus : Ixodes ovatus I. pacificus : Ixodesof pacificus I. persulcatus : Ixodes persulcatus I. ricinus : Ixodes ricinus I. scapularis : Ixodes scapularis I. spinipalpis : Ixodes spinipalpis I. trianguliceps : Ixodes trianguliceps I. turdus : Ixodes turdus I. ventalloi : Ixodes ventalloi i.e., : id est (Latin), that is IFA : immunofluorescent assay IFAT : immunofluorescent antibody test IgG : immunoglobulin G KUniversity : guanine or thymine kb : kilobase kg : kilogram L : left LAMP : loop-mediated isothermal amplification LB : Luria-Bertani LPS : lipopolysaccharide M : adenine or cytosine Mb : megabase MEGA : Molecular Evolutionary Genetics Analysis

mg : milligram min : minute ml : millilitre mm : milimetre mM : millimolar MSP : major surface protein msp4 : major surface protein 4 N : north N : Any nucleotide base n : sample size no. : number nov. : novel nPCR : nested PCR nt : nucleotide ORF : open reading frame PBS : phosphate buffered saline PCR : polymerase chain reaction qPCR : real-time PCR R : right R. annulatus : Rhipicephalus annulatus R. appendiculatus : Rhipicephalus Malayaappendiculatus R. bursa : Rhipicephalus bursa R. camicasi : Rhipicephalus camicasi R. decoloratus : Rhipicephalusof decoloratus R. evertsi : Rhipicephalus evertsi R. microplus : Rhipicephalus microplus R. rickettsii : Rickettsia rickettsii R. sanguineus : Rhipicephalus sanguineus R. simus : Rhipicephalus simus R. turanicus : Rhipicephalus turanicus rDNA : ribosomal nucleic acid RFLP : restriction fragment length polymorphism RLB : reverse line blot hybridisation rpm : revolutions per minute rRNA : ribosomal ribonucleic acid SEAUniversity : Southeast Asia S.O.C : Super optimal broth with catabolite repression s : second sp. : species (singular) spp. : species (plural) ST : sequence type subsp. : subspecies T : thymine Taq : Thermus aquaticus TBF : tick-borne fever

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U : unit U/µl : unit per microlitre USA : United States of America UV : ultraviolet V : voltage VHE : Venezuela human Ehrlichia W : adenine or thymine w/v : weight/volume Y : cytosine or thymine

Malaya of

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LIST OF APPENDICES

Appendix A Source and details of samples collected from livestock farms in this study…………………………………………………. 175 Appendix B Source and details of samples from PERHILITAN in this study………………………………………………………… 184 Appendix C Source and details of tick samples collected from aboriginal villages in this study………………………………………… 190 Appendix D Source and details of animal blood samples provided by VRI, Ipoh, Malaysia for this study………………………………... 192 Appendix E Source and details of samples collected from urban areas (Selangor and Klang Valley) in this study………………….. 197 Appendix F Primers and PCR conditions used in this study…………….. 199 Appendix G Media preparation…………………………………………... 201 Appendix H Published papers……………………………………………. 203 Appendix I Papers presented…………………………………………….. 207 Appendix J GenBank submission of sequences obtained in this study….. 209

Malaya of

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CHAPTER 1: INTRODUCTION

Anaplasmosis and ehrlichiosis are diseases that are transmitted by ticks in both animals and humans (Parola and Raoult, 2001). Anaplasma spp. and Ehrlichia spp. are small Gram-negative, obligate intracellular bacteria in the family of Anaplasmataceae that replicate within eukaryotic host cells (Dumler et al., 2001). According to the latest classification by Dumler et al. (2001), the genus Anaplasma includes Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma marginale, Anaplasma centrale,

Anaplasma ovis and Anaplasma platys whereas the genus Ehrlichia includes Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Ehrlichia muris and Ehrlichia ruminantium. To date, A. phagocytophilum, E. chaffeensis and E. ewingii are the most significant species in the family Anaplasmataceae that cause human infections while all the species in the family Anaplasmataceae can causeMalaya infection in animals. Economic loss due to Anaplasma and Ehrlichia infections in domestic animals has been reported worldwide (Atif, 2016). of Transmission of anaplasmosis and ehrlichiosis to humans are mostly through tick bites or when in contact with the fluids or secretions of infected ticks. The clinical presentation of anaplasmosis and ehrlichiosis are often nonspecific, with patients presenting with febrile illness, headache, myalgia, malaise and complications affecting the respiratory, renal and central nervous systems, especially amongst immunosuppressed patients (Bakken and Dumler, 2015; Dumler, 2005; Dumler et al., 2007; Ismail et al., 2010University). Due to the intracellular nature of the bacteria in the genera Anaplasma and

Ehrlichia, conventional method of culturing on agar media cannot be applied. Hence, detection of Anaplasma spp. and Ehrlichia spp. is usually performed by examination of peripheral blood smears, in vitro cultivation using tissue cultures, serological or molecular diagnostic methods. Molecular techniques including conventional polymerase

chain reaction (PCR), real-time PCR (qPCR), together with direct sequencing are considered as the most rapid and sensitive methods for detection of Anaplasma spp. and

Ehrlichia spp. in human and animal samples (Guillemi et al., 2015). Using direct sequencing technique, amplified PCR amplicons can be sequenced and analysed for comparison with known sequences deposited in the GenBank database.

Most reports on human anaplasmosis and ehrlichiosis are documented in the

United States of America (USA) and European countries (Rar and Golovljova, 2011).

The recent reports of anaplasma and ehrlichiae in humans, animals and ticks in China,

Japan and Korea (Kim et al., 2014; Lee et al., 2009; Ohashi et al., 2005; Ohashi et al.,

2013; Zhang et al., 2008b) suggest the existence of these organisms in the Asia region.

The seroprevalence of canine ehrlichiosis and bovine anaplasmosis have been reported previously (Rahman et al., 2012). A. platys and E. canisMalaya have also been detected in dogs using PCR assays (Mokhtar et al., 2013; Nazari et al., 2013). People who live at the fringe of the offorest or rural areas in Malaysia, including the indigenous community and livestock farm workers, are regarded as populations who are at high risk of acquiring tick-borne diseases (Ghane Kisomi et al., 2016; Kho et al., 2017).

The indigenous community (also referred as Orang Asli or ‘original people’) is a minority group representing only 0.6% of the total population in Malaysia. They stay in huts or settlements surrounded by forest, and engage in activities involving agriculture, hunting and collection of forest products. Their nomadic lifestyle and close contact with peri- domesticUniversity animals have increased the risk of potential tick-borne diseases including anaplasmosis and ehrlichiosis (Masron et al., 2013).

Although anaplasmosis and ehrlichiosis are emerging zoonotic diseases, little information is known in Malaysia. Few studies have investigated the extent of exposure of the local populations to these tick-borne diseases in Southeast Asia (SEA). A recent study reported high seroprevalence of ehrlichiosis in Malaysian indigenous people

(34.3%) and livestock farm workers (29.9%) (Koh et al., 2018). In addition, little information is available on the potential vectors and maintenance hosts of Anaplasma spp. and Ehrlichia spp. in SEA countries, especially in Malaysia. The economic losses caused by anaplasmosis and ehrlichiosis have not been thoroughly investigated in

Malaysia. There is a lack of extensive studies in Malaysia on these animal diseases

(Rahman et al., 2012).

Hence, this study is proposed with the objectives:

i. to identify tick species using morphological and molecular identification

methods.

ii. to identify Anaplasma spp. and Ehrlichia spp. from animals and ticks using

molecular methods. iii. to determine phylogenetic status of AnaplasmaMalaya spp. and Ehrlichia spp. of local strains based on sequence analysis of various genes (16S rDNA, gltA, msp4 and groEL). of

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CHAPTER 2: LITERATURE REVIEW

2.1 Anaplasma spp.

The genus Anaplasma is consisted of a group of tick-associated bacteria belong to the family Anaplasmataceae, order Rickettsiales and class Alphaproteobacteria. The

Anaplasma spp. are small Gram-negative, often pleomorphic, coccoid to ellipsoidal, obligate intracellular bacteria that replicate in membrane-bound vacuoles within the cytoplasm of an eukaryotic host (Dumler et al., 2001; Rikihisa, 2011). According to the reorganisation of genera in the family Anaplasmataceae by Dumler et al. (2001), the species included in the genus Anaplasma are A. phagocytophilum, A. bovis, A. marginale,

A. centrale, A. ovis and A. platys.

2.1.1 Anaplasma marginale A. marginale is the type species for the genusMalaya Anaplasma and the causative agent for bovine anaplasmosis (Dumler et al., 2001; Kocan et al., 2003). The bacterium was first described in 1910 by Sir Arnold Theilerof who noticed the presence of small inclusions near the edge of the erythrocytes of sick cattle (Theiler, 1910). Giemsa stain is usually used for the visualisation of A. marginale where the bacteria appear as rounded and deeply stained inclusion bodies that located at the margin of the infected erythrocytes.

The bacterial size is approximately 0.3 to 1.0 µm in diameter (OIE, 2012).

The genome size of A. marginale is approximately 1.2 Mb (based on St. Maries strain which was isolated from a cow with severe acute anaplasmosis) with unusual high G+CUniversity (guanine + cytosine) content of 49.8% (Brayton et al., 2005). Most of the obligate intracellular bacteria have low G+C content, for example, the G+C content of Rickettsia prowazekii (strain Madrid E), Wolbachia pipientis (strain wMel) and E. ruminantium

(Welgevonden-type strain) are 29.1%, 35.2% and 27.5%, respectively (Andersson et al.,

1998; Collins et al., 2005; Wu et al., 2004). The absence of genes for lipopolysaccharide

(LPS) biosynthesis in A. marginale has resulted with a lack of a cell wall in this bacterium

(Brayton et al., 2005). Unlike other bacteria in the family Anaplasmataceae which are fragile due to the lack of cell wall, A. marginale is able to strengthen its cell wall through the major surface proteins (MSPs), homeric and heteromeric complexes which are present on the cell surface (Brayton et al., 2005; Vidotto et al., 1994).

A. marginale is known to infect erythrocytes of cattle and other ruminants including buffaloes, elk, deer and bison (Kocan et al., 2010). Human infection due to A. marginale has not been reported (OIE, 2012). A. marginale can be transmitted through ticks or biting flies or mechanically through blood-contaminated instruments used for veterinary practice such as needles and ear-tag applicators (Kocan et al., 2015).

Intrastadial transmission (by male ticks) and transplacental transmission (which results in healthy but constantly infected calves) are also important routes that contribute to A. marginale transmission (Kocan et al., 2003). Malaya 2.1.2 Anaplasma phagocytophilum The bacterium was initially knownof as the causative agent of tick-borne fever (TBF) in sheep and cattle with the given name Rickettsia phagocytophila. It was then renamed as Cytoecetes phagocytophila due to its morphological similarity to Cytoecetes microti, a parasite detected in the polymorphonuclear cells of the vole, Microtus pennsylvanicus (Tyzzer, 1938). The bacterium was then grouped in the tribe Ehrlichieae under the order Rickettsiales, as Ehrlichia phagocytophila, together with Ehrlichia equi, the causative agent of equine granulocytic anaplasmosis (EGA) (Woldehiwet, 2010). AfterUniversity the reorganisation of genera based on 16S rRNA and groEL genes by Dumler et al. (2001), E. phagocytophila was grouped under the same clade with E. equi and the human granulocytic ehrlichiosis (HGE) agent (Ehrlichia ‘HGE agent’) is now known as A. phagocytophilum.

The size of A. phagocytophilum is approximately 0.4 to 1.3 µm, but it can be as large as 2.0 µm (Rikihisa, 2011). Due to the lack of contrast against the host cytoplasm,

Gram staining is not suitable for the visualisation of this intracellular bacterium. Instead,

Romanowsky staining is used for staining of the bacterium. Upon staining, purplish mulberry-like bacterial clumps called morulae can be observed in the bacterial smears

(Rikihisa, 2011). Morulae are normally 1.5 to 2.5 µm in diameter but can be up to 6.0 µm

(Rikihisa, 2011).

The genome size of A. phagocytophilum is about 1.47 Mb (based on strain HZ, isolated from a patient in New York State, USA) with 1,369 open reading frames (ORFs) and G+C content of 41.6% (Dunning Hotopp et al., 2006). No plasmids, intact prophages or transposable elements are found in the bacterial genome (Rikihisa, 2011). In addition, the genome of A. phagocytophilum also lacks of genes needed for biosynthesis of LPS and peptidoglycan. As a result, A. phagocytophilum is tightly packed inside inclusions (Dunning Hotopp et al., 2006; Lin and Rikihisa, 2003Malaya). A. phagocytophilum infects granulocytes (neutrophils) and also endothelial cells of humans (Herron et al., 2005) and animalsof such as horses, deer and wild ruminants (Atif, 2016). No transovarial transmission (from adult ticks to eggs) has been reported

(Woldehiwet, 2010) except for Dermacentor albipictus (moose or winter tick) (Baldridge et al., 2009). Hence, a reservoir vertebrate host is important for the maintenance of A. phagocytophilum in nature. The life cycle of A. phagocytophilum starts when adult ticks acquire A. phagocytophilum from infected mammals during blood meals. The bacterium survives in the larva, nymph and adult stage of ticks, and is then transmitted to other mammalsUniversity during the next blood meal (Ogden et al., 1998; Telford et al., 1996; Zhi et al., 2002).

2.1.3 Anaplasma centrale

A. centrale, first described by Sir Arnold Theiler in 1911 (Theiler, 1911), is closely related to A. marginale. A. centrale, also known as A. marginale subsp. centrale

(Dumler et al., 2001; Kocan et al., 2003), causes mild anaplasmosis in ruminants (mostly cattle) (Battilani et al., 2017).

A. centrale infects erythrocytes of animal hosts and the inclusion bodies are located at a more central position within the infected red blood cells when visualised under Giemsa staining. The location of inclusion bodies is a feature that distinguish A. centrale from A. marginale (OIE, 2012). The genome size of A. centrale is approximately

1.21 Mb (based on Israel strain) with a high G+C content of 50.0% (Herndon et al., 2010), similar to that of A. marginale. The transmission of A. centrale normally occurs through ticks as vector (Theiler, 1911). Transovarial transmission has not been reported (Palomar et al., 2015). A. centrale has been used as a live vaccine against A. marginale in Israel,

Australia, Africa and South America (de la Fuente et al., 2005a). 2.1.4 Anaplasma ovis Malaya A. ovis is known to infect erythrocytes of ruminants (mostly goats and sheep) and causes ovine anaplasmosis (Atif, 2016). A.of ovis was first described in sheep in 1912 (Bevan, 1912). A. ovis causes mild infection which is serologically identical to A. marginale (Splitter et al., 1956). Unlike A. marginale which is only found at the edge of erythrocytes and A. centrale which is found at the central region of the infected erythrocytes, A. ovis can be found on both marginal (60.0-65.0%) and submarginal/ central (35.0-40.0%) parts of the erythrocytes (Rymaszewska and Grenda, 2008;

Shompole et al., 1989). UniversitySimilar to other members in Anaplasmataceae , A. ovis is not transovarially transmitted (Palomar et al., 2015) and tick is the main vector (Battilani et al., 2017).

Sheep keds and biting flies may play a role in the transmission of A. ovis (Battilani et al.,

2017; Hornok et al., 2011). A. ovis has not been known to infect human. However, the recent detection of a variant of A. ovis has been detected in a patient in Cyprus suggests the zoonotic potential of this bacterium (Chochlakis et al., 2010).

2.1.5 Anaplasma bovis

A. bovis was first described in cattle in 1936 as Rickettsia bovis (Donatien and

Lestoquard, 1936). It was then renamed as Ehrlichia bovis and later, A. bovis (Dumler et al., 2001). Based on phylogenetic analysis of 16S rRNA, gltA and groEL genes, A. bovis is closely related to A. phagocytophilum and A. platys (Ybañez et al., 2014a).

A. bovis infects the monocytes of multiple animal hosts and causes monocytic anaplasmosis in cattle, deer, goats and cottontail rabbits (Atif, 2016). A. bovis appears in different shapes and sizes, i.e., small (1.0 µm), medium (1.0-3.0 µm) and large (3.0-6.0

µm) forms. The large form is a mature and the least common form (Sreekumar et al.,

1996). A. bovis has not been successfully cultivated in vitro (Rar and Golovljova, 2011).

Vertebrate hosts and tick vectors are crucial in the transmission of A. bovis in the absence of transovarial transmission (Battilani et al., 2017; PalomarMalaya et al., 2015). 2.1.6 Anaplasma platys A. platys, initially known as Ehrlichiaof platys , was first detected in a dog in Florida, USA, with the diagnosis of infectious canine cyclic thrombocytopenia (Harvey et al.,

1978). A. platys has not been cultivated in vitro (Rar and Golovljova, 2011).

A. platys is known to infect platelets of dogs. It has also been detected in animal hosts other than dogs, for example, cats, red foxes and Bactrian camels (Battilani et al.,

2017; Cardoso et al., 2015; Y. Li et al., 2015). Although human infections caused by A. platys have been reported (Arraga-Alvarado et al., 2014; Maggi et al., 2013), the role of A. Universityplatys as the causative agent for human infection still requires further studies. Tick is an important vector for the transmission of A. platys and transmission through infected blood inoculation has been reported (Battilani et al., 2017).

2.2 Ehrlichia spp.

Ehrlichia spp. are tick-associated bacteria belong to the family Anaplasmataceae, order Rickettsiales and class Alphaproteobacteria. The small Gram-negative bacterium inhabits in an intracellular vacuole (morula) that is bounded by a membrane derived from the cytoplasm of an eukaryotic host cell. The bacterium can appear coccoid to pleomorphic in different sizes, i.e., small (0.4 µm), medium (0.7 µm) or large (1.0 µm) forms. Occasionally, bacteria with very large size (≤ 2.0 µm) can be observed (Allsopp and McBride, 2009; Dumler et al., 2001). According to the reorganisation of genera in the family Anaplasmataceae by Dumler et al. (2001), the genus Ehrlichia include E. canis, E. chaffeensis, E. ewingii, E. ruminantium and E. muris.

2.2.1 Ehrlichia canis E. canis is the type species for the genus EhrlichiaMalaya and the causative agent of canine ehrlichiosis (Rikihisa, 1991). E. canis was originally described in dogs in Algeria (Donatien and Lestoquard, 1935). The of disease caused by E. canis was reported throughout the world and had gained extensive attention when a large number of

American military dogs (mostly German shepherds) died from E. canis infection during the Vietnam War (Rikihisa, 1991). Two morphological cell types of E. canis have been observed under electron microscope: reticulate cells and dense-cored cells (Popov et al.,

1998). Reticulate cells are small and oval form of bacteria, with the average diameters around 0.3 to 0.6 µm while dense-cored cells are round with diameters ranging from 0.3 to 0.4University µm (Popov et al., 1998). Morulae appear in different sizes and are filled with large quantities of bacteria measuring 2.0 to 4.0 µm in diameters (Popov et al., 1998).

The genome of E. canis is made up of a single circular chromosome with the size of approximately 1.32 Mb (based on strain Jake) and a G+C content of 29.0%

(Mavromatis et al., 2006). E. canis is an aerobic bacterium incapable of using glucose or fructose as carbon source, therefore the transport systems and essential enzymes

associated with the use of these substrates have not been identified (Mavromatis et al.,

2006). Similar to A. phagocytophilum and E. chaffeensis, enzymes related to the biosynthesis of lipid A and murein sacculus and the metabolism of peptidoglycans and amino sugars have not been reported in E. canis (Mavromatis et al., 2006).

E. canis infects monocytes (macrophages) causing monocytic ehrlichiosis (Rar and Golovljova, 2011). Although E. canis causes infection in dogs and wild canids, it has also been detected in cats and humans (Stich et al., 2008). Tick is an important vector for the maintenance of E. canis through transstadial transmission (Stich et al., 2008).

2.2.2 Ehrlichia chaffeensis

The first case of human infection caused by E. chaffeensis was reported in April

1986 in the USA. The bacterium, initially referred as E. canis, was present as an intracytoplasmic inclusion in the monocytes of a Malaya patient (Maeda et al., 1987). The organism which was first isolated in cell culture in 1991, has been characterised using various molecular techniques (Anderson et ofal., 1991 ; Dawson et al., 1991). E. chaffeensis is a small and nonmotile bacterium that resides and grows in the cytoplasmic vacuole of host cells forming morulae (Paddock and Childs, 2003). Two distinct cell types have been identified under electron microscope: larger reticulate cells (with uniformly dispersed nucleoid) measuring 0.4 to 0.6 µm by 0.7 to 1.9 µm and small dense-cored cells (with dense nucleoid) measuring 0.4 to 0.6 µm in diameters (Paddock and Childs, 2003). The dense-cored cell is the only cell which attaches and enters into the host cell while the reticulateUniversity cell undergoes replication by binary fission. Hence, the dense-cored cell is the mature infectious form and the reticulate cell is the multiplication form of E. chaffeensis

(Zhang et al., 2007).

E. chaffeensis has a single circular chromosome genome approximately 1.18 Mb, based on strain Arkansas which was isolated from a patient in Arkansas (Dunning Hotopp et al., 2006). The G+C content of E. chaffeensis is 30.1%. The 1,115 ORFs is about one

quarter of those found in Escherichia coli (Dunning Hotopp et al., 2006). Similar with A. phagocytophilum, E. chaffeensis also lacks of genes required for the biosynthesis of LPS and peptidoglycans (Dunning Hotopp et al., 2006; Lin and Rikihisa, 2003). The bacterial cell wall may be stabilised with disulphide bonds and non-covalent bonds cross-linking the outer membrane proteins (Vidotto et al., 1994).

E. chaffeensis infects monocytes (macrophgaes) causing monocytic ehrlichiosis in human and a variety of animals including white-tailed deer (primary reservoir), deer, dogs, raccoons and goats (Rar and Golovljova, 2011; Yabsley, 2010). Ticks and vertebrate hosts are important in maintaining E. chaffeensis in nature. Successful transovarial transmission has not been reported and the bacteria is transmitted transstadially during a blood meal (Parola et al., 2005). 2.2.3 Ehrlichia ewingii Malaya E. ewingii was first recognised in 1971 as a new strain of E. canis in a dog in Arkansas, USA (Ewing et al., 1971). The ofbacterium is known to be the causative agent of canine granulocytic ehrlichiosis (Anderson et al., 1992a). Human infections with E. ewingii were first described in four patients from Missouri, USA, using PCR assays

(Buller et al., 1999).

Unlike other Ehrlichia species, E. ewingii infects granulocytes (neutrophils) (Rar and Golovljova, 2011), similar to the genetically distinct A. phagocytophilum (Dumler,

2005). E. ewingii has not been cultivated in cell culture (Ismail et al., 2010), but has recentlyUniversity been isolated and maintained for a short period of time (16 weeks) in human promyeloblast cell line (HL60) (Killmaster and Levin, 2016). E. ewingii is not studied as extensive as other Ehrlichia species.

2.2.4 Ehrlichia ruminantium

Heartwater was recognised as a tick-borne disease during 1900 and until 1925. E. ruminantium is the causative agent which was initially identified as a rickettsia (knowns

as Rickettsia ruminantium) but later was renamed as Cowdria ruminantium (Cowdry,

1925a, 1925b). Based on the analysis of 16S rRNA and groEL genes (Dumler et al.,

2001), C. ruminantium was found in one cluster with other Ehrlichia species, hence it was renamed as E. ruminantium. The bacteria appear as purplish-blue coccoids with various sizes (small- 0.4 µm, medium- 0.76 µm, large- 1.04 µm or very large- >1.04 µm) after Giemsa staining (Allsopp, 2010). Pleomorphic forms (horseshoe, ring and bacillary shaped) have also been reported (Allsopp, 2010).

The genome size of E. ruminantium is approximately 1.52 Mb (based on the South

African Welgevonden isolate- type strain). It has a low G+C content (27.5%) with 920

ORFs (Collins et al., 2005). E. ruminantium genome has a large number of repetitive sequences which make up 8.3% of the chromosomes (Collins et al., 2005). Just like the other members of the family Anaplasmataceae, E.Malaya ruminantium is lack of the genes needed for the biosynthesis of cell wall components, lipid A and murein sacculus (Collins et al., 2005). of E. ruminantium is known to infect endothelial cells, neutrophils and macrophages of domestic and wild ruminants, causing heartwater cowdriosis (Rar and Golovljova,

2011). E. ruminantium is transmitted transstadially and maintained in Amblyomma ticks

(Allsopp, 2010). The report of E. ruminantium being transmitted transovarially

(Bezuidenhout and Jacobsz, 1986) suggests that E. ruminantium may sometimes be carried transovarially. 2.2.5University Ehrlichia muris E. muris is a murine pathogen first isolated from the spleen of a wild mouse,

Eothenomys kageus in Japan in 1983 (Kawahara et al., 1993). The bacterium is pleomorphic with sizes ranging from 0.4 to 1.5 µm long and 0.2 to 1.5 µm in diameters

(Wen et al., 1995). The bacterium is surrounded by cytoplasmic membrane with a rippled outer membrane but without distinct peptidoglycan layer (Wen et al., 1995).

E. muris has a single circular chromosome with the size of approximately 1.2 Mb

(based on the strain AS145T, isolated from the spleen of a wild mouse, E. kageus in Japan) and G+C content of 30.0% (Thirumalapura et al., 2014). Phylogenetic analysis of 16S rRNA gene sequences shown that E. muris is closely related with E. chaffeensis, demonstrating 97.7% sequence similarity (Wen et al., 1995). E. muris infects monocytes

(macrophages) of rodents and is transmitted by ticks (Rar and Golovljova, 2011).

2.3 New Anaplasma and Ehrlichia species

Due to the technological advancement in the diagnosis and intensified surveillance studies, there are increasing reports of new Anaplasma and Ehrlichia species worldwide. For instance, Candidatus Anaplasma camelii and Candidatus Ehrlichia regneryi [first detected in camel, Saudi Arabia, Bastos et al. (2015)]; Candidatus Ehrlichia khabarensis [previously known as EhrlichiaMalaya sp. Khabarovsk, detected in voles, Russia, Rar et al. (2015)]; Anaplasma capra [detected and isolated in goats and humans, China; H. Li et al. (2015)]; Ehrlichia mineirensisof [first detected and isolated from Rhipicephalus microplus (collected from cattle), Brazil, Cabezas-Cruz et al. (2012)];

Ehrlichia minasensis sp. nov. [first detected and isolated from R. microplus (collected from cattle), Brazil, Cabezas-Cruz et al. (2016)]; Candidatus Ehrlichia shimanensis

[previously known as Ehrlichia sp. TS37, first detected in Haemaphysalis longicornis from vegetation, Japan, Kawahara et al. (2006)] and Anaplasma odocoilei sp. nov.

[detected in white-tailed deer, USA, Tate et al. (2013)]. Apart from A. capra, other new speciesUniversity have not been associated with human infections yet.

2.4 Anaplasma, ehrlichiae and their hosts/vectors

2.4.1 Human infections

2.4.1.1 Anaplasmosis

A. phagocytophilum is the only bacterium in the genus Anaplasma which causes human infections, also known as human granulocytic anaplasmosis (HGA) (Dumler,

2005). Most of the reported HGA cases are associated with the exposure to tick-bites.

Hence, a well-established tick bite or tick exposure record is important to establish diagnosis for HGA (Bakken and Dumler, 2015). HGA can also be obtained through alternative routes, for example, perinatal transmission (Dhand et al., 2007), blood transfusion (Annen et al., 2012) and direct exposure to infected animal blood, through skin cuts or inhalation of infected aerosolised blood or infected blood splashed directly on mucous membranes (Bakken and Dumler, 2015).Malaya Several reported cases of human infections caused by A. ovis and A. platys have been documented, however, the potential of roles of these organisms in causing human infections require further investigation. Human infection by A. ovis was first reported in a woman with a history of fever after a tick bite in Cyprus. The organism was detected by PCR assays targeting groEL and msp4 genes (Chochlakis et al., 2010). A case of human infection caused by A. ovis has also been reported based on detection of A. ovis

16S rRNA gene from an Iranian shepherd in the absence of any clinical symptoms

(Hosseini-Vasoukolaei et al., 2014). Infection caused by A. platys was first reported in a veterinarianUniversity from Grenada using PCR assays targeting 16S rRNA and groEL genes (Maggi et al., 2013). A. platys DNA was detected from two family members who were the caretaker of a dog using PCR assays targeting 16S rRNA and p44 genes

(Breitschwerdt et al., 2014), and two women from Venezuela with the detection of intra- platelet inclusion bodies (Arraga-Alvarado et al., 2014). In addition, a newly identified

Anaplasma species known as A. capra has been reported to cause human infection in

China (H. Li et al., 2015).

2.4.1.2 Ehrlichiosis

Human ehrlichiosis is mostly caused by E. chaffeensis (Human monocytic ehrlichiosis, HME) and E. ewingii (Human ewingii ehrlichiosis, HEE) (Dumler et al.,

2007). E. chaffeensis infections are often associated with exposure to ticks and the geographical expansion of animal reservoirs and vectors. Severe HME cases have been reported in organ transplants or immunocompromised patients (Doudier et al., 2010;

Safdar et al., 2002). HEE is understudied due to the lack of a specific diagnostic assay and reporting system for E. ewingii (Ismail et al., 2010). Most of the reported HEE cases occurred in HIV-infected patients or patients with immunosuppression after organ transplantation (Ismail et al., 2010). Malaya Additionally, there are reported cases of human infections caused by E. canis, E. ruminantium, E. muris and E. muris-like (EML)of organism, but their roles as the causative agents remain unknown. E. canis was first isolated from a man in Venezuela in 1996

(Perez et al., 1996). The organism was genetically and antigenically most closely related with E. canis strain Oklahoma and was thus designated as Venezuelan human Ehrlichia

(VHE). Later on, E. canis VHE strain was detected in patients in Venezuela with clinical signs of HME (Perez et al., 2006). Recently, E. canis was detected by molecular and serological techniques in the blood donors in Costa Rica (Bouza-Mora et al., 2017). Three patientsUniversity in South Africa were tested positive for E. ruminantium using PCR assay targeting the 16S rRNA V1 loop region and pCS20 genes (Allsopp et al., 2005).

E. muris was first detected in the spleen of a wild mouse in Japan (Kawahara et al., 1999). An unknown Ehrlichia species was detected in four patients and Ixodes scapularis ticks, from Wisconsin and Minnesota, USA in 2009 (Pritt et al., 2011). Genetic analysis showed that the unknown Ehrlichia species was closely related to E. muris and

it was thus referred as EML agent (Pritt et al., 2011). A recent study reported 69 patients from five different states in USA who were seropositive to the EML pathogen by using

PCR assays targeting groEL gene, thus suggesting human exposure to EML pathogen in

USA (Johnson et al., 2015).

In general, several human populations including farm workers, forestry workers, pet owners (especially cats and dogs), people with exposure to ticks or tick-bites, people infected with Borrelia burgdorferi (causative agent for Lyme disease), campers and hikers have been identified to have higher risk of contracting anaplasmosis and ehrlichiosis (Dinc et al., 2017; Dumler, 2005).

2.4.2 Animal infections

Different species of Anaplasma and Ehrlichia have been shown to infect certain animal hosts (Zobba et al., 2014). A. phagocytophilumMalaya has been reported in a wide range of hosts including horses, cats, canids (dogs and foxes), ruminants (cattle, sheep and goats), birds, rodents, wild boars and reptilesof while the known reservoir is white-tailed deer, Odocoileus virginianus (Stuen et al., 2013). A. marginale and A. centrale are best known to cause infection in cattle, but other ruminants such as water buffaloes, American bison, elk and deer can also be infected with A. marginale (Kocan et al., 2010). A. ovis is a tick-borne bacterium that infects goats, sheep, deer and Mongolian gazelles (Rar and

Golovljova, 2011). Similar to A. phagocytophilum, A. bovis also infects diverse animal hosts including ruminants (cattle, sheep, goats, buffaloes and deer), cottontail rabbits, dogs,University raccoons and cats (Atif, 2016). Dogs are a common host for A. platys, however, A. platys infections have also been reported on deer, cats, camels and red foxes (Atif, 2016).

Table 2.1 shows various animals that have been associated with infections caused by

Anaplasma spp.

Dogs have been reported to acquire E. chaffeensis, E. ewingii and E. canis infections, and are considered as one of the most susceptible animals for Ehrlichia

infection (Chomel, 2011). E. chaffeensis infects most animals including canids (dogs, foxes and coyotes), deer, goats, rodents, raccoons and birds (Yabsley, 2010). E. ewingii infections in dogs, deer and rodents have been reported (Chae et al., 2003; Yu et al.,

2007). E. canis is known to infect dogs, as well as other animals such as cats, wild cats, deer, rodents and wild canids (Li et al., 2016; Stich et al., 2008; Tateno et al., 2013). Wild ruminants and other domestic ruminants such as cattle, sheep, goats and water buffaloes are susceptible to infection caused by E. ruminantium (Allsopp, 2010). E. muris is known to infect rodents including Japanese field mouse, voles, mice and Siberian chipmunks; deer and common shrews (Rar and Golovljova, 2011). Further information on the animals infected by Ehrlichia spp. are summarised in Table 2.1.

2.4.3 Anaplasma and ehrlichiae in ticks Ticks are obligate hematophagous arthropodsMalaya grouped under the family Ixodidae (hard tick) or Argasidae (soft tick), order Parasitiformes, subclass Acari and class Arachnida (Parola and Raoult, 2001). Ixodidof tick has three basic life stages (larva, nymph and adult), with each stage feeding on a different or the same host (Parola and Raoult,

2001). The transmission of blood-borne pathogens may occur from the ticks to either human or animal hosts during blood meals (Fritz, 2009). The major genera of ixodid ticks are Amblyomma spp., Ixodes spp., Dermacentor spp., Haemaphysalis spp., Hyalomma spp. and Rhipicephalus spp. (Parola and Raoult, 2001).

Ticks can be identified morphologically under a stereomicroscope, by referring to specificUniversity taxonomic keys, for example, taxonomic keys as reported by Burridge (2001), Kohls (1957), Walker et al. (2003), Geevarghese and Mishra (2011), Tanskul and Inlao

(1989) and Wassef and Hoogstraal (1984). However morphological identification of ticks to species level may be difficult due to the lack of expertise in tick identification or when a tick has not fully developed (i.e., larva or nymph), or is engorged with blood or physically damaged. Hence, molecular technique (PCR incorporated with sequence

Table 2.1: Animals and ticks associated with various species of Anaplasma spp. and Ehrlichia spp. (Allsopp, 2010; Atif, 2016; Chae et al., 2003; Chastagner et al., 2013; Kocan et al., 2010; Li et al., 2014; Li et al., 2016; Noaman, 2012; Palomar et al., 2015; Rar and Golovljova, 2011; Silaghi et al., 2017; Stuen et al., 2013; Tateno et al., 2013; Yabsley, 2010; Yang et al., 2015b; Yu et al., 2007; Yu et al., 2015).

Species Animal hosts Ticks A. marginale  Deer (Brazilian brown brocket deer, M. gouazoubira; marsh deer, B.  Amblyomma spp. (A. variegatum, A. cajennense, A. maculatum) dichotomus; white-tailed deer, O. virginianus; mule deer, O. hemionus;  Dermacentor spp. (D. andersoni, D. variabilis, D. albipictus, D. reticulatus, black-tailed deer, O. columbianus) D. nitens)  Ruminants (cattle; water buffalo, B. bubalis; American bison, B. bison,  Ixodes spp. (I. ricinus) elk)  Rhipicephalus spp. (R. microplus, R. annulatus, R. simus, R. evertsi evertsi, R. decoloratus, R. bursa) A. centrale  Cattle, goats  RhipicephalusMalaya spp. (R. simus)  Haemaphysalis spp. (H. punctata) A. ovis  Deer (mule deer, O. hemionus; European roe deer, C. capreolus; red  Dermacentor spp. (D. marginatus, D. andersoni, D. occidentalis, D. niveus, deer, C. elaphus; sika deer, C. nippon) D. everestianus)  Mongolian gazelle (P. gutturosa) of Haemaphysalis spp. (H. concinna, H. punctata, H. longicornis)  Ruminants (cattle, goat, sheep; bighorn sheep, O. canadensis)  Ixodes spp. (I. ricinus)  Rhipicephalus spp. (R. bursa, R. sanguineus) A. platys  Canids (dog; red fox, V. vulpes)  Dermacentor spp. (D. auratus)  Camel (C. bactrianus)  Haemaphysalis spp. (H. longicornis)  Cats  Ixodes spp. (I. persulcatus)  Deer (red deer, C. elaphus; sika deer, C. nippon)  Rhipicephalus spp. (R. sanguineus, R. turanicus, R. evertsi, R. camicasi)  Rodents (striped field mouse, A. agrarius) A. bovis  Cottontail rabbit (S. floridanus)  Amblyomma spp. (A. variegatum)  Deer (Korean water deer, H. i. argyropus; roe deer, C. capreolus; red  Dermacentor spp. (D. occidentalis, D. andersoni, D. niveus) deer, C. elaphus; sika deer, C. nippon; Brazilian brown brocket deer,  Haemaphysalis spp. (H. longicornis, H. punctata, H. concinna, H. M. gouazoubira; marsh deer, B. dichotomus) megaspinosa, H. langrangei, H. leporispalustris, H. shimoga)  Dogs  Rhipicephalus spp. (R. sanguineus, R. turanicus, R. evertsi)  Eastern rock sengi (E. myurus)  Mongolian gazelle (P. gutturosa)  Raccoon (P. lotor)  Ruminants (cattle, goat,University sheep, buffalo)  Tsushima leopard cat (P. b. euptilurus)

Table 2.1, continued A. phagocytophilum  Birds (Eurasian collared dove, S. decaocto; Eurasian eagle-owl, B.  Amblyomma spp. (A. americanum) bubo; house sparrow, P. domesticus; Spanish sparrow, P.  Dermacentor spp. (D. variabilis, D. occidentalis, D. silvarum, D. hispaniolensis; blackbird, T. merula; chaffinch, F. coelebs; rock reticulatus, D. marginatus, D. albipictus, D. niveus) bunting, E. cia; woodchat shrike, L. senator; magpie, P. pica)  Haemaphysalis spp. (H. concinna, H. longicornis, H. megaspinosa, H.  Canids (dog; red fox, V. vulpes; timber wolf, C. l. occidentalis) douglasii, H. formosensis, H. hystricis, H. qinghaiensis)  Cats  Hyalomma spp. (H. marginatum, H. detritum)  Common shrew (S. araneus)  Ixodes spp. (I. ricinus, I. persulcatus, I. pacificus, I scapularis, I. ovatus, I.  Deer (white-tailed deer, O. virginianus; roe deer, C. capreolus; red trianguliceps, I. ventalloi, I. spinipalpis, I. nipponensis, I. dentatus, I. deer, C. elaphus; sika deer, C. nippon, fallow deer, D. dama; mule frontalis) deer, O. hemionus)  Rhipicephalus spp. (R. bursa, R. sanguineus, R. turanicus)

 Donkeys

 European brown bear (U. arctos) Malaya  Horses  Mongolian gazelle (P. guturosa)  Ruminants (cattle, sheep, goat, elk, yak, bison) of  Rodents (rat, Rattus spp.; field mice, Apodemus spp.; white-footed mice, P. leucopus; dusky-footed woodrat, N. fuscipes; redwood chipmunk, T. ochrogenys; Eastern chipmunk, T. striatus; Siberian chipmunk, T. sibiricus; voles, M. glareolus, M. rutilus, M. rufocanus, M. arvalis, M. agrestis, M. oeconomus; wood mice, A. sylvaticus; Eastern gray squirrel, S. carolinensis; Northern flying squirrel, G. sabrinus; great long-tailed hamster, T. triton; gray hamster, C. migratorius)  Reptiles (Western fence lizard, S. occidentalis; Northern alligator lizard, E. coeruleus; sage-brush lizard, S. graciosus; Pacific gopher snake, P. catenifer; common garter snake, T. sirtalis)  White-toothed shrew (C. lasiura)  Wild boar (S. scrofa)

University

Table 2.1; continued E. chaffeensis  Birds (common pheasant)  Amblyomma spp. (A. americanum, A. testudinarium, A. parvum)  Canids (dog; red fox, V. vulpes; coyote, C. latrans)  Dermacentor spp. (D. variabilis, D. occidentalis)  Deer (white-tailed deer, O. virginianus; sika deer, C. nippon; Brazilian  Haemaphysalis spp. (H. longicornis, H. yeni, H. flava) brown brocket deer, M. gouazoubira; marsh deer, B. dichotomus)  Ixodes spp. (I. persulcatus. I. pacificus, I. ovatus)  Goats  Rhipicephalus spp. (R. sanguineus)  Opossum (D. virginiana)  Prosimian primates (ring-tailed lemur, L. catta; ruffed lemur, V. variegate)  Rodents (striped field mouse, A. agrarius; wild rat)  Raccoon (P. lotor) E. ewingii  Deer (white-tailed deer, O. virginianus)  AmblyommaMalaya spp. (A. americanum)  Dogs  Haemaphysalis spp. (H. longicornis)  Rodents (striped field mouse, A. agrarius)  Rhipicephalus spp. (R. sanguineus) E. canis  Cats  Dermacentor spp. (D. variabilis)  Dogs of Haemaphysalis spp. (H. longicornis)  Deer (sika deer, C. nippon)  Ixodes spp. (I. turdus)  Rodents (striped field mouse, A. agrarius)  Rhipicephalus spp. (R. sanguineus)  Wild canids (wolf, wolf-dog, coyote, red fox, gray fox)  Wild cats (Tsushima leopard cat, P. b. euptilurus; Iriomote cat, P. iriomotensis) E. ruminantium  Deer (white-tailed deer, O. virginianus; fallow deer, D. dama; Timor  Amblyomma spp. (A. variegatum, A. hebraeum, A. lepidium, A. astrion, deer, C. timorensis) A. pomposum, A. cohaerens, A. gemma, A. marmoreum, A. maculatum)  Ruminants (cattle, sheep, goat, water buffalo)  Wild ruminants (African buffalo, S. caffer; black wildebeest, C. gnou, C. taurinus; blesbuck, D. d. phillipsi; bushbuck, T. scriptus; duiker, Cephalophus sp.; giraffe, G. camelopardalis; steenbok, R. campestris) E. muris  Common shrew (S. araneus)  Haemaphysalis spp. (H. flava)  Deer (sika deer, C. nippon)  Ixodes spp. (I. persulcatus, I. ricinus)  Rodents (mouse, E. kageus, A. speciosus, A. argenteus; vole, M. rutilus, M. rufocanus, M. glareolus, M. agrestis; yellow-necked mice, A. flavicollis; SiberianUniversity chipmunk)

analysis) targeting genes such as cytochrome C oxidase subunit I (COI), internal transcribed spacer 2 (ITS2), 16S rDNA, 12S rDNA and 28S rDNA (Black and Piesman,

1994; Inokuma et al., 2003; Lv et al., 2014a) is a useful tool for tick identification.

The details of ticks associated with Anaplasma spp. and Ehrlichia spp. are shown in Table 2.1. Five genera of ixodid ticks including Amblyomma spp., Ixodes spp.,

Haemaphysalis spp., Dermacentor spp., and Rhipicephalus spp. have been implicated as potential vectors. A. phagocytophilum has been detected in Ixodes ticks (I. scapularis,

Ixodes persulcatus, Ixodes ricinus and Ixodes pacificus) and other tick species including

H. longicornis, Haemaphysalis concinna, Haemaphysalis megaspinosa, Dermacentor nuttalli, Dermacentor variabilis, Dermacentor silvarum, Rhipicephalus sanguineus and

Rhipicephalus bursa (Chastagner et al., 2013; Stuen et al., 2013). Approximately 20 tick species have been reported as potential vectors of A.Malaya marginale, of which D. variabilis, R. microplus and Dermacentor andersoni are most frequently reported (Kocan et al., 2010). of Transmission of A. centrale to animals through Rhipicephalus simus and

Haemaphysalis punctata has been reported (Palomar et al., 2015; Rar and Golovljova,

2011). A. ovis has been detected in various tick species including D. andersoni, R. bursa,

H. punctata, H. concinna, I. ricinus and Dermacentor marginatus (Silaghi et al., 2017).

A. bovis can be transmitted through different tick species including Haemaphysalis spp.

(H. longicornis, H. megaspinosa and Haemaphysalis shimoga), Amblyomma spp. (AmblyommaUniversity variegatum), Rhipicephalus spp. (R. sanguineus) and Dermacentor spp. (D. andersoni and Dermacentor occidentalis) (Silaghi et al., 2017). A. platys has been detected from R. sanguineus, the brown dog tick, as well as other tick species including

H. longicornis, Dermacentor auratus, Rhipicephalus turanicus and Rhipicephalus evertsi

(Rar and Golovljova, 2011; Silaghi et al., 2017).

Amblyomma americanum has been recognised as the main vector for E. chaffeensis and E. ewingii (Parola et al., 2005). Other tick species harbouring E. chaffeensis include R. sanguineus, I. persulcatus, H. longicornis, D. variabilis and

Amblyomma testudinarium. E. ewingii has also been reported in R. sanguineus and H. longicornis (Rar and Golovljova, 2011). R. sanguineus is the primary vector for E. canis, however, transmission of E. canis by D. variabilis, H. longicornis and Ixodes turdus has been reported (Rar and Golovljova, 2011). E. ruminantium has only been reported in ticks from the genus Amblyomma with A. variegatum and Amblyomma hebraeum as the most important vectors (Allsopp, 2010). Meanwhile, E. muris has been reported in I. ricinus,

I. persulcatus and Haemaphysalis flava (Rar and Golovljova, 2011) (Table 2.1).

Even though Anaplasma spp. and Ehrlichia spp. are found mainly in ticks, some species have been detected in other arthropods, forMalaya example, A. marginale has been described in biting flies including stable flies (Stomoxys calicitrans) and horse flies (Tabanidae) (Aubry and Geale, 2011); ofA. ovis has been detected in sheep keds (Melophagus ovinus) (Hornok et al., 2011) and E. ruminantium in tsetse flies (Glossina pallidipes) (Hornok et al., 2016).

2.5 Infections caused by anaplasma and ehrlichiae

2.5.1 Clinical symptoms in humans

The clinical presentations of patients diagnosed with anaplasmosis and ehrlichiosisUniversity are often nonspecific. Patients presenting with febrile illness, headache, myalgia and malaise have been reported (Dumler, 2005). Even though the diseases fatality rates are low (less than 1.0% for HGA and 3.0% for HME), complications such as septic or toxic shock-like syndrome, acute respiratory distress syndrome (ARDS), acute renal failure, meningoencephalitis, brachial plexopathy, demyelinating polyneuropathy, opportunistic infections with both viral and fungal agents, haemorrhage

and acute abdominal syndrome have been documented (Bakken and Dumler, 2015). The respiratory and gastrointestinal systems are often affected but skin rash is generally not often observed in patients with HME and is rare in patients with HGA (Dumler, 2005).

Other clinical symptoms observed in children and pregnant women having HME are altered mental status and abdominal pain that can mimic acute appendicitis (Ismail et al.,

2010).

Only very few HEE cases have been reported. The cases reported in the USA were associated with increasing age, human immunodeficiency virus (HIV) infection and immunocompromised status (undergoing immunosuppression therapy or organ transplantation) (Dumler et al., 2007; Ismail et al., 2010).

The laboratory findings of infections caused by A. phagocytophilum, E. chaffeensis and E. ewingii are almost similar, i.e.,Malaya thrombocytopenia, leukopenia, anaemia, increased serum transaminase activity (proposing mild to moderate liver injury) and lymphopenia (Dumler, 2005). of 2.5.2 Clinical signs in animals

In general, animals infected with Anaplasma spp. and Ehrlichia spp. demonstrate fever, anorexia, apathy, lethargy, reduction in milk production (mostly for ruminants), anaemia, weight loss, pallor mucous membrane and sometimes death (Atif, 2016). A. phagocytophilum is the causative agent of TBF or pasture fever in ruminants (sheep, goats and cattle) (Woldehiwet, 2006). But the fever may vary depending on the age of the infectedUniversity animals, the variant of A. phagocytophilum involved, the host species and immunological status (Stuen et al., 2013). Besides that, there have been cases of abortion and reduced fertility in A. phagocytophilum-infected ewes and rams, respectively (Stuen et al., 2013). Interestingly, dogs naturally infected with A. phagocytophilum often remain healthy and this has result in widespread serological evidence of canine granulocytic anaplasmosis but a lack of history of clinical illness (Carrade et al., 2009).

A. marginale is the major causative agent of bovine anaplasmosis. A. marginale can infect cattle of all ages and the disease severity varies according to the age of cattle.

Calves are usually more resistant to the infection, hence, animals between the age of 6 months and one year develop mild disease while animals between one to two years of age suffer non-fatal but acute disease, and animals more than two years of age suffer from acute disease with mortality risks (Aubry and Geale, 2011). Once cattle infected with A. marginale, they will persistently remain as infected carriers for their whole life (Aubry and Geale, 2011).

Dogs infected by E. canis (canine monocytotropic ehrlichiosis, CME) can be demonstrated in acute, subclinical or chronic forms (Harrus and Waner, 2011). All dog breeds are susceptible to E. canis infection with German shepherd dogs seem to be more susceptible with high morbidity and mortality ratesMalaya (Harrus and Waner, 2011). Acute disease normally develops within two to four weeks following tick transmission with disease manifestations differ depending onof the virulence of E. canis strains and co- infections with other tick-borne pathogens (for example Hepatozoon canis, Babesia canis) (Gal et al., 2007; Little, 2010). Some dogs may enter subclinical phase of infection with the dogs stay chronically infected for months to years without showing much clinical signs (normally only show mild thrombocytopenia) (Little, 2010).

2.6 Anaplasma and ehrlichiae infections in Asian countries UniversityAsia is the largest continent on Earth that is bounded by the Pacific Ocean on the east, the Indian Ocean on the south, the Arctic Ocean on the north and the European countries on the west. Southeast Asia (SEA) lies between the Pacific Ocean and Indian

Ocean and falls within the equatorial and sub-equatorial zones. Both Asia and SEA provide different habitats and microclimates which are conductive for the survival of

various haematophagous arthropods including ticks, fleas, mosquitoes, mites, lice and tabanids (Irwin and Jefferies, 2004).

Tick-borne diseases such as anaplasmosis and ehrlichiosis are reported in many parts of Asian and SEA countries. In Asia, human cases have been reported in China,

Japan, Korea and Mongolia, while ruminants (cattle, sheep, goats and deer), pet animals

(dogs), H. longicornis, R. microplus, R. sanguineus and I. persulcatus are among the most reported animal hosts and ticks associated with anaplasmosis and ehrlichiosis, as shown in Table 2.2. In SEA, limited human cases have been reported (only one publication from

Indonesia and Thailand, respectively) while both ruminant (cattle) and pet animal (dogs) and R. sanguineus ticks are the most reported animal hosts and tick vector for anaplasmosis and ehrlichiosis, respectively. Malaya 2.7 Current status of anaplasmosis and ehrlichiosis in Malaysia The tropical rainforest of Malaysiaof is the habitat for various animals and haematophagous arthropods including ticks. There has been no report of human cases of anaplasmosis and ehrlichiosis in Malaysia. However, anaplasmosis and ehrlichiosis have been reported in dogs, cattle and R. microplus ticks, using either serology or molecular methods. Table 2.3 shows the anaplasma and ehrlichiae infections that have been reported from animal samples in Malaysia.

University

Table 2.2: Prevalence of anaplasma and ehrlichiae infections reported in Asian region, and the method used for detection. Reports involved human subjects are highlighted in bold.

Country Hosts or vectors Anaplasmataceae Prevalence (%) Detection method Reference Bangladesh Cattle Anaplasma spp. 18.5 Blood smear Kispotta et al. (2016) Dogs Anaplasma spp. 28.0 PCR Qiu et al. (2016) R. sanguineus, host: dogs Anaplasma spp. 40.0 H. bispinosa, host: cattle A. bovis 1.2 Cattle Anaplasma spp. 3.9 Blood smear Alim et al. (2012) China Human- patients A. phagocytophilum 22.7 qPCR Dong et al. (2013) E. chaffeensis 25.0 A. capra 5.9 MalayanPCR H. Li et al. (2015) Human- rural residents A. phagocytophilum 18.2 IFA Zhang et al. (2014) E. chaffeensis 9.8 Human- urban residents A. phagocytophilum 1.5 E. chaffeensis 2.4of Human- forest areas residents A. phagocytophilum 7.1 IFA Hao et al. (2013) Human- farmers A. phagocytophilum 33.7 IFA Y. Zhang et al. (2012) Human- patients A. phagocytophilum 9 clinical cases nPCR Zhang et al. (2008a) Human- farm workers A. phagocytophilum 8.8 IFA Zhang et al. (2008b) Dogs A. ovis 4.1 PCR Cui et al. (2017) A. bovis 6.2 nPCR A. phagocytophilum 0.4 Sheep A. phagocytophilum 35.1 nPCR Yang et al. (2016) Goats A. phagocytophilum 26.4 H. longicornis, host: vegetation Ehrlichia spp. 0.4 nPCR Luo et al. (2016) Red deer A. ovis 31.8 nPCR Li et al. (2016) A. bovis 9.0 A. platys 9.0 Sika deer A. ovis 20.0 nPCR UniversityA. bovis 15.0 A. platys 15.0 E. canis 5.0

Table 2.2, continued China Goats A. centrale 16.0 nPCR Ge et al. (2016) H. bispinosa, host: sheep Ehrlichia spp. 2.9 nPCR Yu et al. (2016) D. silvarum, host: cattle Ehrlichia spp. 7.4 D. silvarum, host: vegetation Ehrlichia spp. 3.6 R. microplus, host: cattle Ehrlichia spp. 77.7 R. microplus, host: vegetation Ehrlichia spp. 87.4 H. qinghaiensis, host: vegetation Ehrlichia spp. 12.8 R. sanguineus, host: vegetation Ehrlichia spp. 16.3 R. sanguineus, host: cattle Ehrlichia spp. 28.6 H. longicornis, host: sheep Ehrlichia spp. 70.4 H. longicornis, host: vegetation Ehrlichia spp. 17.5 I. persulcatus, host: vegetation Ehrlichia spp. 2.4 Malaya Eurasian collared doves A. phagocytophilum 3.5 nPCR Yang et al. (2015b) Eurasian eagle owls A. phagocytophilum 15.4 Common pheasants E. chaffeensis 8.3of Bactrian camels A. platys 7.2 nPCR Y. Li et al. (2015) Rhipicephalus sp., host: camels A. platys 9.5 Rhipicephalus sp., host: vegetation A. platys 14.3 Mongolian gazelles A. bovis 78.3 nPCR Li et al. (2014) A. phagocytophilum 65.2 A. ovis 52.2 PCR H. longicornis, host: vegetation, Mongolian A. bovis 66.7 nPCR gazelles A. phagocytophilum 76.7 D. niveus, host: vegetation, Mongolian A. bovis 35.3 nPCR gazelles A. phagocytophilum 88.2 A. ovis 58.8 PCR Wild mouse A. phagocytophilum 3.1 qPCR Dong et al. (2013) E. chaffeensis 6.3 D. silvarum, host: vegetation and animals A. phagocytophilum 8.2 E. chaffeensis 14.8 University

Table 2.2, continued China H. longicornis A. phagocytophilum 1.6 qPCR Dong et al. (2013) E. chaffeensis 6.3 Goats A. bovis 16.0 nPCR Liu et al. (2012) A. ovis 15.3 A. phagocytophilum 6.1 Dogs A. phagocytophilum 10.1 IFA L. Zhang et al. (2012) 10.9 nPCR Goats A. phagocytophilum 3.8 IFA 26.7 nPCR Cattle A. phagocytophilum 0.7 IFA 23.4 nPCR D. silvarum A. phagocytophilum 58.3 MalayanPCR H. longicornis A. phagocytophilum 43.9 I. persulcatus A. phagocytophilum 12.5 R. microplus A. phagocytophilum 7.5of R. sanguineus A. phagocytophilum 5.2 A. testudinarium, host: cattle E. chaffeensis 55.2 nPCR Cao et al. (2000) H. yeni, host: cattle, rabbits E. chaffeensis 8.1 Hong Kong Stray dogs A. platys 8.0 PCR Wong et al. (2011) E. canis 7.0 A. phagocytophilum 6.0 SNAP 4Dx E. canis 1.0 Pet dogs E. canis 6.0 PCR E. canis 7.0 SNAP 4Dx India Dogs E. canis 14.3 Blood smear Kottadamane et al. (2017) E. canis 86.9 ELISA Cattle A. marginale 5.0 ELISA Ahmad Rather et al. (2016) Sheep A. marginale 1.9 Goats A. marginale 1.6 Dogs E. canis 0.4 nPCR Singla et al. (2016) Cattle UniversityA. marginale 16.7 PCR-RFLP Nair et al. (2013) A. bovis 3.3

Table 2.2, continued Japan Human- patients A. phagocytophilum 4 clinical cases Western blot, IFA Yoshikawa et al. (2014) A. phagocytophilum 2 clinical cases nPCR Ohashi et al. (2013) Rodents- mice and voles Anaplasma sp. AP-sd 4.4 PCR-RLB Moustafa et al. (2016) E. muris 1.2 Sika deer Anaplasma sp. AP-sd 51.0 nPCR Moustafa et al. (2015) Brown bears Anaplasma sp. AP-sd 15.0 Rodents Anaplasma sp. AP-sd 2.4 Japanese Iriomote cats E. canis 12.0 nPCR Tateno et al. (2013) Tsushima leopard cats E. canis 8.0 A. bovis 15.0 Deer A. phagocytophilum 15.6 MalayanPCR Masuzawa et al. (2011) A. bovis 21.9 A. centrale 37.5 Wild boars A. phagocytophilum 3.6 nPCR Masuzawa et al. (2011) A. bovis of17.9 A. centrale 3.6 Raccoons A. bovis 5.2 nPCR Sashika et al. (2011) H. megaspinosa, host: vegetation A. bovis 2.1 nPCR Yoshimoto et al. (2010) A. phagocytophilum 12.5 Sika deer E. chaffeensis 31.0 nPCR Kawahara et al. (2009) Deer A. phagocytophilum 19.0 nPCR Kawahara et al. (2006) A. centrale 11.9 A. bovis 8.7 Candidatus E. shimanensis 2.4 H. longicornis, host: vegetation Candidatus E. shimanensis 1.5 H. longicornis, host: deer A. bovis and A. centrale 12.0 Korea Human- patients A. phagocytophilum 1 clinical case PCR Kim et al. (2014) A. phagocytophilum 11.1 IFA Park et al. (2003) E. chaffeensis 14.4 A. phagocytophilum 8.9 Western blot UniversityE. chaffeensis 10.7 A. phagocytophilum 1.8 IFA Heo et al. (2002) E. chaffeensis 0.4

Table 2.2, continued Korea Korean native goats Anaplasma spp. 17.9 PCR Seong et al. (2015) H. longicornis, host: cattle A. phagocytophilum 5.5 nPCR Kang et al. (2013) A. platys 20.1 E. chaffeensis 19.5 E. ewingii 19.4 E. canis 22.4 E. muris 0.5 H. longicornis, host: vegetation A. phagocytophilum 1.9 nPCR Oh et al. (2009) A. bovis 0.4 A. centrale 0.1 E. chaffeensis 0.2 H. longicornis, host: vegetation A. phagocytophilum 1.1 MalayanPCR Chae et al. (2008) A. platys 1.2 E. chaffeensis 9.6 H. flava, host: vegetation A. platys of1.6 E. chaffeensis 2.0 I. nipponensis, host: vegetation and A. phagocytophilum 1.4 animals A. platys 0.5 E. chaffeensis 0.3 Rodents A. phagocytophilum 5.6 E. chaffeensis 35.2 House mouse E. chaffeensis 1 mouse nPCR Chae et al. (2008) Shrews A. platys 35.3 E. chaffeensis 26.5 Mongolia Human- donors A. phagocytophilum 2.4 IFA Walder et al. (2006) I. persulcatus, host: vegetation A. phagocytophilum 6.2 qPCR Karnath et al. (2016) I. persulcatus, host: vegetation A. phagocytophilum 6.2 nPCR Masuzawa et al. (2014a) Cattle A. marginale 8.7 nPCR Ybañez et al. (2013a) North Korea H. longicornis, host: goats A. phagocytophilum 4.1 nPCR Kang et al. (2016) A. bovis 26.4 University

Table 2.2, continued Pakistan Horses A. phagocytophilum 4.3 PCR-RFLP Razzaq et al. (2015) Cattle Anaplasma spp. 15.0 Blood smear Mushtaqa et al. (2015) A. marginale 32.5 nPCR Buffaloes Anaplasma spp. 7.5 Blood smear A. marginale 18.8 nPCR Buffaloes A. marginale 17.4 PCR-RFLP Ashraf et al. (2013) Cattle A. marginale 31.1 ELISA Atif et al. (2013) Cattle A. marginale 22.0 Blood smear Rajput et al. (2005) A. centrale 9.2 Buffaloes A. marginale 13.6 A. centrale 8.4 Malaya Taiwan H. hystricis, host: pangolins Anaplasma spp. 1 tick PCR Khatri Chhetri et al. (2016) Ehrlichia spp. 1 tick Rodents A. phagocytophilum 8.7 nPCR Masuzawa et al. (2014b) A. bovis of18.1 Dogs E. canis 9.9 SNAP 4Dx Yuasa et al. (2012) Anaplasma spp. 14.0 E. canis 20.8 nPCR Hsieh et al. (2010) A. platys 47.5 IFA Chang et al. (1996) Southeast Asia region Cambodia Dogs E. canis 21.8 PCR Inpankaew et al. (2016) Indonesia Human- residents E. chaffeensis 14.6 IFA Richards et al. (2003) Laos A. testudinarium Ehrlichia spp. 0.7 qPCR Andrew et al. (2016) Haemaphysalis spp. Ehrlichia spp. 2.0 H. aborensis Ehrlichia spp. 33.3 Philippines Dogs A. platys 40.0 PCR Ybañez et al. (2016) Cattle A. marginale 95.5 nPCR Ochirkhuu et al. (2015) Cattle A. marginale 14.1 nPCR Ybañez et al. (2014b) Dogs E. canis 2.9 mPCR Corales et al. (2014)

University

Table 2.2, continued Philippines Cattle Anaplasma spp. 54.7 PCR Ybañez et al. (2013c) (Cebu) A. marginale 8 cattle nPCR Ybañez et al. (2013b) R. microplus, host: cattle A. marginale 8 ticks A. centrale 1 tick R. sanguineus, host: dogs E. canis 4.3 nPCR Ybañez et al. (2012) A. platys 0.6 Philippines Military working dogs E. canis 59.0 ELISA Baticados and Baticados (2011) Thailand Human- patients A. phagocytophilum 4.5 IFA Blacksell et al. (2015) E. chaffeensis 36.0 Human- volunteers E. chaffeensis 44.0 IFA Heppner et al. (1997) Dogs with anaemia E. canis 9.9 nPCR Kaewmongkol et al. (2017) A. platys 3.7 Malaya Beef cattle A. marginale 14.5 PCR Jirapattharasate et al. (2017) D. auratus, host: wild boar A. platys 9.0 PCR Sumrandee et al. (2016) H. langrangei, host: deer A. bovis of16.7 A. platys 20.8 H. obesa, host: deer A. bovis 66.7 Stray dogs E. canis 3.9 PCR Liu et al. (2016) A. platys 4.4 Water buffaloes A. marginale 8.0 PCR Saetiew et al. (2015) H. shimoga, host: vegetation A. bovis 1 tick nPCR Malaisri et al. (2015) Domestic dogs E. canis 3.0 PCR Laummaunwai et al. (2014) Domestic cat A. platys 1 cat PCR Salakij et al. (2012) R. sanguineus, host: dogs E. canis 3.3 PCR Foongladda et al. (2011) A. platys 2.3 Rats Anaplasma spp. 46.5 Blood smear Thanee et al. (2009) Tree shrews Anaplasma spp. 6.0 D. auratus, host: dogs Anaplasma spp. 15.0 PCR Parola et al. (2003) A. javanense, host: pangolins Anaplasma spp. 29.6 H. langrangei, host: bears Anaplasma spp. 37.5 R. microplus, host: cattle UniversityEhrlichia spp. 22.0

Table 2.2, continued Thailand Dogs E. chaffeensis 74.0 IFA Suksawat et al. (2001) E. canis 71.0 A. phagocytophilum 58.0 E. canis 20.4 nPCR A. platys 10.2 Vietnam Dairy cattle A. marginale 28.0 ELISA Geurden et al. (2008) H. hystricis, host: wild pigs Ehrlichia spp. 5.3 PCR Parola et al. (2003) PCR: Polymerase chain reaction; qPCR: Real-time polymerase chain reaction; nPCR: Nested polymerase chain reaction; IFA: Immunofluorescent assay; ELISA: Enzyme-linked immunosorbent assay; PCR-RFLP: Polymerase chain reaction- restriction fragment length polymorphism; PCR-RLB: Polymerase chain reaction- reverse line blot hybridisation Malaya Table 2.3: Animal host for Anaplasmataceae, prevalence (%) and detection methods for infections reported in Malaysia.

Animal hosts or vectors Anaplasmataceae Prevalenceof (%) Detection method Reference A. varanense, host: snakes A. phagocytophilum 1 tick PCR Kho et al. (2015b) A. bovis 2 ticks A. platys 2 ticks Ehrlichia spp. 2 ticks A. helvolum, host: snakes A. phagocytophilum 2 ticks Monkeys A. bovis 10.0 PCR Tay et al. (2015) Cattle Anaplasma spp. 84.4 PCR Tay et al. (2014) R. microplus, host: cattle Anaplasma spp. 5.5 Dogs A. platys 13.3 nPCR Mokhtar et al. (2013) Cattle and buffaloes A. marginale 77.6 c-ELISA Premaalatha et al. (2013) Dogs E. canis 2.0 PCR Nazari et al. (2013) Cattle and buffaloes A. marginale 77.6 c-ELISA Rahman et al. (2012) Dogs E. canis 15.0 IFAT Rahman et al. (2010) PCR: Polymerase chain reaction; nPCR: Nested Universitypolymerase chain reaction; c-ELISA: Competitive enzyme-linked immunosorbent assay; IFAT: Immunofluorescent antibody test

2.8 Laboratory diagnosis of anaplasmosis and ehrlichiosis

2.8.1 Histochemistry and immunochemistry

Direct examination of peripheral blood is one of the methods for diagnosis of anaplasmosis and ehrlichiosis. A diagnosis can be established by the identification of morulae in the eosin-azure (Romanowsky)-type stained (including Giemsa’s, Wright’s,

Leishman’s and Diff-Quik) blood smear using light microscopy during the acute phase of the disease (Ganguly and Mukhopadhayay, 2008; Thomas et al., 2009). Even though this method gives fast result, it is relatively insensitive as compared to other available diagnostic assays and an experienced microscopist is required to perform and analyse the result (Dumler et al., 2007; Thomas et al., 2009).

Immunohistochemical (IHC) staining is another method to document the presence of anaplasma or ehrlichiae in patients before the beginningMalaya of antibiotic therapy or within the first 48 hours after antibiotic therapy has been started (Biggs et al., 2016). The method requires the staining of antigens in paraffin-embedded,of formalin-fixed biopsy or autopsy tissues (bone marrow, spleen, liver, lymph node and lung).

2.8.2 Serology methods

Several serological tests including indirect immunofluorescent assay (IFA), enzyme-linked immunosorbent assay (ELISA), complement fixation test (CF) and card agglutination test (CAT) have been employed for the diagnosis of anaplasmosis and ehrlichiosis. IFA is the gold standard used for serological diagnosis of human anaplasmosisUniversity and ehrlichiosis (Ismail et al., 2010). Using IFA, antibodies present in the patient serum samples bind to the anaplasma or ehrlichia antigens (A. phagocytophilum or E. chaffeensis) fixed on a slide and are detected using fluorescein labelled conjugates

(Biggs et al., 2016). In most cases, since antibody has not reached to a detectable level during the first week of infection, paired sera collected 2-3 weeks after illness onset are preferred samples for serological examination (Biggs et al., 2016).

A confirmed case of A. phagocytophilum or E. chaffeensis infection is determined by a fourfold increase in the antibody titers between acute and convalescent sera or a seroconversion to a titer of 128 or higher (Bakken et al., 2002; Thomas et al., 2009). Due to the possibility that antibody titer can last for months or years after initial exposure, a confirmed case should be based on both the evidence of antibody titers and other clinical evidence of infection (Thomas et al., 2009). In addition, non-specificity may occur due to cross-reactive immune responses to antigens which are typically group-specific (Biggs et al., 2016).

Several serological methods are also employed for the detection of exposure to

Anaplasma spp. and Ehrlichia spp. in animals. An ELISA commercial kit that utilises a monoclonal antibody specific for MSP5 [Anaplasma Antibody Test Kit (cELISA); VMRD Inc., Pullman, WA, USA] was used to identifyMalaya Anaplasma-infected cattle. In addition, commercially available SNAP 4Dx® test kit (IDEXX Laboratories, Westbrook, ME, USA) that detect antibodies to specificof peptides of Ehrlichia spp. (E. canis) and Anaplasma spp. (A. phagocytophilum) is used for the diagnosis of canine anaplasmosis and ehrlichiosis and other animals (including cats and horses) (Little, 2010). In addition, an ELISA kit based on the recombinant major antigenic protein 2 (rMAP2) and the

Immunocomb dot-ELISA test which uses a crude ehrlichial extract (Biogal, Galed

Laboratories, Israel) are also used for the detection of E. canis in dogs (causing CME)

(Harrus and Waner, 2011). These serological assays are facing the same limitations as otherUniversity serological tests: low sensitivity for early infection, false negative results (negative serologic results does not exclude diagnosis) and cross-reactivity among Anaplasma spp. and Ehrlichia spp. (Aubry and Geale, 2011).

2.8.3 Isolation of anaplasma and ehrlichiae

Due to the obligate intracellular nature of the bacteria, isolation of Anaplasma spp. and Ehrlichia spp. requires cell culture technique. Members in the genera Anaplasma

and Ehrlichia (except A. bovis and A. platys) have been isolated using different cell lines

(Table 2.4). In vitro cultivation of these pathogens provides an opportunity for better understanding of the pathogens, development of vaccines and serological tests and investigation of pathogen-host cell interaction in a controlled environment (Bell-Sakyi et al., 2000; Bell-Sakyi et al., 2007). This method is more likely to be employed in research laboratories rather than serving as a diagnostic tool in clinical laboratories (Harrus and

Waner, 2011).

Table 2.4: Cell lines used for successful isolation of Anaplasma spp. and Ehrlichia spp.

Species Cell lines for successful isolation Reference(s) A.  Human promyelocytic leukemia cell line (HL-60) Bell-Sakyi et al. phagocytophilum  I. scapularis embryo-derived cell lines IDE8 and (2007); Dyachenko ISE6 et al. (2013);  I. ricinus embryo-derived cell line IRE/CTVM20 Goodman et al. (1996) A. marginale  I. scapularis embryo-derived cell lines IDE8 and Bell-Sakyi et al. ISE6 (2007)  I. ricinus embryo-derived cell line MalayaIRE/CTVM18 A. centrale  R. appendiculatus embryo-derived cell line Bell-Sakyi et al. RAE25 (2015) A. ovis  I. scapularis embryo-derivedof cell line IDE8 Bell-Sakyi et al. (2007) A. bovis Not yet been isolated A. platys Not yet been isolated E. chaffeensis  Canine peritoneal macrophage (DH82 cell line) Bell-Sakyi et al.  I. scapularis embryo-derived cell line ISE6 (2007); Standaert et al. (2000) E. ewingii  Human promyelocytic leukemia cell line (HL-60) Killmaster and (for only about 16 weeks) Levin (2016) E. canis  Canine peritoneal macrophage (DH82 cell line) Bell-Sakyi et al.  I. scapularis embryo-derived cell lines IDE8 and (2007) ISE6  I. ricinus embryo-derived cell line IRE/CTVM18 E. ruminantium  A. variegatum larva-derived cell lines Bell-Sakyi et al. AVL/CTVM13, 17 (2000); Bell-Sakyi University I. scapularis embryo- derived cell line IDE8 et al. (2007)  R. appendiculatus embryo-derived cell lines RAE25 and RAE/CTVM1 E. muris  Canine peritoneal macrophage (DH82 cell line) Wen et al. (1995)

2.8.4 Molecular detection of Anaplasma spp. and Ehrlichia spp.

As compared to other laboratory diagnostic methods, molecular detection of

Anaplasma spp. and Ehrlichia spp. (mostly by PCR) is more sensitive, specific and rapid

(Ismail et al., 2010). Molecular methods are able to give information on the epidemiology of the pathogens and allow the detection of co-infections in the clinical samples (Guillemi et al., 2015). Unlike serological methods which rely on the time taken for antibodies to build to a detectable level, PCR amplification of Anaplasmataceae DNA from the whole blood samples of patients during acute stage of illness (where antibody level is still very low or undetectable) is particular useful for early confirmation of the causative pathogens

(Biggs et al., 2016). Additionally, PCR allows the detection of Anaplasma spp. and

Ehrlichia spp. in various sample types including ticks and organ tissues (Silaghi et al.,

2017).

Conventional PCR assay is the most widely use PCR format for detection of

Anaplasmataceae DNA but other PCR formats have also been developed. For example, qPCR allows quantification of bacterial load in the Malayasamples (Harrus and Waner, 2011) and eliminate the time-consuming gel electrophoresis step by using intercalating fluorescent dyes. Nested PCR (nPCR) is ofa more sensitive approach than conventional PCR where two rounds of amplifications take place by using two different primer sets

(inner and outer primer pairs with different annealing temperatures) (Hunt, 2011).

Multiplex PCR enables the detection of multiple pathogens simultaneously (Hunt, 2011;

Thomas et al., 2009). Loop-mediated isothermal amplification (LAMP) is an isothermal

DNA amplification method that is used for rapid detection of infectious disease especially in poor or underdeveloped countries because of its low cost and easy to operate procedure (KulešUniversity et al., 2017; Ma et al., 2011). PCR incorporated with other techniques such as PCR-RFLP (restriction fragment length polymorphism) which uses specific restriction endonucleases for genotyping purpose (Alberti et al., 2005) and PCR-RLB (reverse line blot hybridisation) which allows simultaneous detection and identification of Anaplasma spp. and Ehrlichia spp. in the samples by using primers (PCR part) and probes (RLB part) have been developed

(Bekker et al., 2002). FRET (fluorescence resonance energy transfer)-qPCR is an advanced method which uses one set of primers and probes to detect and differentiate different Anaplasma spp. and Ehrlichia spp. present in the samples in a single reaction and melting curve is used for quantification analysis of the detected organisms (Zhang et al., 2015). PCR-HRM (high resolution melt analysis) is a rapid and sensitive diagnostic method that allows differentiation of organisms in a sample based on different melting temperatures and fluorescent dyes for real-time monitoring of the process (Krücken et al., 2013). Fluorescence in situ hybridisation (FISH) method uses a DNA probe (with the length between 15-30 nucleotides) which is linked to a fluorescent molecule hybridised to its matching target DNA sequence in situ (Kuleš et al., 2017).

2.8.4.1 Molecular characterisation of Anaplasma spp. and Ehrlichia spp. Many PCR protocols have been designed Malaya to target the 16S rRNA gene of Anaplasma spp. and Ehrlichia spp. (Guillemi et al., 2015). However, since 16S rRNA gene is highly conserved among the familyof members of the same genus, gltA and groEL genes have been proposed as an alternative to 16S rRNA gene for phylogenetic analysis and identification of Anaplasma spp. and Ehrlichia spp. as both genes exhibit high variation, and thus provide better discrimination power among closely related species

(Inokuma et al., 2001a; Sumner et al., 1997). In addition, other genes that have been used as targets for molecular diagnosis include msp1β (major surface protein 1β), msp2 (major surface protein 2), msp4 (major surface protein 4), ankA (ankyrin protein), p28 (28kDa outerUniversity membrane protein) and p30 (30kDa outer membrane protein) (Guillemi et al., 2015; Silaghi et al., 2017; Strašek Smrdel et al., 2015).

Using direct sequencing technique, amplified PCR amplicons can be sequenced and analysed. Multilocus sequence typing (MLST) allows genotypic characterisation of isolates and population studies by analysing DNA sequences of multiple housekeeping genes (Huhn et al., 2014). Phylogenetic analysis enables sequence comparison and

determines the phylogenetic relationships of bacteria (Wen et al., 2002). For example,

Dumler et al. (2001) reorganised the genera in the family Anaplasmataceae based on the sequences of 16S rRNA and groEL genes. In addition, next generation sequencing (NGS) is a reliable DNA sequencing approach that plays an important role for microbial discovery and provides an estimation of the pathogenicity of organisms through sequencing of certain pathogenicity-related genes (Kuleš et al., 2017; Tijsse-Klasen et al., 2014).

2.9 Treatment for anaplasmosis and ehrlichiosis

Most patients respond well to tetracycline (bacteriostatic agent) if treatment is given during the early onset of the disease (Ismail et al., 2010). However, doxycycline (bacteriostatic agent) is preferred over tetracycline becauseMalaya of its twice-daily oral dosage, better patient tolerance and relatively lower risk of adverse drug effects for children under the age of eight. The recommended doxycyclineof dosage for an adult is 100 mg given orally or intravenously every 12 hours; while the dosage for children aged eight years or older is 2.2 mg/kg every 12 hours with a maximum dosage of 100 mg. The recommended tetracycline dosage for adult is 500 mg for every six hours and for children aged eight years or older it is 25-50 mg/kg in four divided doses (Dumler et al., 2007). Despite having the risk of causing dental discoloration in children, doxycycline remains the main treatment of choice for paediatric patients (Ismail et al., 2010). For patients having allergy issueUniversity with tetracycline and children under eight years old or pregnant patients, rifampin is recommended (Dumler et al., 2007; Jin et al., 2012). Treatment for anaplasmosis and ehrlichiosis are normally continued for 5 to 14 days (Dumler et al., 2007).

Oxytetracycline and doxycycline are the two most common antibiotics for horses and other ruminants (including cattle). Doxycycline is effective for cats and dogs (Atif,

2016). Chlortetracycline is proved to be an effective drug for persistent bovine anaplasmosis caused by A. marginale (Reinbold et al., 2010).

2.10 Prevention and control of anaplasmosis and ehrlichiosis

The most effective method of prevention is to avoid exposure to tick and immediate removal of ticks (Ismail et al., 2010). Other methods such as wearing protecting or light coloured clothes and applying repellent sprays [N, N-diethyl-m- toluamide (DEET), permethrin, natural compounds (citriodiol, p-menthane-3,8-diol)] can help in prevent tick attachment (Jin et al., 2012; Piesman and Eisen, 2008). For controlling bovine anaplasmosis, maintenance of Anaplasma-free herd and quarantine of any Anaplasma-positive animals is required as they are the source of infection for other uninfected animals (Aubry and Geale, 2011). Malaya The universal vaccine for any Anaplasma or Ehrlichia species is not yet available. Vaccines have been developed for bovineof anaplasmosis caused by A. marginale. Attenuated strains of A. marginale and live A. centrale are used as vaccine candidates

(Kocan et al., 2010). Besides that, the use of tick vaccines containing tick proteins (Q38,

SUB, SILK, TROSPA, BM86/BM95 and 64P) which interfere with tick vector competence may result in reduced tick infestation (Atif, 2016).

University

CHAPTER 3: MATERIALS AND METHODS

3.1 Sample collection

The samples processed in this study were ticks and animal blood samples obtained from various sources. Animal blood samples were mainly provided by collaborators from the Veterinary Research Institute, Ipoh and Department of Wildlife and National Parks

(PERHILITAN) Peninsular Malaysia from 2013 to 2014 for health screening purpose.

Tick samples were collected from livestock farms (cattle and sheep), the forest areas

(questing ticks and ticks collected from small animals) as well as from peri-domestic animals in aboriginal villages (Appendix A-C). Due to the limited information about the animal hosts and tick vectors for anaplasmosis and ehrlichiosis in Malaysia, samples were collected whenever possible. Ticks were collected from livestock subjected to blood sampling. Malaya 3.1.1 Samples obtained from livestock farms

Sampling of animal blood samplesof and ticks were conducted in eight livestock farms (six cattle, one goat and one sheep farms) located in different regions of Peninsular

Malaysia (Table 3.1) from February to September 2013. Approval (Reference no:

JPV/PSTT/100-8/1) for the sampling has been obtained from the Director, Department of

Veterinary Services, Ministry of Agriculture and Agro-Based Industry, Malaysia, prior to the commencement of the study. A total of 304 blood samples (224 cattle, 40 sheep and 40 goats) were collected in this study (Table 3.1) (Appendix A). Blood sampling was performedUniversity by veterinarians in each farm. Briefly, approximately 1 to 3 ml whole blood sample was collected from each animal via jugular vein in EDTA-coated tube. The samples were then transported in ice to the laboratory. A volume of 200 µl was aliquoted from each blood sample and stored at -20 °C for DNA extraction. The remaining of the blood samples were stored at -80 °C for future work. Ticks were collected from livestock

whenever possible. The ear, eyes, abdomen, tail and perineal regions of the animals were examined for ticks. The collected ticks were preserved in -80 °C prior to processing.

Table 3.1: Sampling sites, date of sampling and number of samples collected from livestock farms in this study.

Sampling sites Date of No. blood sample No. of animals infested (GPS locations) (no. of sampling collected (Animal with ticks (No. of ticks animals sampled, n) breed, n) subjected to molecular analysis) Jelai Gemas, Negeri February 27 cattle bloods 27 (47 ticks) Sembilan 2013 (Nellore, 11; YKK (N2° 40′ 2.273′′ E102 [Yellow cattle cross with °34′ 23.022′′) (n=27) Kedah Kelantan], 15)

Jerantut, Pahang August 38 cattle bloods (Kedah- 9 (14 ticks) (N3° 56′ 48.61′′ E102 2013 Kelantan [Zebu], 38) °22′ 47.57′′) (n=38)

Ulu Lepar, Pahang August 40 cattle bloods 38 (94 ticks) (N3° 45′ 24.059′′ E103 2013 (Nellore, 32; Brahman, °12′ 12.92′′) (n=40) 8) Malaya

Pokok Sena, Kedah September 40 sheep bloods 21 (44 ticks) (N6°9′ 13.478′′ E100 2013 (Damara,of 40) °32′ 3.379′′) (n=40)

Tanah Merah, Kelantan September 40 cattle bloods (Kedah- Not collected (N5° 51′ 48.45′′ E102 2013 Kelantan, 40) °0′ 52.97′′) (n=40)

Kuala Berang, September 40 cattle bloods (YKK, 39 (71 ticks) Terengganu 2013 40) (N5° 4′ 8.926′′ E102 °59′ 49.45′′) (n=40)

Kuala Pah, September 40 goat bloods (Boer, Not collected Negeri Sembilan 2013 32; African dwarf, 5; (N2° 56′ 49.75′′ E102 Savannah, 2; Cashmere, °5′ 7.635′′) (n=40) 1) University Air Hitam, Johore September 40 cattle bloods Not collected (N2° 1′ 43.63′′ E103 2013 (Mafriwal, 36; Jersey, 4) °18′ 55.36′′) (n=40) Total 304 134 (270 ticks)

3.1.2 Samples obtained from Veterinary Research Institute (VRI), Ipoh

A total of 302 animal blood samples (78 deer, 75 goats, 55 buffaloes, 46 horses,

22 cows, 15 pangolins, seven dogs and four rats) collected in EDTA tubes were provided by the Parasitology department of the Veterinary Research Institute (VRI), Ipoh, from

January to October 2013 (Appendix D). Approval to use the sample for molecular analysis has been obtained from the Director of VRI prior to the commencement of the study [Reference no.: JPV: VRI/197/PA/141 Jid.11(48)].

Additionally, 11 animal blood samples obtained from ten dogs and one cat

(Appendix D) which were collected by VRI staff during a field trip at Ipoh, Perak (from

10-13 December 2012) were also included in this investigation. Animal ethical approval from the VRI Animal Care and Use Committee (VRI ACUCVRIpara/1/2014) was obtained prior to the sampling. A volume of 200 Malaya µl of each blood sample has been pipetted to a sterile microcentrifuge and was kept at -20 °C prior to DNA extraction. 3.1.3 Samples obtained from Wildlife ofDepartment (PERHILITAN) Approval was obtained from PERHILITAN [Reference no.: JPHL&TN (IP):80-

4/2 Jilid 15(51)] prior to the commencement of the study. A total of 139 ticks (Appendix

B) were collected from vegetation (n=117) and small animals (n=22) from two sampling trips organised by the Department of Wildlife and National Parks (PERHILITAN),

Peninsular Malaysia in Kuala Lompat, Krau Wildlife Reserve (N 03º50’ E 102º06’),

Pahang (June 2013) and a forest at Sungai Deka Elephant Sanctuary, (N 05º28’ E 102º44’)University Terengganu (October 2012). A total of 36 organ samples (liver, kidney and spleen) from small mammals (i.e., one squirrel, four rats and seven bats) collected from the field trips were also included

(Appendix B). A total of 80 animal blood samples (70 monkeys and ten wild boars) and

71 Whatman® FTA® cards collected from a variety of animals were provided by

PERHILITAN (Appendix B) on March 2013 and April 2015. Each blood sample (200

µl) was aliquoted to a sterile microcentrifuge tube and kept at -20 °C prior to DNA extraction. For the FTA cards, ten discs (2.0 mm in diameter) were excised from each card using a puncher and kept in -20 °C prior to processing.

3.1.4 Tick sampling in aboriginal villages

Sampling for ticks from peri-domestic animals in 12 aboriginal villages located in six states of Peninsular Malaysia was conducted in between September 2012 to January

2013 by research students working under “Tick-borne Emerging Infectious Disease

Program (funded by RP013-2012A)”. Approval has been granted from the Department of Orang Asli Development (JAKAO), Malaysia prior to the study. Of a total of 246 animals (106 dogs, 105 cats, 20 chickens, 11 goats, three cows and one snake) examined, only 88 animals (31 dogs, 31 cats, 13 chickens, nine goats, three cows and one snake) were infected with ticks. All the ticks collected wereMalaya preserved in -80 °C prior to processing. A total of 192 ticks collected from 47of animals (16 cats, 13 dogs, 12 chickens, three goats, two cows and one snake) were grouped into 65 pools (one to ten individuals according to geographical location, tick species and animal host) prior to DNA extraction and molecular analysis (Table 3.2) (Appendix C).

3.1.5 Other samples

Other samples that were investigated in this study include:

i. Twenty-one adult ticks (11 Amblyomma varanense and ten UniversityAmblyomma helvolum ) collected from seven Python molurus snakes from Sepang, Selangor during September 2012 (Appendix E).

ii. Thirty stray dog blood samples collected from an animal shelter in

Klang Valley (Mokhtar et al., 2013) and 33 R. sanguineus ticks

collected from 13 dogs in Klang Valley (Appendix E).

Table 3.2: Sampling sites and samples collected from aboriginal villages in this study.

Sampling Date of No. of animal host examined (n) No. of ticks subjected to molecular analysis sites sampling Dog Cat Chicken Others Negeri September 2012 40 12 1 8 goats 3 individual ticks from 2 dogs; 4 pools of ticks from 1 chicken and 3 Sembilan goats Pahang October- 21 18 5 3 goats 7 pool of ticks from 1 dog, 1 cat and 3 chickens November 2012 Johore November 2012 15 23 2 1 snake 1 pool of ticks from 1 snake; 1 individual tick and 8 pool of ticks from 1 dog, 2 chickens and 6 cats Kedah January 2013 16 17 1 3 cows 3 individualMalaya ticks from 2 cows Perak January 2013 10 8 2 - 12 individual ticks and 3 pool of ticks from 9 dogs; 4 individual ticks and 8 pool of ticks from 3 chickens and 6 cats Kelantan January 2013 4 27 9 - of10 individual ticks and a pool of ticks from 3 cats and 3 chickens Total 106 105 20 11 goats, 3 192 ticks (33 individuals and 32 pools) from 47 animals (1 snake, 2 cows, 1 snake cows, 3 goats, 12 chickens, 13 dogs and 16 cats)

University

3.2 Identification of tick species using morphological identification methods

Ticks collected in this study were identified morphologically based on the taxonomic keys of Burridge (2001), Kohls (1957), Walker et al. (2003) or Geevarghese and Mishra (2011). The tick images were viewed and captured using a stereomicroscope

(Olympus, Japan). Representative ticks of different species were placed individually in microcentrifuge tubes containing 70% ethanol at room temperature as voucher specimens.

3.3 Identification of tick species using molecular identification methods

Molecular identification of ticks was also carried out based on sequence analysis of the tick 16S rRNA or 28S rRNA genes (Black and Piesman, 1994; Inokuma et al.,

2003), whenever the species identification was in doubt. Malaya 3.4 DNA extraction The DNA extraction procedures wereof carried out in a Class II, A2 biological safety cabinet, (NuAire Inc, USA). A QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used for extracting DNA from each sample. The protocols were slightly adjusted for different sample types.

3.4.1 Tick samples

Ticks which had been stored at -80 °C freezer were first thawed, washed in 5% sodium hypochlorite and 70% ethanol for 3 min each and rinsed twice in sterile distilled waterUniversity for 3 min. The ticks were homogenis ed in 180 µl Buffer ATL (tissue lysis buffer) using surgical blades in a microcentrifuge tube. After adding 20 µl Proteinase K, the sample was mixed thoroughly by vortexing for 10 s before incubated at 56 °C for overnight. A volume of 200 µl Buffer AL (lysis buffer) was then added to the sample.

After vortexing for 10 s, the sample was incubated at 70 °C for 10 min before added with

230 µl ethanol (molecular grade). The mixture was transferred to a spin column (provided

by the kit) and centrifuged at 8000 rpm for 1 min. After the centrifugation step, the spin column was placed in a new, clean 2 ml collection tube. A volume of 500 µl Buffer AW1

(wash buffer) was added and the column was centrifuged at 8,000 rpm for 1 min. The spin column was later placed in a new, clean collection tube and added with 500 µl Buffer

AW2 (wash buffer). After centrifugation at 14,000 rpm for 3 min, an additional step of centrifugation (14,000 rpm for 1 min) was added to prevent carryover of Buffer AW2.

The column was placed in a sterile 1.5 ml microcentrifuge tube, added with 60 µl Buffer

AE (elution buffer) and incubated at room temperature for 5 min before centrifuging at

8,000 rpm for 1 min to elute the DNA. Lastly, another 60 µl Buffer AE were added into the column. The column was incubated again at room temperature for 5 min and centrifuged at 8,000 rpm for 1 min. The eluted DNA was collected and stored at -20 °C prior to PCR amplification. Malaya 3.4.2 Whole blood samples A volume of 200 µl whole blood sampleof was added to a 1.5 ml microcentrifuge tube containing 20 µl proteinase K. The mixture was then added with 200 µl Buffer AL and vortexed for 10 s prior to incubation at 56 °C for 10 min. This was then followed by the addition of a volume of 200 µl ethanol (molecular grade) to the mixture. Subsequent extraction steps were performed as described in section 3.4.1. A total of 100 µl DNA solution was eluted from each sample and stored at -20 °C prior to PCR amplification.

3.4.3 Organ samples UniversityThe organ samples were thawed and cut into tiny pieces using sterile surgical blades, prior to DNA extraction. The organ samples (25 mg for liver and kidney; 10 mg for spleen) were homogenised using a hand-held homogeniser in a 1.5 ml microcentrifuge tube containing 80 µl phosphate buffered saline (PBS). A volume of 100 µl Buffer ATL and 20 µl proteinase K were then added to the homogenate. The sample was mixed thoroughly by vortexing for 10 s and incubated at 56 °C for approximately 3 hours. During

incubation, the sample was vortexed occasionally. DNA extraction for the subsequent steps was performed as described in section 3.4.1. A total of 200 µl DNA solution was eluted and stored at -20 °C prior to PCR amplification.

3.4.4 Whatman® FTA® card

Ten discs excised from each FTA card were placed in a sterile 1.5 ml microcentrifuge tube. The discs were washed thrice with 200 µl FTA Purification Reagent

(GE Healthcare, UK) at room temperature for 5 min to remove cell debris and PCR inhibitors. The discs were then washed twice with 200 µl 1X TE buffer at room temperature for 5 min. Lastly, the discs were placed in a sterile 1.5 ml microcentrifuge tube and dried at 50 °C for 15 min. The purified discs were stored at -20 °C. A single disc was used as the DNA template for amplification. Malaya 3.5 Identification of Anaplasma spp. and Ehrlichia spp. from animals and ticks using molecular methods of 3.5.1 Integrity checking of samples for amplification

3.5.1.1 Amplification of the 16S rRNA or 28S rRNA gene from tick DNA samples

The amplification of either 16S rRNA or 28S rRNA gene from tick DNA samples was used as an indicator to show that DNA extraction from ticks was successful and there was no PCR inhibitors in the samples. In this study, primers 16S+2 and 16S-1 or primers

28SF and 28SR (Black and Piesman, 1994; Inokuma et al., 2003) (Appendix F) were used in University the PCR assays. These PCR assays amplified a portion of tick 16S rRNA gene (approximately 300 bp amplicon) and tick 28S rRNA gene (approximately 490 bp amplicon). The thermal cycling profile for amplification of tick 16S rRNA gene consisted of an initial denaturation step at 94 °C for 2 min, 35 cycles of denaturation at 95 °C for

30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, followed by a final extension step at 72 °C for 5 min. The PCR conditions for amplification of tick 28S rRNA

gene included an initial denaturation step at 95 °C for 5 min, 35 cycles of denaturation at

95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension step at 72 °C for 5 min.

3.5.1.2 Amplification of the cytochrome B (cytB) gene of animal blood and organ

samples

A PCR assay using primers cytBFor and cytBRev (Oshaghi et al., 2006)

(Appendix F) was used to check the integrity of the DNA template for animal blood and organ samples. The thermal cycling condition consisted of an initial denaturation step at

95 °C for 3.5 min, 36 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 50 s and extension at 72 °C for 40 s, followed by a final extension step at 72 °C for 5 min. The

PCR assay is expected to amplify a fragment of cytB gene (approximately 358 bp) from animal blood and organ samples. Malaya 3.5.2 Polymerase chain reaction All PCR assays were performed inof a reaction volume of 25 µl containing two microliters of DNA template, 1U GoTaq® Flexi DNA Polymerase (Promega, WI, USA),

® 1X Green GoTaq Flexi Buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.2 µM of forward and reverse primers and 15.4 µl miliQ water. The composition of PCR reaction mixture is shown in Table 3.3. For nested PCR assays, two microliters of the products from the first amplification was used as the DNA template for the nested amplification. Positive (A. phagocytophilum and E. chaffeensis genomic DNA extracted from IFA slides or plasmid DNA)University and negative (miliQ water) controls were included in each PCR assay. Amplification was performed in a MyCycler™ thermal cycler (Bio-Rad Laboratories,

Hercules, CA, USA).

Table 3.3: PCR reaction mixture used for amplification of Anaplasmataceae DNA.

Reagents Initial Final Volume (µl per concentration concentration reaction) miliQ water - - 15.4 Green GoTaq® Flexi Buffer 5X 1X 5.0 dNTPs 10 mM 0.2 mM 0.5

MgCl2 25 mM 1.5 mM 1.5 Forward primer 25 µM 0.2 µM 0.2 Reverse primer 25 µM 0.2 µM 0.2 GoTaq® Flexi DNA Polymerase 5 U/µl 1 U 0.2 DNA template - - 2 Total 25

3.5.3 Preliminary screening of Anaplasmataceae DNA

Ticks, animal bloods, organ samples and FTA cards were subjected to preliminary screening using a PCR assay targeting a partial fragment (345 bp) of the 16S rRNA gene of Anaplasmataceae. The composition of the PCR master mix used in this study is shown in Table 3.3. Two primers, i.e., EHR16SD and EHR16SRMalaya (Parola et al., 2000) (Appendix F) were used in the PCR assay. The amplificationof procedure was performed with an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for

30 s, annealing at 55 °C for 30 s and extension at 72 °C for 90 s, and a final extension step at 72 °C for 5 min.

3.5.4 Further characterisation of Anaplasma spp. and Ehrlichia spp.

Samples that were positive in the screening assays (section 3.4.3) were randomly selected for further characterisation using different PCR approaches. The selection was basedUniversity on sample type, sample source, and the availability of DNA template. 3.5.4.1 Amplification of the full length 16S rRNA, gltA and groEL genes of

anaplasma and ehrlichiae a. Amplification of the full length 16S rRNA gene of Anaplasma spp. and Ehrlichia spp. was performed using the following primers and PCR conditions:

i. Primers fD1 and Rp2 (Weisburg et al., 1991) (Appendix F). The thermal cycling condition consisted of an initial denaturation step at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 2 min, annealing at 58 °C for 30 s and extension at 72 °C for 4 min, followed by a final extension step at 72 °C for 20 min. The PCR assay is expected to amplify a nearly full length fragment of the 16S rRNA gene (1500 bp).

ii. Primers ATT062F and ATT062R (Pinyoowong et al., 2008) (Appendix F). The thermal cycling condition consisted of an initial denaturation step at 94 °C for 4 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s and extension at 72

°C for 1 min, followed by a final extension step at 72 °C for 4 min. The PCR assay is expected to amplify a nearly full length fragment of the 16S rRNA gene (1500 bp). iii. Primers fD1 with EHR16SR and primers Rp2 with EHR16SD. The thermal cycling condition consisted of an initial denaturation stepMalaya at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 90 s, followed by a final extension step at 72 of°C for 5 min. Each PCR assay is expected to amplify a partial fragment of the 16S rRNA gene (760 bp). b. For amplification of the gltA gene of Anaplasma spp. and Ehrlichia spp., a PCR assay was performed using primers EHR-CS136F and EHR-CS778R as previously described by Inokuma et al. (2001a) (Appendix F). The thermal cycling condition consisted of an initial denaturation step at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 90 s, followed by a final extension stepUniversity at 72 °C for 5 min. A high primer concentration (final concentration: 0.5 µM) was used for the PCR assay. The PCR assay is expected to amplify a fragment of 643 bp. c. A nested PCR assay was used to amplify a 1320 bp fragment of the groEL gene of

Anaplasma spp. and Ehrlichia spp. Primers HS1-f and HS6-r (Rar et al., 2010; Sumner et al., 1997) (Appendix F) were used for the first amplification. The amplification procedure was performed with an initial denaturation step at 94 °C for 3 min followed by

35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1.5 min. Primers HS3-f and HSVR (Liz et al., 2000; Rar et al., 2010)

(Appendix F) were used for nested amplification. Two microliters of the first PCR product was used for the nested amplification. The amplification procedure was performed with an initial denaturation step at 94 °C for 3 min followed by 35 cycles of denaturation at 94

°C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 1.5 min. For the

PCR assay, a higher primer concentration (final concentration: 0.5 µM) was used.

3.5.4.2 PCR assays targeting specific organism a. Amplification of A. phagocytophilum DNA i. The 16S rRNA gene of A. phagocytophilum was amplified by using a nested PCR assay as described by Kawahara et al. (2006). The first amplification was performed by using primers EC9 and EC12A (Appendix F). Each of theMalaya 40 amplification cycles was started with denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s and extension at 72 °C for 1 min. The PCR assay produced an approximatelyof 1462 bp amplicon. The nested amplification was performed by using primers SSAP2f and SSAP2r (Appendix F). Each of the 40 amplification cycles was started with denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. The PCR assay amplifies approximately 641 bp of the 16S rRNA gene of A. phagocytophilum. ii. Additionally, a nested PCR was also used to amplify the msp4 gene of A. phagocytophilum. The first amplification was performed by using primers MSP4AP5 and MSP4AP3University (de la Fuente et al., 2005b; Silaghi et al., 2011) (Appendix F). The PCR thermal cycling condition consisted of an initial denaturation step at 95 °C for 10 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 45 s and extension at 72

°C for 1 min, followed by a final extension step at 72 °C for 7 min. A higher primer concentration (final concentration: 0.8 µM) was used in this assay. The PCR produced an approximately 849 bp amplicon. Primers msp4f and msp4r (Bown et al., 2007; Silaghi et

al., 2011) (Appendix F) were used in the second amplification. The thermal cycling condition consisted of an initial denaturation step at 95 °C for 10 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 45 s and extension at 72 °C for 1 min, followed by a final extension step at 72 °C for 7 min. Similarly, a higher primer concentration (final concentration: 0.8 µM) was used in this assay. The PCR assay produces an approximately 343 bp amplicon. iii. A heminested PCR assay was performed to amplify a 573 bp of the groEL gene of A. phagocytophilum, using primers EphplgroEL(569)F and EphplgroEL(1193)R (Alberti et al., 2005; Reye et al., 2010) (Appendix F) for first amplification. The PCR thermal cycling condition consisted of an initial denaturation step at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 61 °C for 30 s and extension at 72 °C for 45 s, followed by a final extension step at 72 °C for 10Malaya min. A higher primer concentration (final concentration: 0.8 µM) was used in this assay. The PCR assay produced an approximately 624 bp amplicon. The heminestedof amplification was performed using primers EphplgroEL(569)F and EphgroEL(1142)R (Alberti et al., 2005; Reye et al.,

2010) (Appendix F). The PCR thermal cycling condition consisted of an initial denaturation step at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 45 s, followed by a final extension step at 72

°C for 10 min. A high primer concentration (final concentration: 0.8 µM) was used in this assay. The PCR assay produces an approximately 573 bp amplicon. b. AmplificationUniversity of A. bovis DNA The 16S rRNA gene of A. bovis was amplified by using a nested PCR assay as described by Kawahara et al. (2006). The first amplification was performed by using primers EC9 and EC12A (Appendix F). Each of the 40 amplification cycles started with denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s and extension at 72 °C for 1 min. The PCR assay produced an approximately 1462 bp amplicon. The following nested

amplification was performed by using primers AB1f and AB1r (Appendix F). Each of the

40 amplification cycles started with denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. This PCR amplifies approximately 551 bp of the 16S rRNA gene of A. bovis. c. Amplification of E. chaffeensis DNA

A nested PCR assay was used to amplify a 389 bp of the 16S rRNA gene of E. chaffeensis. Primers ECB and ECC (Anderson et al., 1992b; Dawson et al., 1994)

(Appendix F) were used for the first amplification. Each of the 35 amplification cycles was started with denaturation at 94 °C for 1 min, annealing at 45 °C for 2 min and extension at 72 °C for 1 min. The PCR assay produces an approximately 450 bp amplicon.

The following nested amplification was performed using primers HE1 and HE3 (Anderson et al., 1992b; Dawson et al., 1994) (AppendixMalaya F). Each of the 35 amplification cycles was started with denaturation at 94 °C for 1 min, followed by annealing at 55 °C for 2 min and extension at 72 °C for 1 min.of d. Amplification of E. canis DNA

The 16S rRNA gene of E. canis was amplified by using a nested PCR assay.

Primers ECB and ECC (Anderson et al., 1992b; Dawson et al., 1994) (Appendix F) were used for the first amplification. Each of the 35 amplification cycles was started with denaturation at 94 °C for 1 min, followed by annealing at 45 °C for 2 min and extension at 72 °C for 1 min. The PCR assay produced an approximately 450 bp amplicon. The nestedUniversity amplification was performed using primers ECA and HE3 (Wen et al., 1997) (Appendix F). The thermal cycling condition consisted of an initial denaturation step at

94 °C for 5 min, 40 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 1 min. The PCR assay amplifies an approximately 389 bp of the 16S rRNA gene of E. canis.

3.5.5 Detection of amplified PCR products generated in the PCR assays

After amplification, agarose gel electrophoresis was used to analyse the amplified products obtained from the PCR assays. The amplified products were electrophoresed in a 1.5 % agarose gel prestained with GelRed (Biotium, CA, USA). The electrophoresis was performed in Tris-Borate-EDTA (TBE) buffer (Vivantis, CA, USA) (Appendix G) at a constant voltage of 100 V for 50 min. 1 µl DNA ladder (100 bp or 1 kb, depending on the expected size of the amplicon) (Solis BioDyne, Estonia) was run in parallel with 5 µl

PCR products added with 1 µl of gel loading buffer. The gels were then visualised and photographed under UV light (G-Box, Syngene, UK). Positive results were indicated by the presence of a band corresponding with the expected size of each PCR assay.

3.6 Generation of positive control for PCR assaysMalaya Positive controls are essential to be included in each PCR assay to avoid false positive or false negative results. In this studyof amplified DNA fragments (Table 3.4) were cloned into pCR4-TOPO vector (Invitrogen, CA, USA). One microliter of the purified plasmids was used as positive controls in each PCR run.

Table 3.4: Details of the DNA fragments cloned as positive control.

Plasmid Label Target gene, organism Length of gene insert (bp) 0088 EHR-1 16S rRNA gene (short), A. bovis 345 bp 0088 ATT-1 16S rRNA gene, A. bovis 1500 bp University 3.6.1 Setting up cloning reaction and transformation of plasmid into competent E.

coli

Cloning was performed using TOPO TA Cloning® Kit for Sequencing (Invitrogen,

CA, USA) according to the manufacturer’s instructions. Table 3.5 shows the reagents used in the TOPO® cloning reaction. All the reagents were mixed thoroughly and

incubated for 30 min at room temperature. Transformation was performed by adding 4 µl of the TOPO® cloning reaction to a vial of One Shot® E. coli competent cells and mixed gently. The reaction was then incubated on ice for 30 min. The cells were subjected to heat-shock for 30 s at 42 °C without shaking and immediately transferred to ice after 30 s of incubation. A volume of 250 µl S.O.C. medium was then added and the culture was incubated horizontally in a shaker (200 rpm) at 37 °C for 1 hour (or until a cloudy solution was observed). The cell suspension (50 µl) was spread on a pre-warmed Luria-Bertani

(LB) agar plate containing 50 µg/ml ampicillin (Appendix G) and incubated overnight at

37 °C. Any colonies growing on the agar plate were considered positive for the cloning reaction. At least one loop full of colonies was preserved in glycerol stock for long term storage at -80 °C. Malaya Table 3.5: Reagents and the volumes required in the TOPO® cloning reaction. Reagents ofVolume (µl) Fresh PCR product 4 Salt solution 1 TOPO® vector 1 Final volume 6

3.6.2 Plasmid extraction

Five single E. coli colonies were picked from an LB-ampicillin agar plate and inoculated into a tube containing 5 ml LB-ampicillin broth (Appendix G). The inoculated brothUniversity was incubated horizontally in a shaker (150 rpm) overnight at 37 °C. Plasmid extraction was performed by using GeneAll Hybrid-Q Plasmid Rapidprep (GeneAll,

South Korea). Briefly, the bacterial culture was harvested in a 15 ml sterile centrifuge tube by centrifugation at 14,000 rpm for 5 min. The supernatant was discarded as much as possible. The bacterial pellet was then resuspended in 250 µl Buffer S1 (provided by the kit and added with RNase) and transferred to a new 1.5 ml microcentrifuge tube. A

volume of 250 µl Buffer S2 was added and mixed by inverting the microcentrifuge tube for 4 times. After the cell suspension had become viscous, 350 µl Buffer G3 was added to the microcentrifuge tube. The mixture was mixed by inverting the microcentrifuge tube for 4 to 6 times and centrifuged at 14,000 rpm for 10 min. The supernatant was then transferred to a spin column (provided by the kit) and centrifuged at 14,000 rpm for 1 min. After the pass-through had been discarded, 500 µl Buffer AW was added to the column. This was followed by a centrifugation step at 14,000 rpm for 1 min. The step was repeated with the addition of 700 µl Buffer PW. Any residual wash buffer was removed by centrifugation at 14,000 rpm for 1 min. The column was then transferred to a new 1.5 ml microcentrifuge tube. Lastly, 50 µl Buffer EB was added to the column and incubated for 1 min before centrifugation at 14,000 rpm. The eluted plasmid DNA was stored at -20 °C prior to use. Malaya 3.6.3 Analysing positive clones To ensure that the gene of interest ofwas successfully inserted into the plasmid as described in section 3.6.1, purified plasmid was used as DNA template for PCR assays targeting the corresponding gene by using respective forward and reverse PCR primers

(sections 3.5.3 and 3.5.4).

3.7 Sequence determination and analysis of selected amplified products

Sequence determination of amplified products was performed to confirm the specificityUniversity of each PCR assay and to identify the organisms amplified from PCR assays. Only selected amplified products (representing different sample types) were subjected to sequence determination.

3.7.1 Purification of PCR products

Amplified PCR products were purified using a GeneAll Expin™ Combo GP kit

(GeneAll, South Korea) prior to sequencing. For PCR product with a single band on

agarose gel, 5 volumes of Buffer PB was added to 1 volume of the PCR product. The mixture was then transferred to a SV column (provided by the kit) prior to centrifugation at 13,000 rpm for 1 min. The pass-through was discarded and the SV column was reinserted back into the same collection tube. A volume of 700 µl Buffer NW was added to the SV column and centrifuged at 13,000 rpm for 1 min. The pass-through was discarded and the SV column was reinserted back into the same collection tube. The SV column was centrifuged for an additional 1 min at 13,000 rpm to remove any residual wash buffer. The SV column was then transferred to a sterile 1.5 ml microcentrifuge tube.

Lastly, 30 µl Buffer EB was added to the column and incubated at room temperature for

1 min prior to centrifugation at 13,000 rpm for 1 min. The eluted purified PCR product was then subjected to sequence determination. For PCR product generating multiple bands onMalaya the agarose gel, the band of interest was excised using a sterile surgical blade on a UV transilluminator (Vilber Lourmat, Germany). The gel slice was weighed in a of1.5 ml microcentrifuge tube and added with 3 volumes of Buffer GB. The agarose gel was incubated at 50 ºC until it was completely melted (required 5-10 min depends on the size of the gel). The mixture was transferred to a SV column (provided by the kit) and centrifuged at 13,000 rpm for 1 min. The pass- through was discarded and the SV column was reinserted back into the same collection tube. A volume of 700 µl Buffer NW was then added to the SV column and centrifuged at 13,000 rpm for 1 min. The pass-through was discarded and the SV column was reinsertedUniversity back into the same collection tube. The SV column was centrifuged for an additional 1 min at 13,000 rpm to remove any residual wash buffer. Lastly, the SV column was added with 30 µl Buffer EB and incubated at room temperature for 1 min prior to centrifugation at 13,000 rpm for 1 min. The purified PCR product was subjected to sequence determination as described above, using primers M13 Forward (-20) and M13

Reverse, provided by the TOPO TA Cloning® Kit for Sequencing (Invitrogen, CA, USA).

3.7.2 Sequence determination and analysis

Sequence determination of amplified products was performed by a service provider (First BASE Laboratories, Malaysia) using a Big Dye Terminator version 3.1

Cycle Sequencing kit (Applied Biosystems, CA, USA) on an ABI PRISM 377 Genetic

Analyzer (Applied Biosystems, CA, USA). Both forward and reverse PCR primers were used as the primers for sequencing. To determine the sequence of the full length 16S rRNA gene of Anaplasma spp. and Ehrlichia spp., primer pairs ATT062F/ATT062R,

ATT066F (5’- CCC TGG TAG TCC ACG CTG -3’) and ATT067R (5’- CAG CGT GGA

CTA CCA GGG -3’) (Pinyoowong et al., 2008) were used. For amplicons derived from nested PCR assays, the primers used in the nested amplification were used for sequencing.

The obtained sequences were aligned with BioEdit Sequence Alignment Editor Software (version 7.0.5.3) or Geneious version R6.1Malaya (Biomatters, New Zealand). The quality of the sequences were checked manually. Sequences with noisy data or ambiguous nucleotide positions were discarded. The samplesof were either re-sequenced or cloned into a plasmid to generate better sequence data. All the aligned sequences were compared for similarity with sequences deposited in the GenBank database using the Basic Local

Alignment Search Tool (BLAST) program (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

(National Center for Biotechnology Information, United States National Library of

Medicine, MD, USA). The phylogenetic status of the anaplasma and ehrlichiae identified in this study were analysed using the neighbour-joining method of the MEGA (Molecular EvolutionaryUniversity Genetics Analysis) 6.0 software (Tamura et al., 2013). 3.7.3 Sequence with ambiguous nucleotide positions

Amplified fragments with low sequence quality, for instance, noisy data or with ambiguous nucleotides were cloned into pCR4-TOPO vector (Invitrogen, CA, USA) according to sections 3.6.1-3.6.3. Plasmids with the amplified PCR products were

subjected to sequence determination, using primers M13 Forward (-20) and M13 Reverse, provided by the TOPO TA Cloning® Kit for Sequencing (Invitrogen, CA, USA).

3.8 Serological detection of selective tick-borne pathogens from dog samples

3.8.1 SNAP 4Dx® kit

A total of 43 dog blood samples (30 blood samples from Klang Valley while 13 blood samples from VRI, Ipoh) were tested for serological response against A. phagocytophilum, E. canis, A. platys and B. burgdorferi using SNAP 4Dx® test kits

(IDEXX Laboratories, ME, USA) in accordance with the manufacturer’s protocol (the kit also includes testing against Dirofilaria immitis, which is a mosquito-borne organism).

Briefly, three drops of each blood sample were added to the provided sample tube together with four drops of conjugate. The mixture was mixedMalaya thoroughly by inverting 3 to 5 times before pipetting into a sample well of a SNAP device. The test result was read after 8 min. A positive reaction was indicated by the appearanceof of color in the activation circle. The result of the test is valid only when the positive control develops colour.

University

CHAPTER 4: RESULTS

4.1 Collection and identification of tick species using morphological

identification method

4.1.1 Livestock farms

During the sampling of animal ticks from eight livestock farms (six cattle, one goat and one sheep farms) located at different regions of Peninsular Malaysia (Table 3.1) from February to September 2013, a total of 226 ticks were collected from four cattle farms and 44 ticks were collected from a sheep farm (Table 4.1). Based on morphological identification, 70 of the cattle ticks were identified as R. microplus while 200 ticks (156 ticks collected from cattle and 44 ticks collected from sheep) were identified as

Haemaphysalis bispinosa (Figure 4.1), as reported by Kho et al. (2015a). The identification of Rhipicephalus (BoophilusMalaya) were based on the previous report by Brahma et al. (2014). The ticks were identified based on small palpal segments, hexagonal basis capitulum, presence of indistinctof eyes, coxa I with paired spur and coxa II to IV without spurs. Festoons were absent in the male Rhipicephalus (Boophilus) ticks.

The caudal appendage in a male tick is the most visible feature used to differentiate R. microplus from R. annulatus (Brahma et al., 2014).

Haemaphysalis ticks were identified based on the presence of small palpal segments, rectangular basis capitulum (straight lateral margins), absence of eyes and presence of festoons (Brahma et al., 2014; Geevarghese and Mishra, 2011). HaemaphysalisUniversity ticks are also characterised by short mouthparts with the anal groove forming a loop posterior to the anus (Walker et al., 2003). Male ticks were also characterised by the absence of ventral plates (Brahma et al., 2014).

The identity for some of the ticks were confirmed using molecular identification method, as described in section 4.2.

Table 4.1: Total number of tick samples collected from different sources in this study.

Location Animal hosts (n) Tick species (n) Livestock farms  Jelai Gemas, Cattle (27) H. bispinosa (41), R. microplus (6) Negeri Sembilan  Jerantut, Pahang Cattle (38) R. microplus (14)  Ulu Lepar, Pahang Cattle (40) H. bispinosa (57), R. microplus (37)  Pokok Sena, Kedah Sheep (40) H. bispinosa (44)  Tanah Merah, Kelantan Cattle (40) 0  Kuala Berang, Cattle (40) H. bispinosa (58), R. microplus (13) Terengganu  Kuala Pah, Goat (40) 0 Negeri Sembilan  Air Hitam, Johore Cattle (40) 0 Total 270 (200 H. bispinosa and 70 R. microplus) Forest areas (PERHILITAN)  Sg. Deka, Terengganu Vegetation areas Dermacentor spp. (29), Haemaphysalis spp. (9), Amblyomma spp. (3)  Kuala Lompat, Pahang Vegetation areas Dermacentor spp. (48), Haemaphysalis spp. (26), Amblyomma spp. (2) Rat (10), bat (1), Haemaphysalis spp. (15), Dermacentor squirrel (1), skink spp. (6), Amblyomma sp. (1) (1) Total 139 (83 Dermacentor spp., 50 HaemaphysalisMalaya spp. and 6 Amblyomma spp.) Aboriginal villages  Negeri Sembilan Dog (2), chickenof R. sanguineus (3 individuals), (1), goat (3) Haemaphysalis spp. (4 pools)  Pahang Dog (1), cat (1), Haemaphysalis spp. (7 pools) chicken (3)  Johore Snake (1), dog (1), Amblyomma spp. (1 pool), Haemaphysalis chicken (2), cat (6) spp. (1 individual and 8 pools)  Kedah Cow (2) R. microplus (3 individuals)  Perak Dog (9), chicken R. sanguineus (1 individual), Dermacentor (3), cat (6) spp. (2 individuals), Haemaphysalis spp. (13 individuals and 11 pools)  Kelantan Cat (3), chicken Haemaphysalis spp. (10 individuals and 1 (3) pool) Total 33 individuals and 32 pools Others  Sepang, Selangor Snake (7) A. varanense (11), A. helvolum (10)  UniversityKlang Valley Dog (13) R. sanguineus (33)

Figure 4.1: Ticks collected from livestock farms in this study. A: Sheep tick, Haemaphysalis sp.; B: Cattle tick, R. microplus; C: Cattle tick, Haemaphysalis sp. Malaya

4.1.2 Forest areas

A total of 117 questing ticks were ofcollected from vegetation from two sampling sites at Kuala Lompat, Krau Wildlife Reserve (N 03º50’ E 102º06’), Pahang and a forest at Sungai Deka Elephant Sanctuary, (N 05º28’ E 102º44’), Terengganu. Based on morphological identification, five, 35 and 77 ticks collected from vegetation (Table 4.1) were identified as Amblyomma spp., Haemaphysalis spp. and Dermacentor spp., respectively (Figure 4.2). Additionally, 22 ticks were collected from one reptile (Eutropis multifasciata) and 12 small mammals (one Lariscus insignis, one Rhinolophus lepidus, oneUniversity Leopoldamys sabanus and nine Maxomys rajah) from a field trip in a forest area

(Kuala Lompat, Pahang) (Table 4.1), through a collaborative effort with Department of

Wildlife Peninsular Malaysia (PERHILITAN), Malaysia. The tick identified from the reptile was identified as Amblyomma sp., while six and 15 ticks collected from small mammals were morphologically identified as Dermacentor spp. and Haemaphysalis spp., respectively.

The Dermacentor ticks were identified based on the presence of basis capitulum which is rectangular dorsally, coxa I have large and equal paired spurs, mouthpart is anterior and anal groove is posterior to the anus (Walker et al., 2003). For male

Dermacentor ticks, festoons are present, coxa IV is enlarged and ventral plates are absence (Geevarghese and Mishra, 2011).

The Amblyomma ticks were characterised by the long and anterior mouthparts, basis capitulum have straight lateral margins, palp articles 2 are longer than article 1 and

3, coxa 1 have unequal paired spurs while coxa 4 are of normal size, the presence of festoons and anal groove is posterior to the anus (Walker et al., 2003). The ventral plates are absent in the male ticks (Geevarghese and Mishra, 2011). Malaya of

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Figure 4.2: Ticks collected from the forest areas in this study. A: Questing ticks (L: Female, R: Male), Dermacentor spp.; B: Questing tick, Amblyomma sp.; C: Questing tick, Haemaphysalis sp. (The female tick was characterised by the small scutum size as compared to the male tick where the scutum covers the entire dorsum of the male idiosoma).

4.1.3 Aboriginal villages

A total of 192 ticks were collected from 12 aboriginal villages located in six states in Peninsular Malaysia (Kho et al., 2017). The ticks were identified based on morphology to genus level and segregated into 65 pools (one to ten individuals of the similar species)

(Table 4.1). As the size of most of the ticks were small, the ticks collected from the same location, genus and animal host were pooled. A total of 55 pools of Haemaphysalis ticks were collected from three goats, 11 dogs, 12 chickens and 16 cats. One pool of

Amblyomma ticks was collected from a snake. Three pools of R. microplus ticks were collected from two cows while four pools of Rhipicephalus ticks were collected from three dogs. Two pools of ticks collected from a dog were not able to be identified morphologically because the ticks were engorged with blood. Hence, they were identified using molecular method (Figure 4.3). Malaya

University

Malaya of

Figure 4.3: Ticks collected from aboriginal villages in this study. A: Chicken tick, Haemaphysalis sp.; B: Dog tick, Rhipicephalus sp.; C: Goat tick, Haemaphysalis sp.; D: Cat tick, Haemaphysalis sp.; E: Dog ticks (L: Male, R: Female), Haemaphysalis spp. (The female tick was characterised by the small scutum size as compared to the male tick where the scutum covers the entire dorsum of the male idiosoma). University

4.1.4 Other samples

Of the 21 ticks collected from seven P. molurus snakes from Sepang, 11 and ten ticks were morphologically identified as A. varanense and A. helvolum, respectively

(Figure 4.4) according to Chao et al. (2013) and Burridge (2001), as reported by Kho et al. (2015b).

All the 33 ticks collected from 13 strayed dogs in Klang Valley were morphologically identified as Rhipicephalus spp. (Figure 4.5).

Figure 4.4: Snake ticks collected from Sepang, Selangor. A: A. helvolum; B: A. varanense Malaya of

Figure 4.5: Rhipicephalus sp. collected from a dog in Klang Valley.

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4.2 Identification of tick species using molecular identification methods

A total of 117 ticks collected from cattle (n=25), sheep (n=5), vegetation (n=49), and other animals (n=38) were subjected to sequence analysis of the 16S rRNA gene region in this study. Table 4.2 shows the results of BLAST analyses of the 16S rRNA

gene sequences obtained from 117 ticks collected from different sources and locations in this study. Of the 73 sequences demonstrating 99-100% similarity with the sequences available in the GenBank database, H. bispinosa (n=25), Haemaphysalis wellingtoni (n=

13), H. shimoga (n=2), Haemaphysalis hystricis (n=4), Dermacentor atrosignatus (n=10),

D. auratus (n=2), R. microplus (n=12), R. sanguineus (n=3) and A. helvolum (n=2) were identified. Additionally, four sequence types (STs) of Dermacentor ticks (n=25), four sequence types (STs) of Haemaphysalis ticks (n=17) and one sequence type (ST) of

Amblyomma ticks (n=2) which did not have any full match (< 99%) with sequences deposited in the GenBank database were identified only up to the genus level.

4.2.1 Livestock farms

The 16S rDNA sequences obtained from 12 representative cattle ticks demonstrated 100% (186 nt/186 nt) similarity to that Malayaof R. microplus collected from cattle in a previous study conducted in this laboratory (isolate A1-A4, GenBank accession nos.: KM246878-KM246882) (Low et al., of 2015). The sequences obtained from 18 representative Haemaphysalis ticks (from 13 cattle and five sheep) demonstrated 100%

(193 nt/193 nt) similarity to several H. bispinosa strains (vouchers TUTEZ-G135,

TUTEZ-R1324 and TUTEZ-R1320; GenBank accession nos.: KC853418-KC853420)

(Brahma et al., 2014) collected from goat and cattle in India. The details of the BLAST results are shown in Table 4.2. University

Table 4.2: Results of BLAST analyses of the 16S rRNA gene sequences obtained from 117 ticks collected from different sources in this study.

Location Animal Tick species (n) BLAST result (nearest match) Sequence type host (morphological (ST) identification) Livestock farms  Jelai Gemas, Cattle Haemaphysalis spp. (2) H. bispinosa voucher TUTEZ-G135 [KC853420; 193 nt/193 nt (100% H. bispinosa Negeri Sembilan identity); goat, India]  Ulu Lepar, Pahang Haemaphysalis spp. (7)  Pokok Sena, Kedah Sheep Haemaphysalis spp. (5)  Kuala Berang, Cattle Haemaphysalis spp. (4) Malaya Terengganu  Jerantut, Pahang R. microplus (5) R. microplus isolate A5 [KM246882; 186 nt/186 nt (100% identity); R. microplus  Ulu Lepar, Pahang R. microplus (5) cattle, Malaysia) of  Kuala Berang, R. microplus (2) R. microplus isolate A5 [KM246882; 186 nt/186 nt (100% identity); Terengganu cattle, Malaysia) Forest areas  Kuala Lompat, Pahang Bat Haemaphysalis sp. (1) H. bispinosa voucher TUTEZ-R1324 [KC853419; 233 nt/233 nt (100% H. bispinosa identity); cattle, India] Rat Haemaphysalis sp. (1) H. bispinosa voucher TUTEZ-R1324 [KC853419; 230 nt/231 nt (99% identity); cattle, India] Vegetation Haemaphysalis spp. (2) H. hystricis [KC170733; 237 nt/239 nt (99% identity); vegetation, H. hystricis Thailand]  Sg. Deka, Terengganu Haemaphysalis sp. (1) H. shimoga [KC170730; 239 nt/239 nt (100% identity); vegetation, H. shimoga Thailand] Haemaphysalis sp. (1) H. shimoga [KC170730; 238 nt/239 nt (99% identity); vegetation, Thailand]  Kuala Lompat, Pahang Vegetation Haemaphysalis spp. (5) H. obesa [KC170732; 223 nt /236 nt (94% identity); vegetation, Haemaphysalis Rat HaemaphysalisUniversity sp. (1) Thailand] ST1

Table 4.2, continued  Kuala Lompat, Pahang Vegetation Haemaphysalis spp. (3) H. obesa [KC170732; 222 nt /236 nt (94% identity); vegetation, Haemaphysalis Thailand] ST1 Haemaphysalis sp. (1) H. obesa [KC170732; 222 nt /237 nt (94% identity); vegetation, Thailand] Squirrel Haemaphysalis sp. (1) H. asiatica [KC170734; 223 nt/239 nt (93% identity); vegetation, Haemaphysalis Thailand] ST3  Sg. Deka, Terengganu Vegetation Haemaphysalis sp. (1)  Kuala Lompat, Pahang Haemaphysalis sp. (1) H. qinghaiensis clone GHQ1 [KJ609201; 220 nt/236 nt (93% identity); Haemaphysalis China] ST4 Dermacentor spp. (2) D. atrosignatus [KC170745; 239 nt /239 nt (100% identity); wild boar, D. atrosignatus Rat Dermacentor spp. (2) Thailand] Malaya  Sg. Deka, Terengganu Vegetation Dermacentor sp. (1)  Kuala Lompat, Pahang Dermacentor spp. (2) D. atrosignatus [KC170745; 238 nt /239 nt (99% identity); wild boar, Thailand] of Rat Dermacentor sp. (1) D. atrosignatus [KC170745; 231 nt /232 nt (99% identity); wild boar, Thailand] Vegetation Dermacentor sp. (2) D. atrosignatus [KC170745; 230 nt /231 nt (99% identity); wild boar, Thailand] Dermacentor spp. (11) D. andersoni haplotype SSCP Van_269 [AF309032; 218 nt /233 nt Dermacentor (94% identity); vegetation, Canada] ST1 Dermacentor spp. (6) D. andersoni haplotype SSCP Van_269 [AF309032; 221 nt /234 nt Dermacentor (94% identity); vegetation, Canada] ST2  Sg. Deka, Terengganu Dermacentor sp. (1)  Kuala Lompat, Pahang Dermacentor sp. (1) D. andersoni haplotype SSCP Van_269 [AF309032; 220 nt /234 nt (94% identity); vegetation, Canada]  Sg. Deka, Terengganu Dermacentor spp. (3)  Kuala Lompat, Pahang Dermacentor sp. (1) D. andersoni haplotype SSCP Van_269 [AF309032; 220 nt /235 nt (94% identity); vegetation, Canada] Dermacentor sp. (1) D. nuttalli isolate XJ088 [JX051114; 222 nt/239 nt (93% identity); Dermacentor UniversityChina] ST3

Table 4.2, continued  Kuala Lompat, Pahang Vegetation Dermacentor sp. (1) - D. nuttalli [KT764942; 210 nt /227 nt (93% identity); China] Dermacentor - D. silvarum [KP258209; 210 nt /227 nt (93% identity); China] ST4 Skink Amblyomma sp. (1) A. helvolum [KC170738; 239 nt/239 nt (100% identity); snake, A. helvolum Thailand] Vegetation Amblyomma spp. (2) A. testudinarium [KC170737; 232 nt/239 nt (97% identity); deer, Amblyomma Thailand ST1 Aboriginal villages  Negeri Sembilan Chicken Haemaphysalis sp. (1) H. bispinosa voucher TUTEZ-R1324 [KC853419; 248 nt/248 nt (100% H. bispinosa Goat Haemaphysalis sp. (1) identity); cattle, India]  Perak Dog Haemaphysalis spp. (2) Malaya  Negeri Sembilan Goat Haemaphysalis sp. (1) H. bispinosa voucher TUTEZ-R1324 [KC853419; 182 nt/182 nt (100% identity); cattle, India]  Perak Dog Haemaphysalis sp. (1) H. hystricis [KC170733; 232 nt/233 nt (99% identity); vegetation, H. hystricis Cat Haemaphysalis sp. (1) Thailand] of Chicken Haemaphysalis spp. (3) H. wellingtoni isolate: HW-A [AB819221; 228 nt/228 nt (100% H. wellingtoni Cat Haemaphysalis spp. (2) identity); grey bunting, Japan]  Pahang Chicken Haemaphysalis spp. (4) Cat Haemaphysalis spp. (2) Dog Haemaphysalis sp. (1)  Negeri Sembilan Goat Haemaphysalis sp. (1) H. wellingtoni isolate: HW-A [AB819221; 288 nt/289 nt (99% identity); grey bunting, Japan]  Perak Dog Haemaphysalis spp. (4) H. hystricis [KC170733; 234 nt/242 nt (97% identity); vegetation, Haemaphysalis Thailand] ST2 Dermacentor spp. (2) D. auratus [KC170746; 288 nt/289 nt (99% identity); wild boar, D. auratus Thailand]  Negeri Sembilan Rhipicephalus spp. (3) R. sanguineus [KC170744; 255 nt/255 nt (100% identity); dog, R. sanguineus UniversityThailand]  Johore Snake Amblyomma sp. (1) A. helvolum [KC170738; 235 nt/235 nt (100% identity); snake, A. helvolum Thailand]

4.2.2 Ticks collected from the forest areas

4.2.2.1 Vegetation i. Dermacentor spp.

Five sequence types of mitochondrial 16S rRNA amplified gene fragments were obtained from 32 representative Dermacentor ticks collected from vegetation. One sequence type (from seven ticks) demonstrated 99-100% sequence similarity to D. atrosignatus from a wild boar in Thailand (GenBank accession no.: KC170745, unpublished). Hence, these ticks were referred as D. atrosignatus in this study. The amplified 16S rRNA gene fragments from 23 ticks (Dermacentor ST1 and ST2) exhibited the highest 94% sequence similarity with D. andersoni from vegetation in Canada

(haplotype SSCP Van_269; GenBank accession no.: AF309032) (Qiu et al., 2002). One sequence type (Dermacentor ST3) obtained from a singleMalaya tick demonstrated the highest 93% sequence similarity with D. nuttalli from China (isolate XJ088; GenBank accession no.: JX051114) (Lv et al., 2014b). Additionally,of the 16S rDNA sequence of a single tick showed the highest 93% sequence similarity with D. nuttalli (GenBank accession no.:

KT764942, unpublished) and D. silvarum from China (GenBank accession no.:

KP258209, unpublished) (Dermacentor ST4). Due to the low sequence similarity with known deposited tick sequences, these ticks (Dermacentor ST1-ST4) were referred as

Dermacentor spp. throughout the study. The details of the BLAST results are shown in

Table 4.2. ii. UniversityHaemaphysalis spp. The mitochondrial 16S rRNA gene fragments amplified from two ticks demonstrated 99% sequence similarity with H. hystricis from vegetation in Thailand

(GenBank accession no.: KC170733, unpublished). Two ticks revealed 99-100% similarity with H. shimoga from vegetation in Thailand (GenBank accession no.:

KC170730, unpublished). The mitochondrial 16S rRNA gene amplified from nine

representative ticks from the vegetation showed the highest 94% sequence similarity to

Haemaphysalis obesa collected from vegetation in Thailand (GenBank accession no.:

KC170732, unpublished) (Haemaphysalis ST1). The mitochondrial 16S rDNA sequence of a single tick demonstrated the highest 93% sequence similarity with Haemaphysalis qinghaiensis from China (GenBank accession no.: KJ609201, unpublished)

(Haemaphysalis ST4). The mitochondrial 16S rDNA sequence of another tick showed the highest 93% sequence similarity to Haemaphysalis asiatica from vegetation in

Thailand (GenBank accession no.: KC170734, unpublished) (Haemaphysalis ST3). Due to low sequence similarity with known tick species, these ticks (Haemaphysalis ST1, ST3 and ST4) are referred as Haemaphysalis spp. The details of the BLAST results are shown in Table 4.2. iii. Amblyomma spp. Malaya This study identified two ticks with mitochondrial 16S rDNA sequences demonstrating the highest 97% sequence similarityof with those of A. testudinarium from deer in Thailand (GenBank accession no.: KC170737, unpublished). The details of the

BLAST results are shown in Table 4.2.

4.2.2.2 Small animals i. Dermacentor spp.

The mitochondrial 16S rRNA gene sequences amplified from three ticks infesting rats demonstrated 99-100% sequence similarity to D. atrosignatus from wild boar in ThailandUniversity (GenBank accession no.: KC170745, unpublished). The details of the BLAST results are shown in Table 4.2. ii. Haemaphysalis spp.

The mitochondrial 16S rDNA sequences of a bat tick and a rat tick showed 99-

100% sequence similarity to H. bispinosa collected from goats in India (voucher TUTEZ-

R1324; GenBank accession no.: KC853419) (Brahma et al., 2014). The mitochondrial

16S rDNA sequence of a rat tick demonstrated the highest 94% sequence similarity to H. obesa from vegetation in Thailand (GenBank accession no.: KC170732, unpublished)

(Haemaphysalis ST1). A tick collected from squirrel demonstrated the highest 93% sequence similarity to H. asiatica from vegetation in Thailand (GenBank accession no.:

KC170734, unpublished) (Haemaphysalis ST3). Due to the low sequence similarity to known tick species, these ticks (Haemaphysalis ST1 and ST3) were referred as

Haemaphysalis spp. The details of the BLAST results are shown in Table 4.2. iii. Amblyomma spp.

The mitochondrial 16S rDNA sequence of a tick collected from a skink from

Kuala Lompat showed 100% sequence similarity with that of A. helvolum from snake in

Thailand (GenBank accession no.: KC170738, unpublished). The detail of the BLAST result is shown in Table 4.2. Malaya 4.2.3 Ticks collected from peri-domestic animals in aboriginal villages The 28S rRNA gene amplified fromof six pools of chicken ticks and eight pools of cat ticks was not discriminative as the sequences demonstrated the highest 99% (396 nt/399 nt) sequences similarity with several Haemaphysalis spp., including H. flava, H. hystricis, Haemaphysalis sulcata, Haemaphysalis formosensis and Haemaphysalis leporispalustris from Australia (GenBank accession nos.: JX573128-JX573130,

JX573132, JX573134) (Burger et al., 2013).

Hence, most of the time, ticks were identified based on sequence analyses of the mitochondrialUniversity 16S rDNA sequences. The mitochondrial 16S rDNA sequences of seven pools of chicken ticks, four pools of cat ticks, one pool of dog ticks and one pool of goat ticks exhibited 99-100% sequences similarity to H. wellingtoni collected from grey bunting in Japan (isolate HWA; GenBank accession no.: AB819221) (Takano et al.,

2014). The mitochondrial 16S rDNA sequences obtained from another pool of chicken ticks, one pool of goat ticks and two pools of dog ticks were 100% identical to H.

bispinosa collected from goats in India (voucher TUTEZ-R1324; GenBank accession no.:

KC853419) (Brahma et al., 2014). While the mitochondrial 16S rRNA gene amplified from a pool of cat ticks demonstrated 99% sequence similarity to H. hystricis collected from vegetation in Thailand (GenBank accession no.: KC170733, unpublished).

Of the 13 representative mitochondrial 16S rDNA sequences obtained from 13 pools of dog ticks, five demonstrated the highest (97-99%) sequences similarity to H. hystricis collected from vegetation in Thailand (GenBank accession no.: KC170733, unpublished) (Haemaphysalis ST2). The mitochondrial 16S rDNA sequences of two pools of dog ticks showed 99% similarity to D. auratus collected from a wild boar in

Thailand (GenBank accession no.: KC170746, unpublished), while the sequences obtained from two pools of dog ticks demonstrated 100% similarity to H. bispinosa collected from goats in India (voucher TUTEZ-R1324;Malaya GenBank accession no.: KC853419) (Brahma et al., 2014). Another pool of ticks demonstrated 100% identity in the 16S rDNA sequence to H. wellingtoni ofcollected from grey bunting in Japan (isolate HW-A; GenBank accession no.: AB819221) (Takano et al., 2014). Three pools of ticks had 100% matching sequences with that of R. sanguineus collected from a dog in

Thailand (GenBank accession no.: KC170744, unpublished). In addition, the mitochondrial 16S rDNA sequence of a pool of snake ticks was 100% identical to A. helvolum collected from a snake in Thailand (GenBank accession no.: KC170738, unpublished). The details of the BLAST results are shown in Table 4.2. 4.2.4University Ticks collected from other sources The 28S rRNA gene sequences amplified from 23 dog ticks collected from dogs in Klang Valley were 100% (386 nt/386 nt) matching with R. sanguineus from USA

(GenBank accession no.: AF120312) (Klompen et al., 2000) while four dog ticks were only 99% (392 nt/393 nt) matching with R. sanguineus from USA (GenBank accession no.: AF120312) (Klompen et al., 2000). Besides that, the 28S rRNA gene amplified from

a dog tick collected from Klang Valley demonstrated 100% (400 nt/400 nt) sequence similarity with several Haemaphysalis spp., including H. flava, H. formosensis, H. hystricis, H. leporispalustris and H. sulcata from Australia (GenBank accession nos.:

JX573128-JX573130, JX573132, JX573134) (Burger et al., 2013).

4.3 Integrity checking of extracted samples prior to amplification for

Anaplasmataceae DNA i. Tick DNA samples

The integrity of the tick DNA samples used in this study were assessed using PCR assays targeting the tick mitochondrial 16S rRNA gene fragment or 28S rRNA gene fragment (refer to section 4.2). The amplification of a ~300 bp of tick 16S rRNA gene fragment (Figure 4.6) or a ~490 bp of tick 28S rRNA gene fragment (Figure 4.7) was suggestive of the absence of PCR inhibitors in a tickMalaya DNA sample. ii. Animal blood DNA samples The integrity of the DNA extractedof from animal blood and organ samples were assessed using PCR assay targeting cytB gene. The amplification of a 358 bp amplicon of cytB gene (Figure 4.8) was suggestive of the absence of PCR inhibitors.

The cytB sequences were also determined for each animal species. Table 4.3 shows the results of the sequence analysis. The cytB sequences for all animals matched

99-100% with their reference strains except for deer (94%) and sheep (90%). This may be due to the sequences for these animals are still not available in the GenBank database. University

Figure 4.6: Amplification of approximately 300 bp of the 16S rRNA gene fragments from tick samples using 16S-1/16S+2 primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-9: tick DNA samples.

Malaya of

Figure 4.7: Amplification of approximately 490 bp of the 28S rRNA gene fragments from tick samples using 28SF/28SR primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-7: tick DNA samples.

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Figure 4.8: Amplification of approximately 358 bp of the cytB gene fragments from animal blood samples using cytBFor/cytBRev primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-8: animal blood DNA samples.

Table 4.3: Sequence analyses of the cytB sequences from one sample of each animal type.

Animal Animal with the closest sequence match, Percentage similarity blood country (Genbank accession no.) (Nucleotide of the sample sample/nucleotide of reference strain) Monkey Macaca fascicularis, Malaysia (KJ592589) 99% (261 nt/262 nt) Buffalo Bubalus bubalis, Japan (AB529514) 100% (273 nt/273 nt) Cow Bos indicus, Malaysia (GU367347) 100% (258 nt/258 nt) Deer Cervus unicolour, India (EU882027) 94% (238 nt/253 nt) Horse Equus caballus, USA (AY819737) 100% (227 nt/227 nt) Dog Canis lupus familiaris, Kenya (KP858493) 100% (244 nt/244 nt) Rat Rattus rattus, Malaysia (JF437010) 100% (271 nt/271 nt) Goat Capra hircus, Switzerland (DQ514548) 100% (249 nt/249 nt) Pangolin Manis javanica, South-East Asia (KP261030) 100% (247 nt/247 nt) Sheep Ovis aries, Japan (AB006800) 90% (292 nt/323 nt)

4.4 Seroprevalence of selective tick-borne pathogens in dog blood samples

A total of 17 (39.5%) of the 43 dog blood samples, of which 14 (46.7%) from Klang Valley and three (23.1%) obtained from VRI, MalayaIpoh, were serologically positive for E. canis. A. phagocytophilum antibodies were only detected from four (9.3%) dogs from Klang Valley. Antibodies to both A. phagocytophilumof and E. canis were detected in four (9.3%) dogs from Klang Valley. None of the dogs were seropositive to B. burgdorferi and D. immitis.

4.5 Identification of Anaplasma spp. and Ehrlichia spp. from ticks using

molecular methods

4.5.1 Preliminary screening UniversityTick samples were subjected to preliminary screening by using a PCR assay targeting a short fragment (345 bp) of the 16S rRNA gene of Anaplasmataceae (Figure

4.9). A high detection rate (61.5%) was obtained from ticks collected from livestock farms, whereby 166 ticks out of 270 ticks (including 103 H. bispinosa from cattle; 22 H. bispinosa from sheep and 41 R. microplus from cattle) collected from five farms (four cattle and one sheep) were positive.

A total of 40 (28.8%) of 139 ticks (including 18 Dermacentor spp., two D. atrosignatus, nine Haemaphysalis spp., two H. hystricis, one H. shimoga and two

Amblyomma spp. from vegetation; two Dermacentor spp., one D. atrosignatus and three

Haemaphysalis spp. from small animals) from the forest areas were positive for

Anaplasmataceae DNA.

Of the ticks collected from 12 aboriginal villages, 37.0% (24 pools/65 pools) of the ticks were positive for Anaplasmataceae DNA. Positive ticks were derived from 13 pools of Haemaphysalis spp. from cats, chickens and dogs; five pools of H. wellingtoni from chickens, cat and goat; three pools of H. bispinosa from goats and chicken; two pools of R. sanguineus from dogs and one pool of R. microplus from a cow. Of the 21 ticks collected from snakes in Sepang, Selangor, 15 snake ticks (nine A. varanense and six A. helvolum) were positive for AnaplasmataceaeMalaya DNA. BLAST analyses were performed on 53 sequences successfully obtained from 19 H. bispinosa (18 individuals and one pool),of nine Haemaphysalis spp. (eight individuals and one pool), one H. hystricis, five Dermacentor spp., two D. atrosignatus, six A. varanense, three A. helvolum, seven R. microplus and one pool of R. sanguineus. BLAST analyses reveal the presence of A. marginale, A. bovis (two sequence variants), A. platys,

A. phagocytophilum (two sequence variants), Anaplasma spp. (two sequence variants) and Ehrlichia spp. (three sequence variants). The results of the BLAST analyses are shown in Table 4.4. UniversityFifteen representative sequences were selected to build a dendrogram (Figure 4.10). Phylogenetic analysis reveals the clustering of A. platys detected from ticks in this study with A. platys detected in dog from a previous study in Malaysia (strain Sarawak

41 UPM; GenBank accession no.: KU500914; unpublished) and Thailand (GenBank accession no.: EF139459) (Pinyoowong et al., 2008). The A. phagocytophilum identified in this study was placed in the same group with A. phagocytophilum detected in a mouse’s

spleen from Korea (strain AAIK4; GenBank accession no.: KR611719; unpublished) and

D. silvarum from China (strain Jilin 5; GenBank accession no.: DQ449948) (Cao et al.,

2006).

Figure 4.9: Amplification of approximately 345 bp of the 16S rRNA gene fragments from samples using EHR16SD/EHR16SR primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-7: EHRPCR-positive samples; 8: positive control (cloned plasmid contain A. bovis). Malaya

Phylogenetic analysis also reveal theof presence of two clusters of A. bovis, of which one is grouped with H. longicornis from Japan (strain NR07; GenBank accession no.:

AB196475) (Kawahara et al., 2006) and A. bovis detected in a wild boar from Malaysia

(this study, section 4.6.2.1) while another cluster is grouped with A. bovis detected in a raccoon from Japan (strain raccoon499; GenBank accession no.: GU937020) (Sashika et al., 2011) and A. bovis detected in monkeys from Malaysia (this study, section 4.6.2.1).

The short sequences of A. marginale detected in cattle ticks are clustered in one group withUniversity A. marginale from Philippines (isolate C6A; GenBank accession no.: JQ839012) (Ybañez et al., 2013b), A. centrale strain vaccine (GenBank accession no.: AF318944)

(Bekker et al., 2002) and A. ovis from South Africa (isolate OVI; GenBank accession no.:

AF414870) (Lew et al., 2003). The Anaplasma sp. detected from a snake tick is grouped with an Anaplasma sp. most closely related to A. phagocytophilum, which has been reported from a dog from South Africa (isolate 1076; GenBank accession no.:

AY570539) (Inokuma et al., 2005). 80

Table 4.4: BLAST analyses of 53 short 16S rRNA gene sequences amplified from ticks from different sources in this study.

Location Animal Tick species (n) BLAST result (nearest match) Representative samples for host construction of dendrogram Livestock farms  Jelai Gemas, Negeri Cattle H. bispinosa (6) A. marginale strain Uganda MT27 [KU686794; 234  X102-1 (KY046278) Sembilan nt/234 nt (100% identity); cattle, Uganda]  EKY4766-F (KY046277)  Jerantut, Pahang R. microplus (1)  Kuala Berang, R. microplus (1) Terengganu  Jelai Gemas, Negeri Cattle H. bispinosa (1) A. bovis clone 85 [KM114612;Malaya 241 nt/241 nt (100%  1699-M1 (KY046270) Sembilan identity); monkey, Malaysia]  Kuala Berang, H. bispinosa (1) (A. bovis type I) Terengganu  Pokok Sena, Kedah Sheep H. bispinosa (2) Anaplasma sp. cloneof WBM1 [KU189194; 243  KM40-F2 (KY046276)  Kuala Berang, Cattle H. bispinosa (1) nt/243 nt (100% identity); wild boar, Malaysia] Terengganu (A. bovis type II)  Jelai Gemas, Negeri Cattle H. bispinosa (3) A. platys strain Sarawak41 UPM (KU500914; 237  WYY1292 (KY046285) Sembilan nt/237 nt (100% identity); dog, Malaysia)  Pokok Sena, Kedah Sheep H. bispinosa (1) A. phagocytophilum isolate HB-G5-goat-China Not selected [KR002112; 238 nt/239 nt (99% identity); goat, China]  Jerantut, Pahang Cattle R. microplus (2) Ehrlichia sp. clone 2-3 [HM486685; 243 nt/243 nt  VKAA024-F (KY046290) (100% identity); cattle, Canada]  Jelai Gemas, Negeri Cattle R. microplus (1) Ehrlichia sp. isolate BL157-9 [KJ410257; 240  UN2-100-F (KY046291) Sembilan nt/240 nt (100% identity); H. asiaticum, China]  Ulu Lepar, Pahang R. microplus (2)  Jelai Gemas, Negeri Cattle H. bispinosa (2) Ehrlichia sp. isolate TC251-2 [KJ410253; 243  UN2-19-M (KY046295) Sembilan Universitynt/243 nt (100% identity); D. nuttalli, China]  Ulu Lepar, Pahang H. bispinosa (1)

Table 4.4, continued Forest areas  Kuala Lompat, Vegetation D. atrosignatus (1) A. platys strain Sarawak41 UPM (KU500914; 237  KLV077 (KY046283) Pahang Haemaphysalis spp. (2) nt/237 nt (100% identity); dog, Malaysia) H. hystricis (1) Rat Haemaphysalis sp. (1) Vegetation Dermacentor spp. (5) A. phagocytophilum strain AAIK4 [KR611719; 238  KLV086 D. atrosignatus (1) nt/238 nt (100% identity); mouse spleen, South Haemaphysalis spp. (5) Korea]  KLV034 (KY046281) Aboriginal villages  Negeri Sembilan Dog R. sanguineus A. bovis clone 85 [KM114612;Malaya 241 nt/241 nt (100%  DK014-1 (KY046272) (1 pool) identity); monkey, Malaysia] Goat H. bispinosa (A. bovis type I) (1 pool)  Perak Cat Haemaphysalis sp. (1 Anaplasma sp. cloneof BJ01 [JN715833; 250 nt/255  SP002-F (KY046288) pool) nt (98% identity); H. longicornis, China] Others  Sepang, Selangor Snake A. varanense (2) A. bovis clone 85 [KM114612; 241 nt/241 nt (100% Not selected identity); monkey, Malaysia] (A. bovis type I) A. varanense (2) A. platys strain Sarawak41 UPM (KU500914; 237 Not selected A. helvolum (2) nt/237 nt (100% identity); dog, Malaysia) A. helvolum (1) Anaplasma sp. isolate 1076 [AY570539; 225 nt/225  S4 nt (100% identity); dog, South Africa] A. varanense (2) Ehrlichia sp. isolate BL157-9 [KJ410257; 249  S3 nt/250 nt (99% identity); H. asiaticum, China]

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Phylogenetic analysis reveals the clustering of Ehrlichia sp. investigated in this study to three groups (Figure 4.10). One group (represented by one tick sample) is closely related to Ehrlichia sp. strain EBm52 from Thailand (GenBank accession no.: AF497581)

(Parola et al., 2003). The other group (represented by one tick sample) is clustered with

Ehrlichia sp. TC251-2 isolate from China (GenBank accession no.: KJ410253) (Kang et al., 2014) and Ehrlichia sp. clone 2-3 from Canada (GenBank accession no.: HM486685)

(Gajadhar et al., 2010) (Figure 4.10). Sequences obtained from two tick samples [SP002-

F and S3] are not clustered with any reference sequences deposited in GenBank, suggesting the existence of potential new strains of Anaplasmataceae in ticks investigated in this study. Malaya of

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A. platys WYY1292 H. bispinosa cattle tick Malaysia (KY046285)* A. platys KLV077 D. atrosignatus questing tick Malaysia (KY046283)* A. platys dog France (AF303467) A. platys dog Thailand (EF139459) A. platys dog Malaysia (KU500914) Anaplasma sp. dog South Africa (AY570539) Anaplasma sp. S4 A. helvolum snake tick Malaysia* A. phagocytophilum strain Webster (U02521) A. phagocytophilum D. silvarum China (DQ449948) A. phagocytophilum mouse organ Korea (KR611719) A. phagocytophilum KLV086 Dermacentor sp. questing tick Malaysia* A. phagocytophilum KLV034 Haemaphysalis sp. questing tick Malaysia (KY046281)* A. bovis wild boar Malaysia (KU189194) A. bovis H. longicornis Japan (AB196475) A. bovis KM40-F2 H. bispinosa sheep tick Malaysia (KY046276)* A. bovis monkey Malaysia (KM114612) A. bovis raccoon Japan (GU937020) Ehrlichia bovis 16S rRNA gene (U03775) A. bovis 1699-M1 H. bispinosa cattle tick Malaysia (KY046270)* A. bovis DK014-1 R. sanguineus dog tick Malaysia (KY046272)* A. marginale R. microplus tick Philippines (JQ839012) A. marginale USA (AF311303) A. centrale vaccine strain (AF318944) A. ovis South Africa OVIMalaya (AF414870) A. marginale X102-1 H. bispinosa cattle tick Malaysia (KY046278)* A. marginale EKY4766-F R. microplus cattle tick Malaysia (KY046277)* Anaplasmaof sp. H.longicornis tick China (JN715833) Anaplasma sp. SP002-F Haemaphysalis sp. cat tick Malaysia (KY046288)* Ehrlichia sp. S3 A. varanense snake tick Malaysia* Ehrlichia sp. D. nuttalli China(KJ410253) Ehrlichia sp. UN2-19-M H. bispinosa cattle tick Malaysia (KY046295)* Ehrlichia sp. cattle R. microplus Thailand (AF497581) Ehrlichia sp. UN2-100-F R. microplus cattle tick Malaysia (KY046291)* Ehrlichia sp. cattle Canada (HM486685) Ehrlichia sp. VKAA024-F R. microplus cattle tick Malaysia (KY046290)* Rickettsia rickettsii (U11021)

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Figure 4.10: Phylogenetic relationships among various Anaplasma spp. and Ehrlichia spp.University based on partial sequences of the 16S rRNA gene (222 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified from ticks in this study.

4.5.2 Further characterisation of Anaplasma spp. and Ehrlichia spp.

4.5.2.1 Amplification of nearly full length 16S rRNA gene of Anaplasma spp. and

Ehrlichia spp. from ticks

Anaplasma or Ehrlichia-positive tick samples were subjected to further amplification to obtain the full length sequences of 16S rRNA gene for analysis. The samples included 118 ticks [85 ticks (57 H. bispinosa and 28 R. microplus) from livestock farms, 21 ticks (eight Dermacentor spp., two D. atrosignatus, nine Haemaphysalis spp., one H. hystricis and one Amblyomma sp.) collected from the forest areas, 11 pools of ticks (four pools of H. wellingtoni, three pools of H. bispinosa, one pool of

Haemaphysalis spp., two pools of R. sanguineus and one pool of R. microplus) from aboriginal villages and a snake tick (A. varanense) from Sepang, Selangor]. DNA fragments of approximately 1500 bpMalaya were obtained from amplification using either primer pairs of fD1/RP2 or ATT062F/ATT062R or fD1/EHR16SR paired with Rp2/EHR16SD in this study (Figureof 4.11). The amplicons were successfully obtained from 46 (38.0%) tick samples [(including 13 R. microplus from cattle, six H. bispinosa from sheep and 16 H. bispinosa from cattle) ticks from livestock farms, nine questing ticks (three Haemaphysalis spp., one H. hystricis and five Dermacentor spp.) collected from the forest areas, one pool of ticks (H. bispinosa from chicken) from aboriginal village and a snake tick (A. varanense) from Sepang, Selangor].

A total of 17 amplicons were selected for sequence determination, of which only tenUniversity sequences were successfully obtained. These amplicons were derived from four R. microplus, two H. bispinosa, one Haemaphysalis sp., one H. hystricis, one A. varanense and one Dermacentor sp. BLAST analyses reveal the identification of Ehrlichia spp. (in five ticks), A. marginale (in one tick), Candidatus E. shimanensis (in one tick) and A. bovis (in three ticks). The results of the BLAST analyses are shown in Table 4.5.

Figure 4.11: Amplification of approximately 1500 bp of the 16S rRNA gene fragments from samples using fD1/Rp2 primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-5: fD1/Rp2-positive samples; 6: positive control (cloned plasmid contain A. bovis).

A phylogenetic tree was constructed to show the genetic relationship between the

Anaplasma spp. and Ehrlichia spp., using sequences derived in this investigation and those sequences of the reference strains (Figure 4.12).Malaya The analysis exhibits that A. marginale detected in ticks in this study were clustered with A. marginale reported from

R. microplus from Philippines (isolate C6A;of GenBank accession no.: JQ839012) (Ybañez et al., 2013b) and cattle from China (isolate ZJ02/2009; GenBank accession no.:

HM439433; unpublished). The phylogenetic analysis in this study also reveals the clustering of Ehrlichia spp. in cattle ticks with Candidatus E. shimanensis from Japan

(GenBank accession no.: AB074459) (Kawahara et al., 2006); Ehrlichia sp. from Japan

(isolate Yonaguni138; GenBank accession no.: HQ697588) (Matsumoto et al., 2011) and

Ehrlichia sp. from Brazil (E. mineirensis) (strain UFMG-EV; GenBank accession no.:

JX629805)University (Cabezas-Cruz et al., 2012 ). Additionally, the Ehrlichia sp. from a R. microplus tick (collected from a cow) is clustered with a few Ehrlichia spp. including

Ehrlichia sp. from Thailand (strain EBm52; GenBank accession no.: AF497581) (Parola et al., 2003); Ehrlichia sp. from China (Fujian; GenBank accession no.: DQ324547; unpublished) and Ehrlichia sp. from China (isolate BL157-9; GenBank accession no.:

KJ410257) (Kang et al., 2014). The 16S rRNA gene sequence obtained from a snake tick

Table 4.5: BLAST analyses of 23 nearly full length 16S rRNA, gltA and groEL gene sequences amplified from ticks from different sources in this study.

Location Animal Tick species (n) Gene used BLAST result (nearest match) host Livestock farms  Jerantut, Cattle R. microplus (2) 16S rRNA Ehrlichia sp. strain UFMG-EV [JX629805; 1294 nt/1308 nt (99% identity); R. microplus, Pahang - H5T056-F Brazil] - VKAA024-F gltA (only Ehrlichia sp. strain UFMG-EV [JX629807; 544 nt/545 nt (99% identity); R. microplus, one tick) Brazil] groEL Ehrlichia sp. strain UFMG-EV [JX629806; 1207 nt/1223 nt (99% identity); R. microplus, Brazil]  Ulu Lepar, H. bispinosa (1) 16S rRNA Ehrlichia sp. Tibet [AF414399;Malaya 1457 nt/1469 nt (99% identity); R. microplus, China] Pahang - UN2-77-F gltA Ehrlichia sp. clone SY36 [KF728364; 497 nt/544 nt (91% identity); H. longicornis, China] of groEL Candidatus E. shimanensis [AB074462; 933 nt/992 nt (94% identity); H. longicornis, Japan]  Ulu Lepar, R. microplus (1) 16S rRNA Ehrlichia sp. clone EBm52 [AF497581; 1335 nt/1335 nt (100% identity); R. microplus, Pahang - UN2-100-F Thailand] gltA Ehrlichia sp. isolate BL157-9(a) [KJ410273; 545 nt/545 nt (100% identity); H. asiaticum, China]  Ulu Lepar, R. microplus (1) groEL Ehrlichia sp. clone Tajikistan-1 [KJ930191; 1249 nt/1254 nt (99% identity); H. Pahang - UN2-100-F anatolicum, Tajikistan] H. bispinosa (1) 16S rRNA Candidatus E. shimanensis [AB074459; 1326 nt/1326 nt (100% identity); H. longicornis, - UN2-19-M Japan] gltA Ehrlichia sp. clone SY130 [KF728365; 517 nt/537 nt (96% identity); H. longicornis, China] groEL Candidatus E. shimanensis [AB074462; 953 nt/1013 nt (94% identity); H. longicornis, Japan] University

Table 4.5, continued  Kuala Berang, Cattle R. microplus (1) 16S rRNA A. marginale strain Uganda MT40 [KU686785; 1364 nt/1366 nt (100% identity); cattle, Terengganu - 1671-F1 Uganda] gltA A. marginale strain Dawn [CP006847; 542 nt/542 nt (100% identity); cattle, USA] groEL Ehrlichia sp. isolate Yonaguni206 [HQ697591; 1143 nt/1187 nt (96% identity); H. longicornis, Japan] H. bispinosa (1) 16S rRNA Noisy data (not able to analyse) - 2023-M1 gltA Ehrlichia sp. clone SY130 [KF728365; 516 nt/534 nt (96% identity); H. longicornis, China] groEL Candidatus E. shimanensis [AB074462; 1129 nt/1196 nt (94% identity); H. longicornis, Japan] Forest areas Malaya  Kuala Lompat, Vegetation Dermacentor sp. 16S rRNA A. bovis clone 85 [KM114612; 934 nt/934 nt (100% identity); monkey, Malaysia] (A. Pahang (1) bovis type I) -KLV086 of Haemaphysalis 16S rRNA A. bovis clone 85 [KM114612; 1008 nt/1010 nt (99% identity); monkey, Malaysia] (A. sp. (1) bovis type I) -KLV032 H. hystricis (1) 16S rRNA - KLV023 Others  Sepang, Snake A. varanense (1) 16S rRNA E. ruminantium isolate Ball3 [U03777; 1303 nt/1325 nt (98% identity), South Africa] Selangor -S3

University

is most genetically (98% sequence similarity) related to that of E. ruminantium (isolate

Ball3; GenBank accession no.: U03777) (Allsopp et al., 1996), as shown in Figure 4.12.

Ehrlichia sp. H5T056-F R. microplus cattle tick Malaysia* Ehrlichia sp. VKAA024-F R. microplus cattle tick Malaysia (KY046297)* Ehrlichia sp. R. microplus Brazil (JX629805)(E.mineirensis) E. canis strain Oklahoma (M73221) Ehrlichia sp. cattle R. microplus Thailand (AF497581) Ehrlichia sp. H. asiaticum China (KJ410257) Ehrlichia sp. R. microplus China (DQ324547) Ehrlichia sp. UN2-100-F R. microplus cattle tick Malaysia (KY046298)* Ehrlichia sp. H. longicornis Japan (HQ697588) Ehrlichia sp. UN2-77-F H. bispinosa cattle tick Malaysia (KY046299)* E. ewingii strain Stillwater (NR044747) Candidatus E. shimanensis H. longicornis Japan (AB074459) Ehrlichia sp. UN2-19-M H. bispinosa cattle tick Malaysia (KY046300)* E. chaffeensis strain Arkansas (NR074500) E. ruminantium (U03777) Ehrlichia sp. S3 A. varanenseMalaya snake tick Malaysia* A. marginale R. microplus Philippines (JQ839012) A. marginale 1671-F1 R. microplus cattle tick Malaysia* A. marginale USAof (AF311303) A. marginale cattle China (HM439433) A. centrale vaccine strain (AF318944) A. ovis South Africa OVI (AF414870) A. phagocytophilum strain Webster (U02521) A. platys dog France (AF303467) A. bovis wild boar Malaysia (KU189194) Ehrlichia bovis (U03775) A. bovis monkey Malaysia (KM114612) A. bovis KLV032 Haemaphysalis sp. questing tick Malaysia* A. bovis KLV086 Dermacentor sp. questing tick Malaysia* University Rickettsia rickettsii 16S rRNA gene (U11021) 0.02

Figure 4.12: Phylogenetic relationships among various Anaplasma spp. and Ehrlichia spp. based on partial sequences of the 16S rRNA gene (887 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified from ticks in this study.

4.5.2.2 Amplification of gltA gene of anaplasma and ehrlichiae

The same 85 ticks (57 H. bispinosa and 28 R. microplus) from livestock farms

(cattle and sheep) as mentioned in section 4.5.2.1 were further subjected to amplification of gltA gene in this study. Amplicons of approximately 641 bp were obtained by using primer pair EHR-CS136F/EHR-CS778R (Figure 4.13). Only 32.9% (14 R. microplus and

H. bispinosa, respectively) ticks were positive for gltA-PCR assays. The gltA sequences were successfully obtained from three R. microplus and H. bispinosa, respectively.

BLAST analyses reveal the presence of Ehrlichia spp. (in five ticks) and A. marginale

(in one tick). The results of the BLAST analyses are shown in Table 4.5.

All the six sequences were used to build a phylogenetic tree (Figure 4.14), together with sequences from the reference strains. The analysis shows the clustering of A. marginale detected in this study with that of A. marginaleMalaya from USA (strain Dawn; GenBank accession no.: CP006847) (Aguilar Pierlé et al., 2014). The five Ehrlichia spp. were clustered separately with those of Ehrlichiaof sp. clone SY130 from China (GenBank accession no.: KF728365) (Dong et al., 2014); Ehrlichia sp. strain UFMG-EV (E. mineirensis) from Brazil (GenBank accession no.: JX629807) (Cabezas-Cruz et al.,

2012); Ehrlichia sp. isolate BL157-9(a) from China (GenBank accession no.: KJ410273)

(Kang et al., 2014) and Ehrlichia sp. clone SY36 from China (GenBank accession no.:

KF728364) (Dong et al., 2014), respectively.

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Figure 4.13: Amplification of approximately 641 bp of the gltA gene fragments from samples using EHR-CS136F/EHR-CS778R primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-5: EHR-CS136F/EHR-CS778R -positive samples; 6: Positive control (A. phagocytophilum genomic DNA extracted from IFA slides); 7: Positive control (E. chaffeensis genomic DNA extracted from IFA slides).

Ehrlichia sp. H. longicornis China (KF728364) Ehrlichia sp. UN2-77-F H. bispinosa cattle tick Malaysia (KY046303)* Ehrlichia sp. D. nuttalli China (KJ410275)Malaya Ehrlichia sp. H. asiaticum China (KJ410273) Ehrlichia sp. UN2-100-F R. microplus cattle tick Malaysia (KY046302)* Ehrlichia sp. H. ofasiaticum China (KJ410269) Ehrlichia sp. H. longicornis China (KF728365) Ehrlichia sp. UN2-19-M H. bispinosa cattle tick Malaysia (KY046304)* Ehrlichia sp. 2023-M1 H. bispinosa cattle tick Malaysia* E. muris (AF304144) E. chaffeensis strain Arkansas (AF304142) E. canis strain Oklahoma (AF304143) Ehrlichia sp. R. microplus Brazil (JX629807)(E. mineirensis) Ehrlichia sp. VKAA024-F R. microplus cattle tick Malaysia (KY046301)* E. ruminantium (AF304146) A. phagocytophilum strain Webster human USA (AF304136) A. centrale strain Aomori (AF304141) A. ovis sheep China (JX559689) A. marginale Florida USA (AF304140) A. marginale strain Dawn cattle USA(CP006847) A. marginale 1671-F1 R. microplus cattle tick Malaysia* University Neorickettsia risticii (AF304147)

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Figure 4.14: Phylogenetic relationships among various Anaplasma spp. and Ehrlichia spp. based on partial sequences of the gltA gene (533 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. Neorickettsia risticii (AF304147) was used as an outgroup. * : Representative sequences amplified from ticks in this study.

4.5.2.3 Amplification of groEL gene of anaplasma and ehrlichiae

The same 85 ticks (57 H. bispinosa and 28 R. microplus) from livestock farms as mentioned in section 4.5.2.1 were further subjected to amplification of groEL gene using established primers. Approximately 1320 bp amplicons were obtained by using nested amplification primer pair HS3-f/HSVR (Figure 4.15). A total of 20 ticks (23.5%; including 11 R. microplus and nine H. bispinosa) were positive for groEL-PCR assays.

As direct sequencing of the amplicons produced noisy sequence data, seven randomly selected amplicons were cloned into pCR4-TOPO vector (Invitrogen, CA, USA) and sequenced. BLAST analyses of seven groEL sequences obtained from four R. microplus and three H. bispinosa ticks confirms the identification of Ehrlichia spp. in all seven ticks in this study. The results of the BLAST analyses are shown in Table 4.5. All the seven groEL sequences were used toMalaya build a phylogenetic tree (Figure 4.16). Phylogenetic analysis shows the clustering of Ehrlichia spp. detected in this study with those of Ehrlichia sp. isolate Yonaguni206of from Japan (GenBank accession no.: HQ697591) (Matsumoto et al., 2011); Ehrlichia sp. strain LCT20 from China (GenBank accession no.: KF977220; unpublished); Candidatus E. shimanensis from Japan

(GenBank accession no.: AB074462) (Kawahara et al., 2006) and Ehrlichia sp. strain

UFMG-EV (E. mineirensis) from Brazil (Genbank accession no.: JX629806) (Cabezas-

Cruz et al., 2012), respectively.

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Figure 4.15: Amplification of approximately 1320 bp of the groEL gene fragments from samples using HS3-f/HSVR nested amplification primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-3: HS3-f/HSVR-positive samples; 4: Positive control (A. phagocytophilum genomic DNA extracted from IFA slides); 5: Positive control (E. chaffeensis genomic DNA extracted from IFA slides).

Ehrlichia sp. H. longicornis Japan (HQ697591) Ehrlichia sp. 1671-F1 R. microplus cattle tick Malaysia* E. ewingii human USA (AF195273)Malaya Ehrlichia sp. H. anatolium Tajikistan (KJ930192) Ehrlichia sp. cattle R. microplus China (KF977220) Ehrlichia sp. UN2-100-Fof R. microplus cattle tick Malaysia (KY046306)* Candidatus E. shimanensis H. longicornis Japan (AB074462) Ehrlichia sp. 2023-M1 H. bispinosa cattle tick Malaysia* Ehrlichia sp. UN2-77-F H. bispinosa cattle tick Malaysia (KY046307)* Ehrlichia sp. UN2-19-M H. bispinosa cattle tick Malaysia (KY046308)* E. chaffeensis strain Arkansas (L10917) E. canis strain Florida (U96731) Ehrlichia sp. R. microplus Brazil (JX629806)(E.mineirensis) Ehrlichia sp. H5T056-F R. microplus cattle tick Malaysia* Ehrlichia sp. VKAA024-F R. microplus cattle tick Malaysia (KY046305)* E. ruminantium strain Umbanein (DQ647005) A. bovis vole Russia (JX092099) A. phagocytophilum strain HZ (CP000235) A. marginale cattle Philippines (JQ839014) University A. centrale R. simus South Africa (AF414866) R. rickettsii strain Hauke (CP003318)

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Figure 4.16: Phylogenetic relationships among various Ehrlichia spp. based on partial sequences of the groEL gene (943 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (CP003318) was used as an outgroup. * : Representative sequences amplified from ticks in this study.

4.5.2.4 Molecular characterisation of A. phagocytophilum based on three genes

(msp4, 16S rRNA and groEL)

Nested PCR assays targeting on three A. phagocytophilum-genes (16S rRNA, msp4 and groEL) were used for detection of A. phagocytophilum in this study. A total of

46 ticks [27 ticks from the forest areas (one Amblyomma sp., eight Haemaphysalis spp., two H. hystricis, one H. shimoga, 13 Dermacentor spp., and two D. atrosignatus) and 19 ticks from aboriginal villages (one R. microplus, two R. sanguineus, eight Haemaphysalis spp., five H. wellingtoni and three H. bispinosa)] were subjected to amplification of msp4 gene of A. phagocytophilum. The nested amplification produced 343 bp amplicon by using primer pair msp4f/msp4r (Figure 4.17). A total of 17 (37.0%) ticks (including two

Dermacentor spp. and two D. atrosignatus from the forest areas; one R. microplus, two H. bispinosa, five H. wellingtoni and five HaemaphysalisMalaya spp. from aboriginal villages) were successfully amplified. BLAST analyses from seven representative sequences show the presence of two sequence variants of A.of phagocytophilum . The results of the BLAST analyses are shown in Table 4.6.

All the seven sequences were used to build a phylogenetic tree, as shown in Figure

4.18. Phylogenetic analysis reveals the differentiation of A. phagocytophilum into two groups. One group was clustered with A. phagocytophilum detected in patient from USA

(strain Webster; GenBank accession no.: EU857674; unpublished) and A. phagocytophilum detected in ticks from France (clone cam3, GenBank accession no.: JX197225;University clone cam5, GenBank accession no.: JX197227) (Chastagner et al., 2013). Another group is clustered with A. phagocytophilum detected in a rat from China (strain

ZJ-China; GenBank accession no.: EU008082) (Zhan et al., 2008).

Of 13 ticks (six Dermacentor spp., four Haemaphysalis spp. and one H. shimoga from the forest areas; one H. bispinosa and one R. sanguineus from aboriginal villages) subjected to amplification targeting 16S rRNA gene using nested PCR assay, only three

ticks (two Haemaphysalis spp. and one Dermacentor sp.) were positive for A. phagocytophilum. However, sequence determination of the 16S rRNA partial gene fragment of A. phagocytophilum-positive samples was not successful due to noisy data generated from the sequencing. All the 13 ticks were negative for the groEL-PCR assay.

Malaya of

Figure 4.17: Amplification of approximately 343 bp of the msp4 gene fragments of A. phagocytophilum from samples using msp4f/msp4r nested amplification primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-5: msp4f/msp4r-positive samples; 6: Positive control (A. phagocytophilum genomic DNA extracted from IFA slides).

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Table 4.6: BLAST analyses of the partial 16S rRNA gene sequences of A. bovis, E. chaffeensis and the msp4 gene sequences of A. phagocytophilum amplified from ticks from different sources in this study.

Location Animal Tick species (n) Gene BLAST result (nearest match) Representative samples for host used construction of dendrogram Forest areas  Kuala Lompat, Vegetation D. atrosignatus (2) msp4 A. phagocytophilum clone cam5 [JX197227; 258 nt  KLV077 Pahang /258 nt (100% identities); R. bursa, France]  KLV046 Dermacentor sp. (1)  KLV039 Haemaphysalis sp. 16S rRNA A. bovis clone 115 [KM114613; 450 nt/450 nt (100%  KL004 (1) identity); monkey, Malaysia]Malaya (A. bovis type I)  Sg. Deka, Dermacentor sp. (1) A. bovis strain SG175_HL 16S [EU181142; 450 nt/450  T020 Terengganu nt (100% identity); H. longicornis, South Korea]  Kuala Lompat, Haemaphysalis sp. - E. chaffeensis isolate ES 113 [KM009066; 261 nt/261  KLV004 Pahang (1) nt (100% identitofy), R. microplus, Colombia] D. atrosignatus (1) -E. ewingii isolate PB91 [KM009067; 261 nt/261 nt  KLV046 (100% identity), R. microplus, Colombia] Aboriginal villages  Kelantan Chicken Haemaphysalis spp. msp4 A. phagocytophilum clone cam5 [JX197227; 258 nt  SW003 (2) /258 nt (100% identities); R. bursa, France]  SW004-C  Negeri Sembilan Goat H. wellingtoni (1)  UK020  Perak Cat Haemaphysalis sp. msp4 A. phagocytophilum strain ZJ-China [EU008082; 272  SP002-F (1) nt/273 nt (99% identities); rat, China]

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SW003 Haemaphysalis sp. chicken tick Malaysia* UK020 H. wellingtoni goat tick Malaysia* SW004-C Haemaphysalis sp. chicken tick Malaysia* KLV077 D. atrosignatus questing tick Malaysia* KLV046 D. atrosignatus questing tick Malaysia* KLV039 Dermacentor sp. questing tick Malaysia* Anaplasma sp. dog Malaysia (KR920045) A. phagocytophilum D. marginatus France (JX197225) A. phagocytophilum R. bursa France (JX197227) A. phagocytophilum strain Webster (EU857674) A. phagocytophilum deer USA (AY530197) A. phagocytophilum red deer Slovakia (EU180061) A. phagocytophilum bison Poland (AY706388) A. phagocytophilum horse Germany (AY706390) A. phagocytophilum roe deer Spain (AY829457) A. phagocytophilum bovine Switzerland (AY530198) A. phagocytophilum ovine Norway (AY706391) A. phagocytophilum roe deer Germany (AY706386) A. phagocytophilum rat China (EU008082) SP002-F HaemaphysalisMalaya sp. cat tick Malaysia* A. centrale (AF428090) A. marginale strain Oklahoma (AY010252) of A. ovis strain Idaho (AF393742) 0.05

Figure 4.18: Phylogenetic relationships among various A. phagocytophilum based on partial sequences of the msp4 gene (252 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. * : Representative sequences amplified from ticks in this study.

4.5.2.5 Molecular characterisation of A. bovis in tick samples

The DNA of 13 ticks (including six Dermacentor spp. and five Haemaphysalis spp.University from the forest areas, one H. bispinosa and one R. sanguineus from aboriginal villages) were subjected to amplification using nested PCR assays targeting on 16S rRNA gene of A. bovis. The nested amplification produced a 551 bp DNA fragment, by using primer pair AB1f/AB1r (Figure 4.19). Five ticks (three Haemaphysalis spp. and two

Dermacentor spp.) were positive while all the two ticks from aboriginal villages were negative for the nested PCR. BLAST analyses of two amplified 16S rDNA sequences

reveal the presence of two genetic variants of A. bovis. The results of the BLAST analyses are shown in Table 4.6. All the two sequences were used to build a phylogenetic tree

(Figure 4.20). Phylogenetic analysis shows the separation of the sequences, with one grouped with A. bovis detected in monkey from Malaysia (this study, section 4.6.2.1,

GenBank accession no.: KM114613) and another sequence grouped with A. bovis detected in a wild boar in Malaysia (this study, section 4.6.2.1, GenBank accession no.:

KU189194) and A. bovis detected in a H. longicornis tick in Korea (strain SG175_HL;

GenBank accession no.: EU181142) (Lee and Chae, 2010).

Malaya of

Figure 4.19: Amplification of approximately 551 bp of the 16S rRNA gene fragments of A. bovis from samples using AB1f/AB1r nested amplification primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-5: AB1f/AB1r-positive samples; 6: Positive control. University

A. bovis cattle H. longicornis Korea (AF470698) Ehrlichia bovis 16S rRNA gene (U03775) Anaplasma sp. sika deer Japan (LC068733) A. bovis monkey Malaysia (KM114613) KLV004 Haemaphysalis sp. questing tick Malaysia* A. bovis deer Japan (AB454073) A. bovis H. longicornis Japan (AB196475) A. bovis wild boar Malaysia (KU189194) A. bovis H. longicornis Korea (EU181142) T020 Dermacentor sp. questing tick Malaysia* A. bovis cattle China (KX115423) A. bovis goat China (KU509996) A. bovis deer Japan (AB211163) Candidatus Anaplasma pangolinii pangolin Malaysia(KU189193) A. marginale strain Dawn (CP006847) A. phagocytophilum strain Webster (U02521) E. chaffeensis strain Arkansas (NR074500)

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Figure 4.20: Phylogenetic relationships among variousMalaya A. bovis based on partial sequences of the 16S rRNA gene (450 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrappingof with 1,000 replicates. * : Representative sequences amplified from ticks in this study.

4.5.2.6 Molecular characterisation of E. chaffeensis in tick samples

A total of 51 tick DNA samples (11 Haemaphysalis spp., two H. hystricis, one H. shimoga, 15 Dermacentor spp., three D. atrosignatus and one Amblyomma sp. from the forest areas; eight Haemaphysalis spp., four H. wellingtoni, three H. bispinosa, two R. sanguineus and one R. microplus from aboriginal villages) were subjected to amplificationUniversity by using a nested PCR assay targeting the 16S rRNA gene of E. chaffeensis (Figure 4.21). A total of 15 ticks were positive, with the generation of a DNA fragment of 389 bp. Amplified fragments were derived from one R. sanguineus tick from an aboriginal village; five Dermacentor spp., two D. atrosignatus, six Haemaphysalis spp. and one Amblyomma sp. from the forest areas.

BLAST analyses of two representative sequences reveal high similarity (100%,

261 nt/261 nt) to those of the reference strains of E. chaffeensis detected in R. microplus from Colombia (isolate ES 113; GenBank accession no.: KM009066) (Miranda and

Mattar, 2015) and E. ewingii detected in R. microplus from Colombia (isolate PB91;

GenBank accession no.: KM009067) (Miranda and Mattar, 2015) (Table 4.6). The phylogenetic tree (Figure 4.22) shows the grouping of the sequences with those of

Ehrlichia sp. detected in Hyalomma anatolicum from Tajikistan (clone Tajikistan-5;

GenBank accession no.: KP059122; unpublished), and R. microplus from Thailand

(strain EBm52; GenBank accession no.: AF497581) (Parola et al., 2003) and Tibet, China

(strain Tibet; GenBank accession: AF414399) (Wen et al., 2002).

Malaya of

FigureUniversity 4.21: Amplification of approximately 389 bp of the 16S rRNA gene fragments of E. chaffeensis from samples using HE1/HE3 nested amplification primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-5: HE1/HE3-positive samples; 6: Positive control (E. chaffeensis genomic DNA extracted from IFA slides).

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E. ewingii R. microplus Colombia (KM009067) E. chaffeensis R. microplus Colombia (KM009066) Ehrlichia sp. dog R. sanguineus Malaysia (KR920047)* E. ewingii strain Stillwater (NR044747) E. chaffeensis strain Arkansas (NR074500) Ehrlichia sp. R. microplus Tibet China (AF414399) Ehrlichia sp. cattle R. microplus Thailand (AF497581) Ehrlichia sp. H. anatolicum Tajikistan (KP059122) KLV004 Haemaphysalis sp. questing tick Malaysia* KLV046 D. atrosignatus questing tick Malaysia* Ehrlichia sp. H. asiaticum China (KJ410257) E. muris wild mouse Japan (U15527) A. phagocytophilum strain Webster (U02521) E. canis strain Oklahoma (M73221) Cowdria ruminantium (U03777) R. rickettsii 16S rRNA gene (U11021)

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Figure 4.22: Phylogenetic relationships among various E. chaffeensis based on partial sequences of the 16S rRNA gene (261 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping Malaya with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified from ticks in this study. of

4.6 Identification of Anaplasmataceae from animal samples using molecular

methods

4.6.1 Preliminary screening

DNA extracts from animal samples (in bloods, organ samples and FTA cards) were subjected to preliminary screening by using PCR assays targeting a short fragment

(345University bp) of the 16S rRNA gene of Anaplasmataceae (Figure 4.9). High detection rates of

Anaplasmataceae DNA were obtained in cattle (136/224, 60.7%) and sheep (32/40,

80.0%) from livestock farms. None of the 40 goats from livestock farm investigated in this study were positive for Anaplasmataceae DNA.

Of the 296 animal blood samples provided by VRI, 27.7% (82/296) animal blood samples were positive for the EHR-PCR assays. Positive blood samples were derived

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from the blood of deer (46/78, 59.0%), goats (2/75, 2.7%), buffaloes (12/55, 21.8%), cattle (14/22, 63.6%) and pangolins (8/15, 53.3%) while negative blood samples were derived from 46 horses, four rats and a cat. Of the samples collected from the trapped animals from the forest areas, 8.6% (6/70) monkeys, 70.0% (7/10) wild boars and 28.2% blood samples stored in FTA cards (20/71; ten from monitor lizards, eight from bats, one from primate and one from cat) were positive. Besides that, 22 organ samples (eight kidneys, seven liver and spleen, respectively) from six bats, two rats and a squirrel were positive for Anaplasmataceae DNA.

BLAST analyses were performed on 84 sequences successfully obtained from 40 cattle, 20 deer, five bats, five monkeys, four pangolins, two buffaloes, two goats and a squirrel. The details of the BLAST analyses are shown in Table 4.7. BLAST analyses reveal the presence of A. marginale, A. phagocytophilumMalaya (three sequence variants), A. bovis (two sequence variants), A. platys (two sequence variants) and one uncharacterised Anaplasma sp. of The findings obtained from the phylogenetic analysis (Figure 4.23) are summarised as below:- i. The Anaplasma spp. from the pangolins is grouped in the same cluster with the

Anaplasma sp. detected in Amblyomma javanense ticks from Thailand (strain AnAj360;

GenBank accession no.: AF497580) (Parola et al., 2003). ii. A. phagocytophilum DNA that was amplified from animal blood samples can be groupedUniversity into three clusters, of which one is closely related to A. phagocytophilum strain Webster (GenBank accession no.: U02521) (Chen et al., 1994) and A. phagocytophilum in mouse’s spleen from Korea (strain AAIK4; GenBank accession no.: KR611719; unpublished); one is grouped with A. phagocytophilum in goat from Korea (strain Hubei

E4; GenBank accession no.: KF569909; unpublished), and another is grouped with A.

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phagocytophilum reported from a goat in China (isolate HB-G5-goat-China; GenBank accession no.: KR002112; unpublished). iii. A. bovis detected in this study grouped into two clusters based on minor sequence variation, of which one is grouped with A. bovis detected in H. longicornis from Japan

(strain NR07; GenBank accession no.: AB196475) (Kawahara et al., 2006) and another cluster was grouped with A. bovis detected in H. concinna from China (strain hc-hlj209;

GenBank accession no.: KU921422; unpublished). iv. Two clusters of A. platys were observed, with one closely related to A. platys detected in deer from China (isolate 2ax1; GenBank accession no.: KJ659044; unpublished) and another with A. platys detected in dog from Malaysia (strain Sarawak41 UPM; GenBank accession no.: KU500914; unpublished). v. A. marginale in cattle is clustered with A. marginaleMalaya from Philippines (isolate C7D; GenBank accession no.: JQ839011) (Ybañez et al., 2013b), A. centrale from Philippines (isolate C4B; GenBank accession no.: JQ839010)of ( Ybañez et al., 2013b) and A. ovis from South Africa (isolate OVI; GenBank accession no.: AF414870) (Lew et al., 2003). vi. One sequence (Anaplasma sp. 0105) derived from the blood sample of a monkey is placed on a single branch. Further investigation is warranted to determine whether it is genetically related to other Anaplasma species.

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Table 4.7: BLAST analyses of 84 short 16S rRNA gene sequences amplified from animal samples from different sources in this study.

Location Animal host (n) BLAST result (nearest) Representative samples for construction of dendrogram Livestock farms  Jelai Gemas, Cattle (2) A. platys strain Sarawak41 [KU500914; 237 nt/237 nt (100% identity); dog, Not selected Negeri Sembilan Malaysia]  Ulu Lepar, Pahang Cattle (3)  Jerantut, Pahang Cattle (1) A. phagocytophilum strain AAIK4 [KR611719; 237 nt/237 nt (100%  EKP1246 identity); mouse spleen, South Korea]  Jelai Gemas, Cattle (17) A. marginale strain Uganda MT27 [KU686794; 225Malaya nt/225 nt (100%  WYY1474 Negeri Sembilan identity); cattle, Uganda]  Jerantut, Pahang Cattle (3) Not selected  Ulu Lepar, Pahang Cattle (3) of Not selected  Jelai Gemas, Cattle (3) A. marginale strain Uganda MT27 [KU686794; 234 nt/237 nt (99%  WYX1096 Negeri Sembilan identity); cattle, Uganda]  Jerantut, Pahang Cattle (1) Not selected  Ulu Lepar, Pahang Cattle (1)  UN2-109  VRI, Ipoh Buffalo (2) A. platys strain Sarawak41 [KU500914; 237 nt/237 nt (100% identity); dog,  BU10698-2 Deer (16) Malaysia]  DE25208 Deer (1) A. platys isolate 2ax1 [KJ659044; 255 nt/255 nt (100% identity); deer,  DE5582 China] Goat (1) A. phagocytophilum isolate HB-G5-goat-China [KR002112; 223/226 (99%  13997 identity); goat, China] Cattle (6) A. marginale strain Uganda MT27 [KU686794; 225 nt/225 nt (100% Not selected identity); cattle, Uganda] Pangolin (4) Anaplasma sp. AnAj360 [AF497580; 251 nt/251 nt (100% identity); A.  T7 javanense, Thailand] Goat (1) UniversityAnaplasma sp. clone WBM1 [KU189194; 238 nt/238 nt (100% identity);  G4699 Deer (3) wild boar, Malaysia] (A. bovis type II)  DE25230

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Table 4.7, continued Forest areas  Kuala Lompat, Bat’s liver (3) A. phagocytophilum strain AAIK4 [KR611719; 237 nt/237 nt (100% identity); Not selected Pahang Squirrel’s liver (1) mouse spleen, South Korea]  S0040-L Bat’s kidney (3) Not selected Bat’s spleen (3) Not selected Bat’s kidney (1) A. phagocytophilum strain Hubei E4 [KF569909; 242 nt/242 nt (100%  B0037-K identity); goat, China]  Perhilitan Monkey (1) A. phagocytophilum strain Hubei E4 [KF569909; 235 nt/238 nt (99% identity);  0105 provided goat, China] Monkey (4) A. bovis strain hc-hlj209 [KU921422; 238 nt/238 nt (100% identity); H.  0078 concinna, China] Malaya

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A. phagocytophila I. persulcatus Korea (AF470701) A. phagocytophilum EKP1246 cattle Malaysia* A. phagocytophilum mouse organ Korea (KR611719) A. phagocytophilum strain Webster (U02521) A. phagocytophilum S0040-L squirrel liver Malaysia* A. platys deer China (KJ659044) A. platys DE5582 deer Malaysia* A. platys dog Malaysia (KU500914) A. platys DE25208 deer Malaysia* A. platys BU10698-2 buffalo Malaysia* A. phagocytophilum goat China (KR002112) A. phagocytophilum 13997 goat Malaysia* A. bovis H. longicornis Japan (AB196475) A. bovis WBM1 wild boar Malaysia (KU189194)* A. bovis G4699 goat Malaysia* A. bovis DE25230 deer Malaysia* A. bovis H. concinna China (KU921422) A. bovis 0078 monkey Malaysia* A. phagocytophilumMalaya goat China (KF569909) A. phagocytophilum B0037-K bat kidney Malaysia* A. marginale UN2-109 cattle Malaysia* A.of marginale WYX1096 cattle Malaysia* A. marginale USA (AF311303) A. marginale R. microplus Philippines (JQ839011) A. centrale R. microplus Philippines(JQ839010) A. ovis South Africa OVI (AF414870) A. marginale WYY1474 cattle Malaysia* Anaplasma sp. 0105 monkey Malaysia* Anaplasma sp. pangolin A. javanense Thailand (AF497580) Anaplasma sp. T7 pangolin Malaysia* E. ewingii strain Stillwater (NR044747) E. chaffeensis str Arkansas (NR074500) Rickettsia rickettsii 16S rRNA gene (U11021)

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Figure 4.23: Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the 16S rRNA gene (224 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified in this study.

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4.6.2 Further characterisation of Anaplasmataceae

4.6.2.1 Amplification of the nearly full length 16S rRNA gene of Anaplasma spp.

and Ehrlichia spp.

A total of 150 animal samples [78 cattle and 16 sheep from livestock farms; 17 deer, eight pangolins, six cows, six buffaloes and two goats from VRI, Ipoh; 12 organ samples (from five bats and a squirrel), three wild boars and two monitor lizards blood samples stored in FTA cards] were subjected to amplification to determine the full length

16S rRNA gene of Anaplasma spp. or Ehrlichia spp. The 1500 bp amplicon was obtained by PCR assays using primer pairs of fD1/Rp2 or ATT062F/ATT062R or fD1/EHR16SR paired with Rp2/EHR16SD (Figure 4.11). All the 94 cattle and sheep bloods selected from the livestock farms were positive while only 33 animal blood samples provided by VRI (13 deer, eight pangolins, five cows, five buffaloesMalaya and two goats) were positive. The nearly full length 16S rRNA genes were also amplified from six samples [three wild boars (provided by PERHILITAN) and threeof organ samples from three bats (from field trips)].

Of 39 amplicons selected for sequencing, only 28 sequences were successfully obtained from 15 cattle, five monkeys, three pangolins, two deer, two wild boars and a goat (Table 4.8). BLAST analyses reveal the identification of A. marginale, A. centrale,

A. platys and A. bovis (three sequence variants). Referring to the phylogenetic tree (Figure

4.24), the findings are summarised as below: i. A.University centrale (represented by a sample labelled as Y9) obtained from three cattle in this study was grouped in one cluster with A. centrale from Japan (GenBank accession no.:

AF283007) (Inokuma et al., 2001c). A. marginale obtained from cattle from different sources (represented by samples labelled as WYY1474, 1958, EKV4995 and B90046) was clustered with A. marginale from USA (GenBank accession no.: AF311303; unpublished) and A. marginale detected in R. microplus from Philippines (isolate C7D;

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Table 4.8: BLAST analyses of nearly full length 16S rRNA and gltA gene sequences amplified from animal samples from different sources in this study.

Location Animal Gene used BLAST result (nearest match) Representative samples for host (n) construction of dendrogram Livestock farms  Jelai Gemas, Negeri Sembilan Cattle (1) 16S rRNA A. platys 16S ribosomal RNA gene, [EF139459; 1064  Y28  Jerantut, Pahang Cattle (1) nt/1066 nt (99% identity); dog, Thailand]  VKAB001  Jelai Gemas, Negeri Sembilan Cattle (3) A. centrale 16S ribosomal RNA gene [AF283007; 1313  Y9 nt/1313 nt (100% identity); cattle, Japan]  Jelai Gemas, Negeri Sembilan Cattle (2) A. marginale strain Uganda MT40 [KU686785; 1313  WYY1474  Jerantut, Pahang Cattle (1) nt/1313 nt (100% identity), cattle,Malaya Uganda] Not selected  Tanah Merah, Kelantan Cattle (2)  EKV4995  Kuala Berang, Terengganu Cattle (1)  1958  Air Hitam, Johore Cattle (1) of Not selected  Jelai Gemas, Negeri Sembilan Cattle (1) A. marginale strain Uganda MT40 [KU686785; 1167 Not selected nt/1172 nt (99% identity), cattle, Uganda]  Jelai Gemas, Negeri Sembilan Cattle (1) gltA A. marginale strain Dawn [CP006847; 493 nt/493 nt  WYY1474  Jerantut, Pahang Cattle (3) (100% identity); cattle, USA]  EKY4774  EKY4762  EKP1246  Ulu Lepar, Pahang Cattle (1)  UN2-88  Tanah Merah, Kelantan Cattle (1)  EKV4995  Kuala Berang, Terengganu Cattle (1)  1958  Air Hitam, Johore Cattle (1)  I6T-3760

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Table 4.8, continued  VRI, Ipoh Deer (2) 16S rRNA A. platys 16S ribosomal RNA gene, [EF139459; 1064  DE5576 Cow (1) nt/1066 nt (99% identity); dog, Thailand]  BPK2-6219 Cow (1) A. marginale strain Uganda MT40 [KU686785; 1167  B90046 nt/1172 nt (99% identity), cattle, Uganda] Goat (1) A. bovis isolate tick 17/China/2013 [KP314250; 1312  G4699 nt/1314 nt (99% identity); H. longicornis, China] Pangolin (3) A. bovis isolate tick 17/China/2013 [KP314250; 1284  T7 nt/1314 nt (98% identity); H. longicornis, China]  T14  T15  Perhilitan provided Wild boar (2) 16S rRNA A. bovis strain NR07 [AB196475; 1313/1313 (100%  WBM1 identity); H. longicornis, Japan]Malaya Monkey (5) E. bovis 16S rRNA gene [U03775; 1305 nt/1315 nt  0115 (99% identity)]  0088 of  0085

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GenBank accession no.: JQ839011) (Ybañez et al., 2013b). ii. A. platys detected in animal from this study (represented by samples labelled as Y28,

VKAB001, DE5576 and BPK2-6219) was grouped in one cluster with A. platys in dog from Thailand (GenBank accession no.: EF139459) (Pinyoowong et al., 2008) and sika deer from China (isolate 2ax1; GenBank accession no.: KJ659044; unpublished). iii. A. bovis is separated into three clusters: A. bovis detected in wild boars (represented by a sample labelled as WBM1) was in one cluster with A. bovis detected in H. longicornis from Japan (strain NR07; GenBank accession no.: AB196475) (Kawahara et al., 2006); A. bovis detected in a goat (represented by sample labelled as G4699) was in one cluster with Anaplasma sp. detected in R. sanguineus from Bangladesh (closely related to A. bovis) (isolate tick6; GenBank accession no.: LC066136) (Qiu et al., 2016); A. bovis detected in three monkeys (represented by Malayasamples labelled as 0115, 0085 and 0088) was clustered with A. bovis type strain (GenBank accession no.: U03775; unpublished). of iv. The Anaplasma spp. detected from three pangolins (represented by samples labelled as T7, T14 and T15) were segregated on a distinct branch amongst members of

Anaplasmataceae, with a high bootstrap value (98.0%). This finding suggests that the organisms could belong to a new species of Anaplasma. The pangolin-associated

Anaplasma was proposed as “Candidatus Anaplasma pangolinii” in accordance with the host where it was derived. UniversitySeveral almost full length 16S rDNA sequences, i.e., KU189194 (A. bovis, wild boar, clone WBM1); KM114611-KM114613 (A. bovis, monkey, clone 88, 85 and 115, respectively); KU189193 (Candidatus A. pangolinii, pangolin, clone T7) have been deposited in the GenBank database.

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A. bovis H. longicornis Japan (AB196475) A. bovis WBM1 wild boar Malaysia (KU189194)* Anaplasma sp. dog R. sanguineus Bangladesh (LC066136) A. bovis G4699 goat Malaysia* Ehrlichia bovis 16S rRNA gene (U03775) A. bovis 0115 monkey Malaysia (KM114613)* A. bovis 0088 monkey Malaysia (KM114611)* A. bovis 0085 monkey Malaysia (KM114612)* Candidatus A. pangolinii T7 pangolin Malaysia (KU189193)* Candidatus A. pangolinii T14 pangolin Malaysia* Candidatus A. pangolinii T15 pangolin Malaysia* A. phagocytophilum strain Webster (U02521) A. platys Y28 cattle Malaysia* A. platys dog Thailand (EF139459) A. platys sika deer China (KJ659044) A. platys VKAB001 cattle Malaysia* A. platys DE5576 deer Malaysia* A. platys BPK2-6219 cow Malaysia* A. centrale cattle Japan (AF283007) A. centrale Y9 cattle Malaysia* A. centrale vaccine strain (AF318944) A. ovis South Africa OVI (AF414870) A. marginale cattle Uganda (KU686794) A. marginale WYY1474 cattle Malaysia* A. marginale 1958 cattle Malaysia* A. marginale USA (AF311303)Malaya A. marginale R. microplus Philippines (JQ839011) A. marginale EKV4995 cattle Malaysia* A. marginaleof B90046 cow Malaysia* E. chaffeensis strain Arkansas (NR074500) E. ewingii strain Stillwater (NR044747) E. canis strain Oklahoma (M73221) Rickettsia rickettsii 16S rRNA gene (U11021)

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Figure 4.24: Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the 16S rRNA gene (1068 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified from animal samples in this study.

University 4.6.2.2 Amplification of gltA gene of Anaplasma spp. and Ehrlichia spp.

A total of 86 animals (70 cattle and 16 sheep) from livestock farms were subjected to further characterisation by amplification of gltA gene, using primer pair EHR-

CS136F/EHR-CS778R (Figure 4.13). An approximately 641 bp amplicon was obtained from 70 (81.4%; 69 cattle and a sheep) blood samples.

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The sequences for ten amplicons were sequenced, of which eight were successfully obtained from (all cattle) for analysis. BLAST analyses reveal the identification of A. marginale (strain Dawn; GenBank accession no.: CP006847) (Aguilar

Pierlé et al., 2014) (Table 4.8), based on the clustering of the sequences with the type strain (strain Dawn) detected in cattle from USA (GenBank accession no.: CP006847)

(Aguilar Pierlé et al., 2014).

A. marginale 1958 cattle Malaysia* A. marginale I6T-3760 cattle Malaysia* A. marginale EKV4995 cattle Malaysia* A. marginale UN2-88 cattle Malaysia* A. marginale EKP1246 cattle Malaysia* A. marginale EKY4762 cattle Malaysia* A. marginale EKY4774 cattle Malaysia* A. marginale strain Dawn (CP006847) A. marginale WYY1474 cattle Malaysia* A. marginale Florida USA (AF304140) A. ovis sheepMalaya China (JX559689) A. centrale strain Aomori (AF304141) A. capra human China (KM206274) Ehrlichia sp. HGE agent strain Webster (AF304136) Anaplasmaof sp. sike deer Japan (JN055362) R. risticii (AF304147) E. chaffeensis strain Arkansas (AF304142) E. canis R. sanguineus Philippines (JN391409) E. ewingii A. americanum USA (DQ365879)

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Figure 4.25: Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the gltA gene (469 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. * : Representative sequences amplified from animal samples in this study. University 4.6.2.3 Molecular characterisation of A. phagocytophilum from animal blood DNA

samples

Several nested PCR assays targeting on three genes (16S rRNA, msp4 and groEL) of A. phagocytophilum were used for detection of A. phagocytophilum from animal blood

DNA samples in this study. A total of 35 animal samples from VRI (23 deer, six

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buffaloes, four cows and two goats) and 17 animal samples from the forest areas [12 organ samples (four kidneys, three livers and three spleens from five bats; one liver and one spleen from a squirrel), three wild boars and two monitor lizard blood samples stored in FTA cards] were subjected to amplification of the 16S rRNA, msp4 and groEL genes of A. phagocytophilum. The highest detection rate (68.6%) was observed with the 16S rRNA gene PCR assay, followed by the PCR assays targeting msp4 gene (55.6%) and groEL gene (34.3%).

A total of 24 animal blood samples from VRI (19 deer, three buffaloes and two cows) and 13 animal samples from the forest areas [11 organ samples (three kidneys, three livers and three spleens from five bats; one liver and one spleen from a squirrel) and two wild boars] were positive for the nested PCR assays which amplified the 641 bp of the 16S rRNA gene of A. phagocytophilum, using primerMalaya pair SSAP2f/SSAP2r (Figure 4.26). A total of 13 amplicons were sequenced, however; only 11 sequences were successfully determined. BLAST analysesof show that six sequences obtained from one cow, two buffaloes and three deer (GenBank accession nos: MG988297-MG988299) demonstrated 100% (520 nt/520 nt) similarity with that of Candidatus Anaplasma boleense (strain WHBMXZ-139; GenBank accession no.: KX987335). Additionally, another five sequences matched 99-100% (520 nt/520 nt or 519 nt/520 nt) with an

Anaplasma sp. detected in a goat from China (clone Ap20-5a; GenBank accession no.:

KX272643; unpublished). The sequences demonstrated only 96.9-97.6% similarity with A. Universityphagocytophilum type strain (strain Webster; GenBank accession no.: U02521) (Chen et al., 1994). The results of the BLAST analyses for A. phagocytophilum are shown in

Table 4.9.

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Figure 4.26: Amplification of approximately 641 bp of the 16S rRNA gene fragments of A. phagocytophilum from samples using SSAP2f/SSAP2r nested amplification primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-5: SSAP2f/SSAP2r-positive samples; 6: Positive control (A. phagocytophilum genomic DNA extracted from IFA slides).

The dendrogram (Figure 4.27) reveals the clustering of the sequences with Anaplasma sp. detected in goat from China (cloneMalaya Ap20-5a; GenBank accession no.: KX272643; unpublished) and A. phagocytophilum detected in sheep from China (isolate YC38; GenBank accession no.: KJ782381)of (Yang et al., 2015a) but not in one cluster with the A. phagocytophilum type strain (strain Webster; GenBank accession no.:

U02521) (Chen et al., 1994).

For msp4-PCR assay, 20 animal samples obtained from VRI (14 deer, four buffaloes, a goat and a cow) and nine animal samples from the forest areas [four organ samples (two kidney and one spleen from bats; one liver from squirrel), three wild boars and two monitor lizard blood samples stored in FTA cards] were positive, producing approximatelyUniversity 343 bp amplicons (Figure 4.17). Eight amplicons were selected for sequence determination but only five sequences were successfully obtained. BLAST analyses of all five sequences show the identification of two sequence variants of A. phagocytophilum (Table 4.9) demonstrating 100% and 99% identity with A. phagocytophilum clone cam5 from France and A. phagocytophilum strain ZJ-China, respectively.

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Table 4.9: BLAST analyses of the partial 16S rRNA, msp4 and groEL gene sequences of A. phagocytophilum amplified from animal samples from different sources in this study.

Location Animal host (n) Gene used BLAST result (nearest match) Representative samples for construction of dendrogram  VRI, Ipoh Deer (2) 16S rRNA Candidatus A. boleense strain WHBMXZ-139 [KX987335; 520  DE2064 nt/520 nt (100% identity); R. microplus, China]  DE25241 Cow (1)  BG538 Buffalo (2)  BU10698-12  BU10698-28 Deer (1) Malaya  DE0345 groEL Anaplasma sp. clone SY49 [KF728361; 464 nt/480 nt (97% identity); H. longicornis, China] Deer (1) msp4 A. phagocytophilum strain ZJ-China [EU008082; 267 nt/268 nt (99%  DE25206 identity); rat, China] of Deer (2) groEL Anaplasma sp. clone SY49 [KF728361; 447 nt/463 nt (97% identity);  DE25208 H. longicornis, China]  DE5576 Forest areas  Kuala Lompat, Bat’s kidney (1) 16S rRNA Anaplasma sp. clone Ap20-5a [KX272643; 520 nt/520 nt (100%  B0014-K Pahang Squirrel’s liver (1) identity); goat, China]  S0040-L Bat’s kidney (1) Anaplasma sp. clone Ap20-5a [KX272643; 519 nt/520 nt (99%  B0037-K identity); goat, China] Bat’s kidney (1) msp4 A. phagocytophilum clone cam5 [JX197227; 261 nt/261 nt (100%  B0014-K Squirrel’s liver (1) identity); R. bursa, France]  S0040-L Bat’s kidney (1) A. phagocytophilum strain ZJ-China [EU008082; 267 nt/268 nt (99%  B0037-K identity); rat, China]  Perhilitan Wild boar (2) 16S rRNA Anaplasma sp. clone Ap20-5a [KX272643; 520 nt/520 nt (100%  WBM3 provided identity); goat, China]  WBK21 Monitor lizard mUniversitysp4 A. phagocytophilum strain ZJ -China [EU008082; 267 nt/268 nt (99%  0284 (FTA card) (1) identity); rat, China]

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A. phagocytophilum sheep China (KJ782381) Anaplasma sp. WBK21 wild boar Malaysia* Anaplasma sp. WBM3 wild boar Malaysia* Anaplasma sp. S0040-L squirrel liver Malaysia* Anaplasma sp. B0014-K bat kidney Malaysia* Anaplasma sp. BU10698-12 buffalo Malaysia* Anaplasma sp. BG538 cow Malaysia* Anaplasma sp. DE25241 deer Malaysia* Anaplasma sp. DE2064 deer Malaysia* Anaplasma sp. DE0345 deer Malaysia* Anaplasma sp. goat China (KX272643) Anaplasma sp. B0037-K bat kidney Malaysia* Anaplasma sp. BU10698-28 buffalo Malaysia* A. phagocytophilum deer Japan (AB196720) A. phagocytophilum strain Webster (U02521) A. phagocytophilum goat China (JN558811) A. phagocytophilum goat China (KR002112) Anaplasma sp. dog South Africa (AY570539) A. bovis monkey Malaysia (KM114612)* A. marginale strain DawnMalaya (CP006847) A. capra goat China (KJ700627) E. ewingii strain Stillwater (NR044747) of E. chaffeensis strain Arkansas (NR074500) Rickettsia rickettsii 16S rRNA gene (U11021)

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Figure 4.27: Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the 16S rRNA gene (434 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified from animal samples in this study.

UniversityThe phylogenetic tree (Figure 4.28) reveals the grouping of the short gene fragments of msp4 detected in this study into two clusters whereby one group (dog, bat and squirrel) is clustered with A. phagocytophilum type strain (strain Webster; Genbank accession no.: EU857674; unpublished) and another group (deer, monitor lizard and bat) with A. phagocytophilum detected in a rat from China (strain ZJ-China; GenBank accession no: EU008082) (Zhan et al., 2008).

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A. phagocytophilum human USA (JQ522935) A. phagocytophilum donkey Italy (AY702925) A. phagocytophilum strain Webster(EU857674) Anaplasma sp. dog Malaysia (KR920045)* B0014-K bat kidney Malaysia* S0040-L squirrel liver Malaysia* A. phagocytophilum strain AP-V1 deer USA (AY530197) A. phagocytophilum sheep China (GQ412346) A. phagocytophilum bison Poland (AY706388) A. phagocytophilum bovine Spain (AY829456) Anaplasma sp. cattle France (KJ832669) A. phagocytophilum roe deer Spain (AY829457) A. phagocytophilum sheep France (EU857673) A. phagocytophilum roe deer Germany (AY706386) DE25206 deer Malaysia* 0284 water monitor lizard Malaysia* A. phagocytophilum rat China (EU008082) B0037-K bat kidney Malaysia* A. centrale Israel (AF428090)Malaya A. marginale strain Oklahoma USA (AY010252) of A. ovis strain Idaho USA (AF393742) 0.05

Figure 4.28: Phylogenetic relationships among various A. phagocytophilum based on partial sequences of the msp4 gene (259 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. * : Representative sequences amplified from animal samples in this study.

For groEL-PCR assay, 12 deer from VRI and seven animal samples from the forestUniversity areas [five organ samples (two kidneys, one spleen and one liver from three bats; one spleen from a squirrel), one monitor lizard blood sample stored in FTA card and a wild boar)] were positive, producing approximately 573 bp amplicons by using heminested primer pair EphplgroEL(569)F/ EphgroEL(1142)R (Figure 4.29). Five amplicons were selected for sequencing but only three sequences were successfully obtained for analysis. BLAST analyses of three sequences show the identification of

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Anaplasma sp. (clone SY49; GenBank accession no.: KF728361) (Dong et al., 2014)

(Table 4.9). All three sequences were used to construct a dendrogram (Figure 4.30). All the short fragments were in one cluster with Anaplasma spp. detected in ticks from China

(isolate BL126-13; GenBank accession no.: KJ410303) (Kang et al., 2014); (isolate

TC250-2; GenBank accession no.: KJ410304) (Kang et al., 2014); (clone SY49;

GenBank accession no.: KF728361) (Dong et al., 2014).

Malaya of

Figure 4.29: Amplification of approximately 573 bp of the groEL gene fragments of A. phagocytophilum from samples using EphplgroEL(569)F/EphgroEL(1142)R nested amplification primer pair. M: 100 bp DNA markers (Solis BioDyne, Estonia); 1: Negative control (miliQ water); 2-3: EphplgroEL(569)F/EphgroEL(1142)R-positive samples; 4: Positive control (A. phagocytophilum genomic DNA extracted from IFA slides).

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Anaplasma sp. D. nutalli China (KJ410304) Anaplasma sp. H. asiaticum China (KJ410303) Anaplasma sp. H. longicornis China (KF728361) Anaplasma sp. DE25208 deer Malaysia* Anaplasma sp. DE5576 deer Malaysia* Anaplasma sp. DE0345 deer Malaysia* A. phagocytophilum cattle China (KF569919) Anaplasma sp. raccoon Japan (JN588562) A. phagocytophilum moose Sweden (KC800986) A. marginale cattle R. microplus Philippines (JQ839003) A. centrale R. simus South Africa (AF414866) E. ruminantium strain Umbanein (DQ647005) E. ewingii human USA (AF195273) E. canis strain Florida (U96731) E. chaffeensis strain Arkansas (L10917) E. muris I. persulcatus Russia (GU358686)

0.05 Malaya

Figure 4.30: Phylogenetic relationships among various Anaplasma spp. based on partial sequences of the groEL gene (463 bp). The dendrogram was constructed using the neighbour-joining method of the MEGAof software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. * : Representative sequences amplified from animal samples in this study.

4.6.2.4 Molecular characterisation of A. bovis from animal blood DNA samples

A total of 36 animal samples from VRI (24 deer, six buffaloes, four cows and two goats) and 17 animal samples from the forest areas [12 organ samples (four kidneys, three livers and three spleens from five bats; one liver and one spleen from a squirrel), three wildUniversity boars and two monitor lizard blood samples stored in FTA cards] were subjected to amplification of 551 bp of the 16S rRNA gene of A. bovis by using primer pair

AB1f/AB1r (Figure 4.19). A total of 32 animal samples from VRI (24 deer, five buffaloes, two goats and a cow) and 16 animal samples from the forest areas [12 organ samples (four kidneys, three livers and three spleens from five bats; one liver and one spleen from a squirrel), three wild boars and a monitor lizard blood sample stored in FTA

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card] were positive. BLAST analyses of 12 sequences reveal the presence of three sequence variants of A. bovis. The details of the BLAST analyses are shown in Table

4.10. All the sequences were used to build a dendrogram (Figure 4.31). The phylogenetic analysis shows that the A. bovis detected can be grouped into three clusters: A. bovis detected in monkey from Malaysia (this study, section 4.6.2.1), A. bovis detected in wild boar from Malaysia (this study, section 4.6.2.1) and A. bovis detected in H. longicornis from China (isolate tick 17/China/2013; GenBank accession no.: KP314250) (Sun et al.,

2015).

Table 4.10: BLAST analyses of partial 16S rRNA gene sequences of A. bovis amplified from animal samples from different sources in this study.

Location Animal host (n) BLAST result (nearest match) Representative samples for Malayaconstruct of dendrogram  VRI, Ipoh Goat (1) A. bovis isolate Ab-YN219  G4699 Buffalo (1) [KU509996; 437 nt/437 nt (100%  BU10698-2 Deer (3) identity);of goat, China]  DE25211  DE25230  DE0345 Cow (1) Anaplasma sp. clone WBM1  BPK2-6219 [KU189194; 450 nt/450 nt (100% identity); wild boar, Malaysia] (A. bovis type II) Forest areas  Kuala Bat’s kidney (2) A. bovis clone 115 [KM114613;  B0014-K Lompat 447 nt/447 nt (100% identity);  B0037-K Bat’s spleen (1) monkey, Malaysia] (A. bovis type  B0032-S Squirrel’s liver (1) I)  S0040-L  Perhilitan Monitor lizard A. bovis clone 115 [KM114613;  0257 provided (FTA card) (1) 447 nt/447 nt (100% identity); Universitymonkey, Malaysia] (A. bovis type I) Wild boar (1) Anaplasma sp. clone WBM1  WBM3 [KU189194; 450 nt/450 nt (100% identity); wild boar, Malaysia] (A. bovis type II)

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A. bovis monkey Malaysia (KM114612)* 0257 FTA card monitor lizard Malaysia* S0040-L squirrel liver Malaysia* B0037-K bat kidney Malaysia* B0032-S bat spleen Malaysia* B0014-K bat kidney Malaysia* A. bovis monkey Malaysia (KM114613)* A. bovis monkey Malaysia (KM114611)* Ehrlichia bovis 16S rRNA gene (U03775) WBM3 wild boar Malaysia* A. bovis H. longicornis Korea (EU181142) BPK2-6219 cattle Malaysia* A. bovis wild boar Malaysia (KU189194)* A. bovis H. longicornis China (KP314236) A. bovis H. longicornis Japan (AB196475) A. bovis H. longicornis China (KP314250) A. bovis cattle Japan (EU368731) A. bovis goat China (KU509996) A. bovis cattle China (KX115423) DE25211 deerMalaya Malaysia* DE25230 deer Malaysia* of DE0345 deer Malaysia* BU10698-2 buffalo Malaysia* G4699 goat Malaysia* A. centrale vaccine strain (AF318944) A. ovis South Africa OVI (AF414870) A. marginale USA (AF311303) A. marginale R. microplus Philippines (JQ839011) A. phagocytophilum strain Webster (U02521) E. chaffeensis strain Arkansas (NR074500) Rickettsia rickettsii 16S rRNA gene (U11021)

0.02 University Figure 4.31: Phylogenetic relationships among various A. bovis based on partial sequences of the 16S rRNA gene (433 bp). The dendrogram was constructed using the neighbour-joining method of the MEGA software with the maximum composite likelihood substitution model, and bootstrapping with 1,000 replicates. R. rickettsii (U11021) was used as an outgroup. * : Representative sequences amplified from animals in this study.

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4.6.2.5 Molecular characterisation of E. chaffeensis from animal blood DNA

samples

A total of 35 animal samples from VRI (23 deer, six buffaloes, four cows and two goats), 17 animal samples from the forest areas [12 organ samples (four kidneys, three livers and three spleens from five bats; one liver and one spleen from a squirrel), three wild boars and two monitor lizard blood samples stored in FTA cards] were subjected to

PCR assays targeting the approximately 389 bp partial fragment of 16S rRNA of E. chaffeensis, using primer pair HE1/HE3 (Figure 4.21). Only three samples (two cattle and a goat) from VRI and two animal samples from the forest areas (one kidney and one liver from a bat) were positive. Since very faint band was generated from the positive samples, sequence determination of these positive samples were not able to be performed. Malaya 4.7 Molecular detection of Anaplasma spp. and Ehrlichia spp. in stray dogs and dog ticks of A total of 47 dog samples (30 from Klang Valley and 17 from VRI, Ipoh) and 33

R. sanguineus collected from 13 dogs in Klang Valley were subjected to amplification by nested PCR assays targeting the 16S rRNA genes of E. canis (389 bp), E. chaffeensis

(389 bp) and A. phagocytophilum (641 bp).

DNA of E. canis was amplified from 12 (25.5%, 10 from Klang Valley and 2 from VRI, Ipoh) dog blood samples and 17 (51.5%) R. sanguineus. Sequence analysis of 305University nucleotides of five positive samples (three dog blood samples and two R. sanguineus) confirmed 100% similarity with E. canis (strain Jake; GenBank accession no.: CP000107; unpublished) and many other E. canis sequences deposited in the GenBank database.

Furthermore, the 1433 bp full length sequence of the 16S rRNA gene amplified from one of the positive samples (designated as 002, deposited at GenBank as KR920044) demonstrated 99% similarity (three nucleotides difference) with that of the E. canis type

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strain (strain Oklahoma; GenBank accession no.: M73221) (Anderson et al., 1991) and one nucleotide difference with E. canis (strain ECAN_Bkk_07; GenBank accession no.:

EU263991; unpublished). E. chaffeensis 16S rRNA gene was amplified from two (6.0%)

R. sanguineus ticks (deposited at GenBank as KR920047). Sequence analysis revealed

100% (318 nt/318 nt) similarity of the amplified fragments with E. chaffeensis (strain

Arkansas; GenBank accession no.: CP000236) (Dunning Hotopp et al., 2006). No amplification was obtained using the nested PCR assay targeting E. chaffeensis for dog blood samples.

The 16S rRNA genes of A. phagocytophilum were amplified from six (12.8%) dog blood samples and one (3.0%) R. sanguineus. Sequence analysis of the partial fragment of the positive tick sample (deposited at GenBank as KR920046) revealed 99% (562 nt/566 nt) similarity with A. phagocytophilum (strainMalaya Dog2; GenBank accession no.: CP006618; unpublished) but sequence determination of the 16S rRNA gene from A. phagocytophilum-positive dog blood samplesof was not successful due to generation of noisy data. Amplification and sequence analyses of msp4 gene (270 nucleotides) from two 16S rRNA positive-dog blood samples (deposited at GenBank as KR920045) demonstrated 100% similarity to A. phagocytophilum (strain Dog2; GenBank accession no.: CP006618; unpublished) while msp4 gene of A. phagocytophilum was not amplified from 16S rRNA A. phagocytophilum-positive tick.

Table 4.11 summarises the results obtained from the molecular detection of AnaplasmaUniversity spp. and Ehrlichia spp. in animal and tick samples in this study.

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Table 4.11: Summary of the identification of Anaplasma spp. and Ehrlichia spp. DNA in different animal and tick samples collected in this study.

Species Animal hosts Ticks (hosts) A. marginale (A. centrale Cattle R. microplus (cattle) and A. ovis) H. bispinosa (cattle) A. phagocytophilum Bat H. bispinosa (sheep) Cattle H. wellingtoni (goat) Goat D. atrosignatus (vegetation) Monkey R. sanguineus (dog) Monitor lizard Dermacentor spp. (vegetation) Squirrel Haemaphysalis spp. (vegetation, chicken, Deer cat) Dog A. bovis Bat H. bispinosa (cattle, goat, sheep) Buffalo H. hystricis (vegetation) Deer R. sanguineus (dog) Goat A. varanense (snakes) Monkey Dermacentor spp. (vegetation) Wild boar Haemaphysalis spp. (vegetation) Squirrel A. platys Cattle H. bispinosa (cattle) Deer H. hystricis (vegetation) D. atrosignatus (vegetation) A. varanense (snake) A. helvolumMalaya (snake) Haemaphysalis spp. (vegetation, rat) Candidatus A. pangolinii Pangolin Not detected Candidatus A. boleense Cattle ofNot detected Deer Buffalo E. canis Dog R. sanguineus (dog) New sequence variants: Anaplasma spp. (closely Bat related to A. Buffalo phagocytophilum) Cow Deer Squirrel Anaplasma spp. Not detected A. helvolum (snake) (uncharacterised) Haemaphysalis sp. (cat) Ehrlichia spp. Not detected D. atrosignatus (vegetation) (undifferentiated between Haemaphysalis spp. (vegetation) E. chaffeensis and E. R. sanguineus (dog) ewingiiUniversity) Ehrlichia sp. (closely Not detected A. varanense (snake) related to E. ruminantium) Ehrlichia sp. (closely Not detected R. microplus (cattle) related to E. mineirensis) Ehrlichia sp. (closely Not detected R. microplus (cattle) related to Ehrlichia sp. clone EBm52) Ehrlichia sp. (closely Not detected H. bispinosa (cattle) related to Candidatus E. shimanensis)

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CHAPTER 5: DISCUSSION

Limited data is available on the disease burden of anaplasmosis and ehrlichiosis in Malaysia and most SEA countries. Previous investigations showed the presence of antibodies to E. chaffeensis in 44.0% and 14.6% of healthy individuals from Thailand and Indonesia, respectively (Heppner et al., 1997; Richards et al., 2003).

In our recent investigation, IgG antibodies against E. chaffeensis were detected in

34.3%, 29.9% and 9.8% of Malaysian indigenous people, farm workers and blood donors, respectively (Koh et al., 2018). This serological data was consistent with the findings reported in Thailand and Indonesia whereby high antibody prevalences against E. chaffeensis were reported. Comparatively, the seropositivity to A. phagocytophilum was relatively low in the indigenous people (6.9%) and none of the farm workers investigated was seropositive in the recent Malaysian study (KohMalaya et al., 2018). This observation was different from those reported in China whereby higher antibody prevalences against A. phagocytophilum have been reported in farmersof (8.8-33.7%), rural residents (15.4%) and forest area residents (7.1%) have been reported (Hao et al., 2013; Zhang et al., 2008b;

Zhang et al., 2014; X. C. Zhang et al., 2012; Y. Zhang et al., 2012). The variation observed in the seropositivity to A. phagocytophilum could be due to many reasons, for instance, climate and geographical differences can influence the type and distribution of ticks and animal hosts for transmission of A. phagocytophilum (Stuen et al., 2013)

5.1University Identification of ticks 5.1.1 Haemaphysalis spp.

Haemaphysalis was the main genus of ticks identified in a variety of sources, including sheep, cattle, goat, dog, rat, bat, cat and vegetation in this study (Table 4.1).

This finding is consistent with a study carried out by Petney et al. (2007), who described the identification of about 104 species of ticks from 12 genera in SEA countries. In that

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study, Haemaphysalis was the most species-abundant genus comprising 52 species. In this study, BLAST analyses show that tick mitochondrial 16S rRNA gene is a better DNA marker than 28S rRNA gene for differentiation amongst the species of Haemaphysalis ticks, as sequence analysis of the gene allows a better discrimination of ticks to the species level (Table 4.2). Comparatively, the gene encoding the 28S rRNA is more conservative than 16S rRNA gene and BLAST analyses of the sequences obtained only enable the identification of ticks up to genus level (Sections 4.2.3 and 4.2.4).

The main Haemaphysalis species identified in ticks collected from cattle and sheep investigated in this study was H. bispinosa. Besides that, H. bispinosa has also been detected in ticks collected from bat (n=1), rat (n=1), goats (n=2), chicken (n=1) and dogs

(n=2) from aboriginal villages and Kuala Lompat reserve forest in this study (Table 4.2). H. bispinosa was previously reported in Peninsular MalaysiaMalaya in ticks collected from wild animals (bearcat, dhole, Asian leopard cat, Malayan weasel, Malayan civet, toddy cat and Indian boar) and domestic animals (sheep,of goat and dog) (Hoogstraal et al., 1969). Recently, H. bispinosa has also been detected in ticks collected from dogs in Sabah,

Malaysia (Wells et al., 2012) and various sources (including vegetation, goats, cattle and dogs) in Thailand, India and Bangladesh (Brahma et al., 2014; Fuehrer et al., 2012;

Malaisri et al., 2015). H. bispinosa has a three host life cycle which requires a single host during each feeding stage (larva, nymph and adult) (Parola and Raoult, 2001) and is associated with tick-borne pathogens such as Bartonella bovis in Malaysia (Kho et al., 2015aUniversity); Rickettsia spp. in Thailand (Malaisri et al., 2015); B. burgdoferi, Babesia bigemina, Rickettsia spp. and Theileria sergenti in China (Yu et al., 2016; Yu et al., 2015) and A. bovis in Bangladesh (Qiu et al., 2016). In this study, H. bispinosa has been shown to harbour A. marginale, A. bovis, A. platys, A. phagocytophilum and Ehrlichia sp.

(closely related to Candidatus E. shimanensis) (Tables 4.4 and 4.5).

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H. wellingtoni was the main species detected from the peri-domestic animals

(dogs, cats and chickens) in aboriginal villages in this study (Table 4.2). H. wellingtoni has been previously reported in ticks collected from jungle fowls in Peninsular Malaysia

(Konto et al., 2015), chickens from Thailand (Parola et al., 2003) and chickens, rats and birds from Indonesia (Durden et al., 2008). The free-range system of animals rearing in the aboriginal villages might increase the susceptibility of animals to tick infestation. H. wellingtoni has been reported to be associated with Coxiella spp. and Rickettsia spp. in

Malaysia (Khoo et al., 2016) and Kyasanur forest disease virus in China (Yu et al., 2015).

In this study, A. phagocytophilum has been detected in H. wellingtoni ticks (Table 4.6).

In this study, some of the ticks collected from vegetation and peri-domestic animals (dog and cat) were identified as H. hystricis (Table 4.2). H. hystricis has been previously collected from dogs, rats and tree shrewsMalaya in Peninsular Malaysia (Ernieenor et al., 2017), vegetation in Thailand (Malaisri et al., 2015); cattle, dogs and vegetation in Laos (Vongphayloth et al., 2016) and dogs, ofdeer, rats and wild boars in Indonesia (Durden et al., 2008). H. hystricis has been associated as a vector for diseases including ehrlichiosis (Ehrlichia spp.), human granulocytic anaplasmosis (A. phagocytophilum) and Japanese spotted fever (R. japonica) (Parola et al., 2003; Yu et al., 2015). In this study, H. hystricis ticks harbouring A. platys and A. bovis have been detected (Tables 4.4 and 4.5).

Two questing ticks collected from the vegetation of a forest at Sg. Deka Elephant Sanctuary,University Terengganu were identified as H. shimoga in this study (Table 4.2). There is no previous report on distribution of H. shimoga in Malaysia. H. shimoga has been reported in questing ticks from Thailand (Malaisri et al., 2015). H. shimoga harbouring

Rickettsia spp., Anaplasma spp. and Coxiella-like endosymbiont has been reported in

Thailand (Ahantarig et al., 2011; Arthan et al., 2015; Malaisri et al., 2015). The distribution of H. shimoga and its capability in carrying different pathogens have not been

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widely reported in Malaysia. Anaplasma spp. and Ehrlichia spp. have not been detected from two H. shimoga identified in this study.

A number of ticks collected from vegetation (n=10), rat (n=1), squirrel (n=1) and dog (n=4) in aboriginal villages and the forest areas were only recognised as

Haemaphysalis spp. as there are no matching sequences of the mitochondrial 16S rRNA gene in the GenBank database (Table 4.2). Based on sequence analyses of tick DNA, four sequence types demonstrating the highest sequence similarity to either H. obesa (94%),

H. hystricis (97%), H. asiatica (93%) or H. qinghaiensis (93%) to their closest relatives were recognised in this study. The Haemaphysalis ticks were found to carry various

Anaplasma spp. (A. phagocytophilum, A. bovis, A. platys and uncharacterised Anaplasma sp.) and Ehrlichia spp. (E. chaffeensis/E. ewingii) (Tables 4.4-4.6). Further identification of these ticks to the species level should be carried outMalaya to provide a better understanding on the distribution and the role of Haemaphysalis ticks in disease transmission. 5.1.2 Dermacentor spp. of A number of ticks from the vegetation (n=7) and ticks collected from rat (n=3) in the forest areas were identified as D. atrosignatus in this study (Table 4.2). The distribution of D. atrosignatus has been previously reported in various sources including vegetation, pigs, dogs, pangolins, monitor lizards, buffaloes and snakes in Malaysia (both

Peninsular Malaysia and Sarawak) (Hoogstraal and Wassef, 1985a; Mariana et al., 2011).

Besides that, D. atrosignatus has also been reported in a few SEA countries including ThailandUniversity (vegetation), Indonesia (vegetation, pigs, bats, pangolins and shrews) and Philippines (vegetation and pigs) (Durden et al., 2008; Hoogstraal and Wassef, 1985a;

Malaisri et al., 2015). To the best of our knowledge, there is no report of D. atrosignatus harbouring pathogens in Malaysia or other Asian countries. However in this study, D. atrosignatus ticks were found to carry A. platys, A. phagocytophilum and Ehrlichia spp.

(E. chaffeensis/E. ewingii) (Tables 4.4 and 4.6).

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Two ticks collected from a dog in aboriginal village were identified as D. auratus in this study (Table 4.2). The ticks have previously been identified from various sources including vegetation, snakes, civets and pigs in Peninsular Malaysia and Sarawak

(Hoogstraal and Wassef, 1985b). The distribution of D. auratus has also been recorded in a few Asian countries including Thailand, Myanmar, Vietnam, Laos, Indonesia, Sri

Lanka, India, Nepal and Bangladesh from the vegetation and a variety of animal hosts including dogs, bears, pigs, rats, deer, squirrels toddy cats and tree shrews (Hoogstraal and Wassef, 1985b; Malaisri et al., 2015; Parola et al., 2003; Vongphayloth et al., 2016).

D. auratus has been reported as a vector harbouring pathogens such as Anaplasma spp.,

Rickettsia spp., Francisella spp. and Hepatozoon spp. (Parola et al., 2003; Sumrandee et al., 2015, 2016). Anaplasma spp. and Ehrlichia spp. have not been detected from two D. auratus identified in this study. Since D. auratus is widelyMalaya distributed in Asian countries and is capable to transmit potential human pathogens, more attention should be paid on the distribution of this tick in Malaysia andof understanding its role in spreading diseases. Additionally, a high number of questing ticks (n=25) collected from the Kuala

Lompat reserve forest and a forest at Sg. Deka Elephant Sanctuary, Terengganu were recognised as Dermacentor spp. only in this study (Table 4.2) due to the lack of matching sequences to the mitochondrial 16S rRNA gene in the GenBank database. Sequence analyses of the tick 16S rRNA gene reveal the presence of four sequence types demonstrating the highest similarity with D. andersoni (94%), D. nuttalli (93%) and D. silvarumUniversity (93%), with their closest relatives in the GenBank database, respectively. A. bovis and A. phagocytophilum were detected in Dermacentor spp. ticks in this study

(Tables 4.4-4.6). Further identification of these ticks to the species level should be carried out.

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5.1.3 Amblyomma spp.

In this study, three types of Amblyomma ticks have been identified from ticks collected from vegetation (Amblyomma spp.), a skink (A. helvolum) and snakes (A. helvolum and A. varanense) (Table 4.2). Amblyomma ticks have been reported infesting reptilian hosts, for example, monitor lizards (A. helvolum and A. varanense), skinks (A. helvolum), snakes (A. helvolum, A. varanense and Amblyomma cordiferum) as reported by previous investigation from various Asian countries (Auffenberg, 1988; Chao et al.,

2013; Durden et al., 2008; Sumrandee et al., 2014; Vongphayloth et al., 2016). In addition, Amblyomma ticks have also been identified from vegetation (A. americanum,

A. helvolum, A. testudinarium, Amblyomma spp. and Amblyomma integrum) and animal hosts other than reptiles, such as bats (A. cordiferum), rats (Amblyomma spp.), squirrels (Amblyomma spp.), tree shrews (Amblyomma spp.),Malaya deer (A. americanum), cattle (A. testudinarium), dogs (A. testudinarium) and pigs (A. helvolum and A. testudinarium) (Ahamad et al., 2013; Durden et al., 2008of; Malaisri et al., 2015; Mariana et al., 2011; Rudenko et al., 2016; Vongphayloth et al., 2016). Amblyomma ticks have been reported as a vector for tick-borne pathogens including E. ruminantium, A. phagocytophilum, B. burgdorferi, Rickettsia rickettsii and Rickettsia spp. (Malaisri et al., 2015; Oliveira et al.,

2010; Rudenko et al., 2016; Teshale et al., 2015). The distribution of Amblyomma ticks in other animal species in Malaysia is yet to be explored. In this study, A. bovis, A. platys, uncharacterised Anaplasma sp. and Ehrlichia sp. (closely related to E. ruminantium) were detectedUniversity in Amblyomma ticks (A. helvolum and A. varanense) (Tables 4.4 and 4.5). 5.1.4 Rhipicephalus spp.

R. microplus is another species of ticks besides H. bispinosa, which was collected from cattle in livestock farms in this study (Table 4.2). The tick has been reported in ruminants (mainly cattle) from SEA countries including Thailand, Philippines, Laos,

Indonesia and Malaysia (Changbunjong et al., 2009; Durden et al., 2008; Tay et al., 2014;

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Vongphayloth et al., 2016; Ybañez et al., 2013b). R. microplus is known to transmit some bovine diseases including anaplasmosis, babesiosis and theileriosis (Kocan et al., 2010;

Yu et al., 2015). In this study, A. marginale, Ehrlichia sp. (closely related to E. mineirensis) and Ehrlichia sp. (closely related to Ehrlichia sp. clone EBm52) were detected in R. microplus (Tables 4.4 and 4.5).

R. sanguineus is a common tick vector found in dogs from many parts of SEA countries including Thailand, Philippines, Laos and Indonesia (Changbunjong et al.,

2009; Durden et al., 2008; Vongphayloth et al., 2016; Ybañez et al., 2012). In addition,

R. sanguineus had been reported to harbour pathogens such as E. canis, A. platys, H. canis, R. rickettsii and Rickettsia conorii (Latrofa et al., 2014). It is the main species of ticks collected from dogs in this study. A. bovis, A. phagocytophilum, E. canis and Ehrlichia spp. (E. chaffeensis/ E. ewingii) were detectedMalaya in R. sanguineus in this study (Table 4.4 and Section 4.7). of 5.2 Detection and identification of Anaplasmataceae DNA in ticks

This study shows that a total of 61.5% ticks infesting livestock (cattle and sheep),

28.8% ticks collected from vegetation and small animals in the forest areas and 37.0% ticks infesting peri-domestic animals in aboriginal villages were PCR-positive for

Anaplasmataceae DNA. Of the 17 amplified fragments obtained from 14 cattle ticks (12

H. bispinosa and two R. microplus) and three H. bispinosa sheep ticks, sequences matchingUniversity to those of A. marginale, A. bovis, A. platys and A. phagocytophilum were obtained. Additionally, a representative of one, two and one 16S rDNA sequences obtained from cattle ticks were found to match with those of Ehrlichia sp. strain EBm52

(Parola et al., 2003), E. mineirensis (Cabezas-Cruz et al., 2012) and Candidatus E. shimanensis (Kawahara et al., 2006), respectively. A. bovis was identified from ticks infesting peri-domestic animals in aboriginal villages, while both A. phagocytophilum

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and A. platys were identified from questing ticks and a tick collected from a rat in the forest areas (Table 4.4). The significance of the detection of each Anaplasma spp. and

Ehrlichia spp. in ticks and animals is discussed in the following sections.

5.3 Molecular detection of A. marginale in tick and animal samples

A. marginale is known to infect cattle and R. microplus ticks from various parts of the world (Aubry and Geale, 2011; Kocan et al., 2010). In this study, sequence analyses of representative PCR-positive samples confirmed the presence of A. marginale in cattle,

R. microplus and H. bispinosa ticks (Tables 4.4 and 4.7). However, the presence of other

Anaplasma spp. closely related with A. marginale such as A. centrale and A. ovis cannot be ruled out as these organisms share similar 16S rRNA sequences (Figures 4.10 and 4.23). This result is in agreement with a pilot study carriedMalaya out previously whereby cattle blood samples and R. microplus ticks collected from an livestock farm were tested positive for A. marginale and other closely ofrelated species (Tay et al., 2014). In addition, the presence of A. marginale (in cattle and R. microplus tick) and A. centrale (in cattle only) are further confirmed by the sequence analyses of the long 16S rDNA fragments

(Figures 4.12 and 4.24). The findings in this study are thus consistent with reported cases of A. marginale in ruminants (cattle and water buffaloes) and R. microplus ticks from

Philippines (Ochirkhuu et al., 2015; Ybañez et al., 2013b) and Thailand (Saetiew et al.,

2015). UniversityFor the first time in Malaysia, A. marginale (potentially A. centrale and A. ovis) has been detected in H. bispinosa ticks collected from cattle. Previously, B. bovis (another tick-borne pathogen) had been reported in H. bispinosa collected from cattle in Malaysia

(Kho et al., 2015a). But the role of H. bispinosa as a vector requires further investigation as the DNA detected might have come from host blood ingested by the ticks.

Experimental transmission studies are needed to confirm the vector competent of H.

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bispinosa for transmission of A. marginale. Besides that, phylogenetic analyses (Figures

4.10, 4.12 and 4.23-4.24) based on 16S rRNA gene also showed that only one sequence type of A. marginale was detected in this study and the organism was grouped in one cluster with A. marginale in the R. microplus ticks from Philippines. This suggests that a similar strain of A. marginale is circulating in SEA countries.

Cattle is known to develop persistent A. marginale infections which may explain for the high detection rate of Anaplasmataceae DNA (60.7%) in the blood samples of apparently healthy cattle examined in this study (Table 4.7). Persistently infected cattle

(also known as carrier cattle) are characterised by a low level of bacteraemia in blood, life-long immunity and the cattle usually do not develop clinical disease except under stress or immunosuppressed (Kocan et al., 2010). Due to the high prevalence of anaplasmosis detected in cattle and ticks, implementationMalaya of tick control or vaccination for cattle is important as the animal health and production can be affected by bovine anaplasmosis. of

5.4 Molecular detection of A. bovis in tick and animal samples

A. bovis has been reported to infect a wide variety of mammalian hosts and tick vectors in various parts of the world (Table 2.1) (Atif, 2016; Battilani et al., 2017). In

Asia, A. bovis has been detected in animals (including dogs, deer, goats, cattle, cats, rodents and wild boars) and ticks (including H. longicornis, H. shimoga, H. megaspinosa, H. Universitybispinosa, Haemaphysalis langrangei, Dermacentor niveus and R. sanguineus) from China, Bangladesh, Japan, Korea, India, Taiwan and Thailand (Table 2.2). Based on sequence analyses of the short 16S rDNA sequences in this study, A. bovis DNA has been detected in a variety of animals (including goats, deer, monkeys, wild boar, cow, monitor lizard, buffalo, bats and squirrel (Tables 4.7 and 4.10); ticks [H. bispinosa (collected from cattle, goat and sheep), R. sanguineus (collected from dog) and A. varanense (collected

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from snakes)] and questing ticks (H. hystricis, Haemaphysalis spp. and Dermacentor spp.) (Tables 4.4-4.6). These findings imply that A. bovis is a common species spreading across animals and ticks in Malaysia.

In recent years, A. bovis DNA has been detected in various Haemaphysalis species especially in Asian countries, suggesting the potential role of Haemaphysalis ticks as vector for A. bovis. A. bovis has been detected in H. bispinosa cattle tick and R. sanguineus dog tick in Bangladesh (Qiu et al., 2016). In this study, a pool of R. sanguineus dog ticks collected from an aboriginal village was positive for A. bovis (Table

4.4). R. sanguineus ticks are commonly known as the vector for E. canis (and potentially

A. platys) which cause infection in dogs (Dantas-Torres, 2008). Their role in transmitting

A. bovis is unknown. As dogs have been reported to be infected by A. bovis in Japan (Sakamoto et al., 2010), it is postulated that R. sanguineusMalaya ticks might acquire the organism during blood feeding on infected dog hosts. Another interesting finding in this studofy is the presence of A. bovis DNA in the A. varanense snake ticks. This represents the first detection of A. bovis in Amblyomma ticks in Malaysia and Asia. A. bovis has been previously reported to infect Amblyomma ticks in Africa (Dumler et al., 2001). As Anaplasma spp. have been reported in reptiles including sand lizard, Northern alligator lizard, Pacific gopher snake and common garter snake (Ekner et al., 2011; Nieto et al., 2009), it is speculated that reptiles may play a role in the transmission cycle of A. bovis. Sequence analysis of Anaplasma 16S rRNA gene alsoUniversity confirmed the presence of A. bovis in several species of questing ticks (H. hystricis, Haemaphysalis spp. and Dermacentor spp.) collected from the forest areas, thus providing evidence on the existence of Anaplasma spp. in ticks collected in our forest. It is possible for livestock and wildlife to get infected upon exposure to these Amblyomma ticks.

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Based on the analysis of the amplified 16S rRNA partial gene fragments from different animal and tick samples, A. bovis can be differentiated into two sequence types

(A. bovis type I and A. bovis type II), based on one nucleotide difference in the sequences

(Figures 4.20 and 4.31). A. bovis type I had a sequence variation (A →T) in the amplified fragments of 16S rRNA gene region as compared to A. bovis type II. Preliminary results showed that A. bovis type I was detected in mostly monkeys, monitor lizard, organ tissues of small mammals collected from the forest areas and ticks collected from the rural and the forest areas while A. bovis type II was more prevalent in domestic animals and ticks collected from domestic animals (goat, deer, cow) and ticks collected from sheep and cattle (livestock farms) (Tables 4.4-4.7, 4.10). The significance noted in the strain differentiation of A. bovis (two sequence types) is yet to be determined. As A. bovis has not been successfully cultivatedMalaya in vitro, little is known about its biology and virulence properties. Hence, further investigation on the zoonotic potential (despite no human infections has been reportedof yet) of A. bovis is needed.

5.5 Molecular detection of A. platys in tick and animal samples

A. platys has been known to show unique tropism for dog platelets, and hence, is the aetiological agent for infectious canine cyclic thrombocytopenia (Rar and Golovljova,

2011). In this study, A. platys was detected from cattle, deer and ticks (H. bispinosa, A. helvolum, A. varanense, D. atrosignatus, H. hystricis and Haemaphysalis spp. collected fromUniversity cattle, snake, rat and questing ticks in the forest areas) (Tables 4.4 and 4.7). A. platys has been previously reported in Bactrian camels and red deer in China; red foxes in

Portugal; cat in Thailand (Cardoso et al., 2015; Y. Li et al., 2015; Li et al., 2016; Salakij et al., 2012) while a number of Anaplasma species closely related to A. platys have been reported in ruminants (cattle, goats and sheep) from Italy and China (Liu et al., 2012;

Zobba et al., 2014). These findings suggest that A. platys has the ability to infect a wider

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range of hosts than is initially thought. The role of vertebrate species either as accidental hosts or maintenance hosts requires future investigation through experimental transmission studies.

Apart from the report in R. sanguineus ticks, A. platys has also been reported in

Haemaphysalis ticks (H. longicornis and H. flava), D. auratus and Ixodes nipponensis ticks from Thailand and Korea (Chae et al., 2008; Parola et al., 2003). As human infection caused by A. platys has been reported in two women from Venezuela (Arraga-Alvarado et al., 2014), further studies are required to determine the zoonotic potential of A. platys.

5.6 Molecular detection of A. phagocytophilum in tick and animal samples

A. phagocytophilum has been documented as the causative agent for human granulocytic anaplasmosis (HGA) (Dumler, 2005).Malaya Previously, a low seroprevalence (6.9%) of A. phagocytophilum has been detected in the indigenous people in Malaysia (Koh et al., 2018). At the moment there isof no report on the molecular detection of A. phagocytophilum in Malaysia.

In this study, A. phagocytophilum DNA was detected in various animal blood samples (cattle, deer, dog, monkey, goat and monitor lizard and organ samples of small mammals collected from the forest areas including bats and a squirrel using PCR assays targeting the short 16S rRNA and msp4 genes of Anaplasma spp. (Tables 4.7 and 4.9;

Section 4.7). A. phagocytophilum was also detected from ticks infesting dog, chicken, sheep,University goat and cat (R. sanguineus, H. bispinosa , H. wellingtoni and Haemaphysalis spp.) and questing ticks (D. atrosignatus, Dermacentor spp. and Haemaphysalis spp.) (Tables

4.4 and 4.6).

This study presents the first report of A. phagocytophilum in animal and tick samples in Malaysia. Although the presence of A. phagocytophilum has not been reported in other SEA countries, evidences of its presence are available in animals and ticks

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collected from China, Japan, Korea and Taiwan (Table 2.2). Previously, Anaplasma spp.

(undifferentiated between A. platys and A. phagocytophilum) have been detected from cattle and ticks in a pilot study in Malaysia (Tay et al., 2014) and cattle from Philippines

(Ybañez et al., 2013c). Interestingly, this study also confirms the presence of A. phagocytophilum in questing ticks collected from the forest areas and ticks collected in animals from livestock farms and aboriginal villages (Table 4.4 and 4.6). Ixodes ticks

(especially I. ricinus, I. pacificus, I. scapularis and I. persulcatus) have been shown to be competent vectors of A. phagocytophilum and play a role in the transmission of the organism to humans and animals (Rar and Golovljova, 2011). In this study, A. phagocytophilum has been detected in Dermacentor and Haemaphysalis ticks collected from vegetation and animals (Table 4.4), however the role of these ticks in carrying pathogenic A. phagocytophilum strains requires furtherMalaya investigation. In recent years, A. phagocytophilum has been detected in ruminants including cattle, sheep and goats (Rar and Golovljova,of 2011 ). In this study, only one H. bispinosa (collected from sheep), one H. wellingtoni (collected from goat), a cattle and a goat were positive for A. phagocytophilum DNA (Tables 4.4, 4.6-4.7). Majority of the Anaplasma spp. detected in ruminants (mostly cattle) was A. marginale. This might be due to PCR bias as only the abundant species in a mixed DNA sample would be amplified.

It was unfortunate that only short fragments of 16S rRNA and msp4 genes were available for the identification of A. phagocytophilum in this study, as the attempts of amplifyingUniversity longer 16S rRNA and msp4 genes from A. phagocytophilum-positive samples were not successful. Troubleshooting of the relevant PCR assays should be carried out to solve this problem, especially for further conformation and characterisation of A. phagocytophilum detected in this study.

Previous studies reported that A. phagocytophilum strains might have preferential hosts for multiplication and survival. For example, A. phagocytophilum strain (Ap-

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Variant 1) detected in white-tailed deer has not been associated with human disease and it cannot cause an infection in mice, while human pathogenic variant (Ap-ha) can cause infection in human, cattle, sheep, horses, dogs, I. ricinus, I scapularis, I. persulcatus and

Ixodes ovatus (Jin et al., 2012; Rar and Golovljova, 2011; Stuen et al., 2013). This information will be beneficial for public health authority and epidemiologist in the investigations of outbreaks caused by A. phagocytophilum.

5.7 Molecular detection of Candidatus A. pangolinii in pangolins

To the best of our knowledge, this is the first evidence of the detection of

Anaplasma DNA in pangolins (M. javanica). Anaplasma spp. have previously been detected in both A. javanense ticks from Thailand (Parola et al., 2003) and H. hystricis ticks from Taiwan (Khatri Chhetri et al., 2016) collectedMalaya from pangolins. The nearly full length 16S rDNA sequences (1439 bp; GenBank accession no.: KU189193) obtained from the pangolin blood samples in this studyof demonstrated the closest similarity with A. bovis (97.7%) and next closest to A. phagocytophilum (97.6%) and A. platys (97.5%).

The sequence divergence of 2.3% (in comparison with A. bovis) is greater than that 1.0% of the cut off value required for delineation of a new species based on 16S rRNA gene

(Janda and Abbott, 2007). In addition, phylogenetic analysis also showed that the pangolin-associated Anaplasma sp. is segregated on a distinct branch amongst members of the genus Anaplasma and Ehrlichia (Figure 4.24). It is believed that it could be a potentialUniversity novel Anaplasma sp. and the species was tentatively named as “Candidatus Anaplasma pangolinii” in accordance with the host where it was derived.

Interestingly, a portion of the 16S rRNA gene of Candidatus A. pangolinii

(GenBank accession no.: KU189193) was found to be 99.9% matching (954 nt/955 nt) with the 16S rRNA gene of Anaplasma sp. strain AnAj360 (amplified from A. javanense collected from a pangolin in Thailand, a neighbouring country of Malaysia) (Parola et al.,

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2003). A. javanense has been reported to infest three Asian pangolins (M. javanica, Manis pentadactyla and Manis crassicaudata) (Mohapatra et al., 2015). Hence, there is a high possibility that A. javanense tick is the maintenance vector for Candidatus A. pangolinii detected in this study. However, there are also possibility that the organism was merely present in the blood of the infected pangolins. Further investigation is necessary to establish the potential host-vector relationship of A. javanense ticks, pangolins and

Candidatus A. pangolinii. Further studies on the pathogenicity and zoonotic potential of this organism merits further investigation.

5.8 Detection of A. phagocytophilum and E. canis in ticks and dog blood samples

Little is known about anaplasma and ehrlichial infections in animals in Malaysia.

Serological detection of E. canis in dog samples in Malaysia was reported by Rahman et al. (2010) where 15.0% of dogs tested were serologically positive by using indirect fluorescent antibody test (IFAT). A recent study reported that 56.7% of dogs from East

Malaysia investigated were seropositive to A. platys and E. canis by using a commercial test kit (SNAP 4Dx) (Mohammed et al., 2017). In this current study, serological analysis of dog blood samples was carried out by using a commercial test kit (SNAP 4Dx, IDEXX,

Laboratories, ME, USA). Antibodies against E. canis were detected in 39.5% of the dogs

(Section 4.7). This finding suggested that E. canis is a common vector-borne pathogen found in dogs investigated in this study. E. canis has also been investigated in other Asian countriesUniversity including Thailand (IFA; 71.0%), of Philippines Malaya (ELISA; 59.0%), Taiwan (SNAP 4Dx; 9.9%), Hong Kong (SNAP 4Dx; 4.0%), India (dot-ELISA; 86.9%), China (SNAP

4Dx; 2.2%) and Russia (SNAP 4Dx; 2.4%) with variable seropositivities (Baticados and

Baticados, 2011; Kottadamane et al., 2017; Suksawat et al., 2001; Volgina et al., 2013;

Wong et al., 2011; Xia et al., 2012; Yuasa et al., 2012).

139 A low seroprevalence of A. phagocytophilum was detected in four (9.3%) dogs which were also co-infected with E. canis (Section 4.7). This is the first report on the seroprevalence of A. phagocytophilum in Malaysian dogs. Previously, serological evidences of A. phagocytophilum have been reported in dogs from Thailand (IFA;

58.0%), Korea (SNAP 4Dx; 15.6%), China (SNAP 4Dx; 0.5%) and Russia (SNAP 4Dx;

0.6%) (Suh et al., 2017; Suksawat et al., 2001; Volgina et al., 2013; Xia et al., 2012).

The detection of A. phagocytophilum and E. canis DNA in this study suggests the need for appropriate measures for prevention and control of tick-borne diseases in dogs as A. phagocytophilum and E. canis may potentially cause human infections (Perez et al.,

2006).

Canine ehrlichiosis is one of the most reported tick-borne diseases in SEA countries. E. canis has been reported in dogs andMalaya ticks from Malaysia, Thailand, Philippines and Cambodia (Table 2.3). The high detection rate of E. canis DNA in dog blood samples suggests that canine ehrlichiosisof might be prevalent in this region. E. canis DNA was obtained in dogs from this study with a higher detection rate (25.5%) as compared to a previous study by Nazari et al. (2013) whereby only 2.0% of the tested dogs from private veterinary clinics and a veterinary teaching hospital were positive.

Additionally, E. canis has been detected in R. sanguineus ticks collected from dogs in this study for the first time in Malaysia (Section 4.7). This finding provides evidence that dogs can act as a host and R. sanguineus is a tick vector for E. canis in Malaysia. UniversityE. canis has been reported in patients and blood bank donors from Venezuela and Costa Rica, respectively (Bouza-Mora et al., 2017; Perez et al., 2006). Hence, the zoonotic potential of E. canis should not be overlooked as R. sanguineus can bite human and play a role in the transmission of tick-borne pathogens to human (Gray et al., 2013).

As dogs are pet animals in most family in Malaysia and have close contact with their

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owners, a nationwide investigation of ehrlichiosis should be carried out in order to assess the impact of the disease to general population.

5.9 Molecular detection of new variants of Anaplasma spp. and Ehrlichia spp.

in tick and animal samples

Due to the advancement in molecular techniques, new genetic variants of

Anaplasma and Ehrlichia have been detected in ticks and animal samples recently (Table

2.2). A few new genetic variants or potentially new species have been detected in ticks

(R. microplus, H. bispinosa, A. varanense and Haemaphysalis sp.) and animal bloods and organ samples (deer, cow, buffaloes, wild boars, bat and squirrel) based on sequence analysis of the 16S rRNA and groEL genes in this study. Interestingly, BLAST analysis of sequence obtained from a Haemaphysalis cat Malaya tick (labelled as SP002-F) in an aboriginal village shows low sequence similarity to the Anaplasma reference strains. The amplified 16S rDNA sequence of the Anaplasmaof sp. demonstrated 98% (250 nt/255 nt; GenBank accession no.: KY046288) similarity with Anaplasma sp. detected in H. longicornis tick from China (GenBank accession no.: JN715833) (Table 4.4 and Figure

4.10). Another interesting finding is the identification of an Anaplasma sp. from an A. helvolum snake tick (labelled as S4). BLAST analysis of this amplified 16S rDNA sequence showed 100% identity (225 nt/225 nt) with Anaplasma sp. detected in a dog from South Africa (GenBank accession no.: AY570539) but only 99% similarity with the A. Universityphagocytophilum type strain (GenBank accession no.: U02521) (Table 4.4 and Figure 4.10).

Phylogenetic analysis based on partial Anaplasma sp. 16S rRNA gene sequences

(approximately 520 bp) amplified from various wild animals (wild boar, bat and squirrel)

(Section 4.6.2.3) (Table 4.9) in this study showed clustering of the sequences with

Anaplasma sp. detected in goat from China (clone Ap20-5a; GenBank accession no.:

141

KX272643) and A. phagocytophilum detected in sheep from China (isolate YC38;

GenBank accession no.: KJ782381) (Figure 4.27) but only 96.9-97.6% similarity with A. phagocytophilum type strain (GenBank accession no.: U02521). Analyses of the 16S rDNA sequences (approximately 530 bp) from three deer, a cow and two buffaloes

(GenBank accession nos.: MG988297-MG988299) in this study reveal the identification of Candidatus A. boleense which was first identified in ticks from Bole in the Xinjiang

Uygur Autonomous Region, China (Kang et al., 2014) and later in the mosquitoes in

China (Guo et al., 2016). To the best of our knowledge, this is the first report of

Candidatus A. boleense infection in animals. Besides cattle, matching sequence of

Candidatus A. boleense was also found in buffalo and deer samples in our sequence database (Table 4.9). This finding suggests that Candidatus A. boleense is able to infect a variety of domestic animals in the tropics. The clinicalMalaya relevance of this strain in animals merits further investigation. Through sequence analysis, the Ehrlichiaof species obtained from cattle ticks in this study can be grouped into three groups with each group demonstrating close genetic relatedness with Ehrlichia sp. strain EBm52 (amplified from R. microplus), E. mineirensis (amplified from R. microplus) and Candidatus E. shimanensis (amplified from H. longicornis, respectively (Table 4.5). To the best of our knowledge, this is the first report of three Ehrlichia species in cattle ticks (R. microplus and H. bispinosa) in

Malaysia based on analyses of 16S rRNA, gltA and groEL gene sequences. UniversityIn the past, there were reports on new Ehrlichia spp. strains detected in R. microplus ticks from Thailand (Ehrlichia sp. strain EBm52), China (Ehrlichia sp. Fujian) and Tibet (Ehrlichia sp. Tibet) (Parola et al., 2003; Wen et al., 2002). New Ehrlichia spp. strains have also been reported in different Haemaphysalis ticks, for example, H. longicornis, H. hystricis and Haemaphysalis japonica (Rar and Golovljova, 2011). These findings proved that R. microplus and Haemaphysalis ticks might be able to harbour

142

different Ehrlichia spp. Additionally, a nearly full length (1325 bp) sequence of 16S rDNA fragment obtained from an A. varanense tick collected from a snake (labelled as

S3) demonstrated the highest sequence identity of 98.5% (1303 nt/1325 nt) with a E. ruminantium isolate Ball3 from South Africa (Table 4.5 and Figure 4.12). The < 99% sequence similarity suggests that the Ehrlichia strain could be a new genetic variants of

E. ruminantium. The zoonotic or veterinary potential of these Ehrlichia spp. is yet to be explored. The presence of various Anaplasma spp. and Ehrlichia spp. in various ticks may pose a potential health threat to domestic animals and possibly humans as well.

5.10 Limitations of this study and future investigation

Ticks were not collected from all livestock farms as some farms had undergone deticking program prior to tick sampling. Only a smallMalaya subset of blood samples from stray dogs were subjected for screening of Anaplasma spp. and Ehrlichia spp. in this study (Section 3.1.5), hence, the true disease prevalencesof of anaplasmosis and ehrlichiosis in stray dogs are still not known.

The primer sets used for PCR assays in this study were not specific enough to differentiate between species of Anaplasma spp. and Ehrlichia spp., for example A. marginale, A. centrale and A. ovis; E. chaffeensis and E. ewingii are closely related species that shared almost similar sequences in the 16S rDNA fragments. Additionally, due to potential PCR bias, PCR assays tend to amplify the most abundant organism presentUniversity in a mixed sample. As a result, some other organisms can become undetectable in the samples (those with lower abundance). A PCR incorporated with DNA hybridisation assay, for example PCR-RLB (Bekker et al., 2002) will be useful in this case where more than one Anaplasma spp. or Ehrlichia spp. in the tick or animal blood samples can be detected simultaneously. Besides that, morphological identification of ticks to the species level is difficult and challenging without the aid of molecular tools.

143

Hence, there is a need to build expertise in this area to provide better morphological identification of ticks in the future.

A. bovis has been detected in a wide range of animals and tick samples collected in this study. Future work in cultivation of A. bovis strain is needed for a better understanding of the pathogenicity of the bacterium. In view of the high detection rate

(60.7%) of A. marginale in cattle blood samples, development of vaccines (which is important to reduce anaplasmosis in livestock) will be desirable. In addition, next generation sequencing approach can be applied for whole genome determination of

Anaplasma spp. and Ehrlichia spp. This will provide further insight on the biology and pathogenicity of these interesting tick-borne pathogens.

Malaya

University

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CHAPTER 6: CONCLUSION

The molecular investigation in this study show that Anaplasma spp. and Ehrlichia spp. are potential tick-borne pathogens circulating in Malaysia as the organisms were detected in various animal and tick samples from different sources (livestock farms, the forest areas and aboriginal villages). The use of molecular techniques (PCR followed by sequencing approach) has enabled the identification of various Anaplasma spp. and

Ehrlichia spp. in animal and tick samples in this study. With the detection of Ehrlichia spp. from various ticks (R. microplus and H. bispinosa from cattle ticks; D. atrosignatus and Haemaphysalis sp. from vegetation), this study provides some explanation on the high seropositivity to Ehrlichia spp. noted amongst indigenous villagers and livestock farm workers in a recent serological study (Koh et al., 2018). Based on literature review, A. phagocytophilumMalaya is the most important human pathogen amongst Anaplasma spp. The detection of A. phagocytophilum in the infected dog bloods and various tick species collectedof from the forest areas and aboriginal villages suggest possible risk to people who are exposed to tick bites. A. platys is another emerging human pathogen which could be transmitted through ticks infesting dogs and other animal hosts. The detection of A. bovis from a wide variety of animal and tick samples also raises concern on its potential role as a human or animal pathogen. A. phagocytophilum, E. canis and Ehrlichia spp. (E. chaffeensis/ E. ewingii) are detected from infected dogs in this study. Additionally, findings are also obtained in this study regarding new sequence variantsUniversity of Ehrlichia spp. in cattle ticks (H. bispinosa and R. microplus), a novel Anaplasma sp. in the blood samples of pangolins and a closely related species of E. ruminantium in snake tick. The high infection rates of A. marginale in cattle and ticks may affect the meat and milk production in livestock farms. As ticks play an important role as a maintenance host for anaplasmosis and ehrlichiosis, appropriate measures should be instituted to reduce tick populations and to prevent exposure to ticks.

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LIST OF PUBLICATIONS AND PAPERS PRESENTED

List of published papers:

 Koh Fui Xian, Kho Kai Ling, Masoumeh Ghane Kisomi, Wong Li Ping, Awang Bulgiba, Tan Poai Ean, Yvonne Lim Ai Lian, Quaza Nizamuddin hassan Nizam, Chandrawathani Panchadcharam and Tay Sun Tee. (2018). Ehrlichia and Anaplasma infections: Serological evidence and tick surveillance in Peninsular Malaysia. Journal of Medical Entomology, 55 (2): 269-276.  Koh Fui Xian, Kho Kai Ling, Chandrawathani Panchadcharam, Frankie Thomas Sitam and Tay Sun Tee. (2016). Molecular detection of Anaplasma spp. in pangolins (Manis javanica) and wild boars (Sus scrofa) in Peninsular Malaysia. Veterinary Parasitology, 227:73-76.  Koh Fui Xian, Chandrawathani Panchadcharam and Tay Sun Tee. (2016). Vector- Borne Diseases in Stray Dogs in Peninsular Malaysia and Molecular Detection of Anaplasma and Ehrlichia spp. from Rhipicephalus sanguineus (Acari: Ixodidae) Ticks. Journal of Medical Entomology, 53:183-187.  Kho Kai Ling, Koh Fui Xian and Tay Sun Tee. (2015). Molecular evidence of potential novel spotted fever group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks parasitizing wild snakes. Parasites & Vectors, 8:112.  Tay Sun Tee, Koh Fui Xian, Kho Kai Ling and Frankie Thomas Sitam. (2015). Rickettsial infection in Monkeys, Malaysia.Malaya Emerging Infectious Diseases, 21(3):545-547. of List of papers presented:

 Koh Fui Xian, Chandrawathani Panchadcharam and Tay Sun Tee. (2015). Molecular and serological detection of selective tick-borne pathogens in Malaysian dogs. Infection 2015, Putrajaya, Malaysia, 7-8 April 2015, pg. 32 (National).  Koh Fui Xian, Ong Bee Lee, Chandrawathani Panchadcharam and Tay Sun Tee. (2013) Molecular detection of Ehrlichia and Anaplasma species in domestic animals and tick vectors in Malaysia. 24th International Conference of the World Association for the Advancement of Veterinary Parasitology, Australia, pg. 644 (International). University

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