SURVEILLANCE OF RICKETTSIAL INFECTIONS AND WHOLE GENOME ANALYSIS OF A Rickettsia felis-LIKE ORGANISM IN MALAYSIA
KHO KAI LING Malaya of
FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR
University
2017 SURVEILLANCE OF RICKETTSIAL INFECTIONS AND WHOLE GENOME ANALYSIS OF A Rickettsia felis- LIKE ORGANISM IN MALAYSIA
KHO KAI LING Malaya
THESIS SUBMITTED IN FULFILMENTof OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR
University
2017
UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION
Name of Candidate: Kho Kai Ling Registration/Matric No: MHA130012 Name of Degree: Doctor of Philosophy Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): Surveillance of rickettsial infections and whole genome analysis of a Rickettsia felis- like organism in Malaysia Field of Study: Medical 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 knowledge nor do I ought reasonably to know that the making of this work constitutes anof 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.
Candidate’s Signature Date: UniversitySubscribed and solemnly declared before, Witness’s Signature Date:
Name: Designation:
ii ABSTRACT
Rickettsioses are emerging zoonotic diseases which are often underdiagnosed due to
nonspecific clinical manifestations, unavailability of appropriate diagnostic tools and a
lack of awareness in developing countries. Information on the clinical, epidemiology and
ecology of rickettsioses are scarce. The objectives of this study are to investigate selected
cases of rickettsioses in febrile patients in University of Malaya Medical Center and to
determine the seroprevalence of Malaysian populations against spotted fever group (SFG)
rickettsiae. This study also aimed at determining the occurrences of rickettsiae in various
arthropod and animal samples, and investigating the growth characteristics and genomic
features of Rickettsia sp. TH2014, a rickettsial endosymbiont of C. felis orientis flea. This
study describes the DNA detection of Rickettsia typhi, rickettsia closely related to Rickettsia raoultii and Rickettsia sp. RF2125 from Malaya the blood samples of four febrile patients and their clinical presentations. Analysis using indirect immunofluorescence
assays showed significantly higher seropositivityof rates to SFG Rickettsia amongst
indigenous community [R. conorii (50.0 %) and R. felis (22.5 %)] and animal farm
workers [R. conorii (13.8 %) and R. felis (16.1 %)] as compared to urban blood donors.
Rickettsiae were detected up to 66.7 % in ticks and 96.9 % in fleas across different
geographical locations. Sequence analyses of gltA, ompA and ompB partial genes revealed
the identification of SFG rickettsiae closely related to R. raoultii, R. tamurae, R. conorii,
R. heilongjiangensis, R. felis, Rickettsia sp. TCM1 and R. felis-like organism in ticks. UniversityRickettsia closely related to R. raoultii and Rickettsia sp. RF2125 are the predominant species circulating in Peninsular Malaysia since they were detected in human, ticks, fleas
and wildlife. Some potentially novel rickettsiae were also identified for future
investigation. Transmission electron microscope analysis of Rickettsia sp. TH2014
growing in Aedes albopictus (C6/36) cells revealed spherical or elongated intracytosolic
rickettsia which eventually caused the rupture of the host cells. The gltA (307 bp), ompB
iii (793 bp) and 16S rRNA (1368 bp) of the strain exhibited 98.3 %, 94.8 % and 99.6 %
similarity to R. felis type strain (URRWXCal2). The genome of Rickettsia sp. TH2014
was generated from Hi-seq sequencing and assembled with ABySS software to a depth
of 954X coverage. The estimated genome size and GC content of the rickettsia are 1.37
Mb and 32.9 %, respectively. RAST analysis shows 1, 469 coding sequences, 32 tRNA
genes and 3 rRNA genes. Genes associated with actin-based motility, adherence and
invasion, T4SS secretion system and toxin-antitoxin system are annotated in the genome.
Comparative whole-genome single nucleotide polymorphism analysis confirms the
closely genetic relatedness of Rickettsia sp. TH2014 with R. felis type strain
(URRWXCal2). In conclusion, based on the serological and molecular findings obtained
in this study, rickettsioses should be considered as one of the differential diagnoses for unknown febrile illness in our local setting. The highMalaya detection rates of rickettsiae in a large collection of ticks and fleas in this study suggest potential exposure of local population to rickettsioses. The pathogenicof role of Rickettsia sp. TH2014 in causing animal and human infections is yet to be investigated.
University
iv ABSTRAK
Riketsioses merupakan penyakit zoonotik baru bangkit yang kurang didiagnos
disebabkan oleh gejala klinikal yang tidak khusus, ketiadaan alat diagnosis yang sesuai
dan kekurangan kesedaran di negara yang sedang membangun. Informasi mengenai
klinikal, epidemiologi dan ekologi yang berkaitan dengan riketsiosis juga kekurangan.
Objektif kajian ini bertujuan untuk menyiasat kes riketsiosis di kalangan pesakit demam
di Pusat Perubatan Universiti Malaya dan untuk menentukan seroprevalens populasi
Malaysia terhadap riketsia kumpulan ‘spotted fever’ (SFG). Kajian ini juga bertujuan
untuk menentukan kejadian riketsia pada pelbagai sampel artropod dan haiwan, serta
mengaji ciri-ciri pertumbuhan dan genomik untuk Rickettsia sp. TH2014, satu
endosimbion riketsia kepada Ctenocephalides felis orientis. Kajian ini menghuraikan pengesanan DNA untuk Rickettsia typhi, riketsia berkaitMalaya rapat dengan Rickettsia raoultii dan Rickettsia sp. RF2125 daripada empat sampel darah pesakit demam dan penampilan klinikal. Analisa imunopendarflour secaraof tidak langsung menunjukkan kadar seropositiviti yang tinggi untuk riketsia SFG di kalangan orang asli [R. conorii (50.0 %)
and R. felis (22.5 %)] dan pekerja ladang ternakan haiwan [R. conorii (13.8 %) and R.
felis (16.1 %)] berbanding dengan penderma darah dari bandar. Kadar pengesanan untuk
riketsia adalah sebanyak 66.7 % untuk sengkenit dan 96.9 % untuk kutu merentasi lokasi
geografi yang berbeza. Analisis jujukan berdasarkan gen separa gltA, ompA dan ompB
menunjukkan pengenalpastian riketsia SFG yang merangkumi riketsia yang berkait rapat
dengan R. raoultii, R. tamurae, R. conorii, R. heilongjiangensis, R. felis, Rickettsia sp. UniversityTCM1 dan ‘R. felis-like organism’ dalam sengkenit. Riketsia berkait rapat dengan R. raoultii dan Rickettsia sp. RF2125 merupakan species pradominan yang beredar di
Semenanjung Malaysia memandangkan pengesanan mereka dalam manusia, sengkenit,
kutu dan haiwan liar. Beberapa spesies riketsia baru yang berpotensi telah dikenalpastikan
untuk kajian masa depan. Analisis mikroskop elektron penghantaran untuk Rickettsia sp.
v TH2014 yang dikultur dalam sel Aedes albopictus (C6/36) menunjukkan riketsia yang
berbentuk sfera atau memanjang di dalam sitosol sel yang akhirnya menyebabkan sel
perumah pecah. Gen gltA (307 bp), ompB (793 bp) dan 16S rRNA (1368 bp)
menunjukkan persamaan jujukan sebanyak 98.3 %, 94.8 % dan 99.6 % kepada Rickettsia
felis “type strain” URRWXCal2. Genom Rickettsia sp. TH2014 telah dihasilkan daripada
jujukan ‘Hi-seq’ dan dihumpun dengan perisian AbySS meliputi kedalaman 954X.
Anggaran saiz genom dan kandungan GC untuk riketsia tersebut ialah 1.37 Mb dan 32.9
%, masing-masing. Analisis RAST menunjukkan 1, 469 pengekodan jujukan, 32 gen
tRNA, dan 3 gen rRNA. Gen yang berkaitan dengan ‘actin-based motility’, ‘adherence
and invasion’, kompleks kepelbagaian protein T4SS dan sistem toxin-antitoxin telah
dianotasikan daripada genom. Analisa perbandingan genome berasaskan polimorfisme nukleotida tunggal mengesahkan bahawa Rickettsia Malaya sp. TH2014 adalah berkait rapat secara genetik dengan Rickettsia felis type strain (URRWXCal2). Kesimpulannya, berdasarkan kepada kajian serologi dan of molekul yang diperolehi, riketsioses perlu dipertimbangkan sebagai salah satu diagnosis pembezaan untuk penyakit deman yang
tidak diketahui di tempatan. Pengesanan agen riketsia dalam koleksi yang besar untuk
sengkenit dan kutu dalam kajian ini mencadangkan potensi pendedahan penduduk
tempatan kepada riketsioses. Peranan patogenik Rickettsia sp. TH2014 dalam
menyebabkan jangkitan haiwan dan manusia masih belum ditentukan.
University
vi ACKNOWLEDGEMENTS
I would like to sincerely thank my supervisor, Prof. Dr. Tay Sun Tee from Department
of Medical Microbiology, Faculty of Medicine, University of Malaya, for her
immeasurable amount of guidance, encouragement and constructive ideas provided
throughout this study. I am grateful for the research grants, i.e., University Malaya
Research Grant (RP013-2012A), High Impact Research [E000013-20001
(subprogramme 4)], Postgraduate Research Fund (PG006-2013B), and Ministry of
Science, Technology and Innovation, Malaysia, for providing E-science Fund (SF014-
2015) that enable me to pursue this study.
I would like to acknowledge Department of Veterinary Services, Ministry of
Agriculture and Agro-Based Industry, Department of Wildlife and National Parks (PERHILITAN), Department of Orang Asli DevelopmentMalaya (JAKOA) and Dewan Bandaraya Kuala Lumpur (DBKL) for the approval and arrangement of samplings conducted for this study. Thank you to ourof collabrators who have contributed to the success of this study: Assoc. Prof. Dr. Sasheela Sri La Sri Ponnampalavanar, Mdm.
Harvinder Kaur Lakhbeer Singh, Mr. John Jeffery, Prof. Datin Dr. IndraVythilingam,
Prof. Dr. Yvonne Lim Ai Lian, and Assoc. Prof. Dr. Chan Yoke Fun from Faculty of
Medicine, University Malaya; Dr. Low Van Lun from Faculty of Science, University
Malaya; Dr. Chandrawathani Panchadcharam from Veterinary Research Institute, Ipoh.
Special thanks are extended to my parents, Kho Chor Nang and Kua Chiew Tiang, as well
as my brother, Kho Chin Ngan, and other family members for their unconditional support Universitythroughout my life. Lastly, my sincere appreciation to my colleagues: Aida Syafinaz, Azzy Iyzati, Alice Toh Yue Fen, Fatin Izzati, Grace Tan Min Yi, Hou Siau Li, Koh Fui
Xian, Lailatul Insyirah, Lum Kah Yean, and Nur Wahida from the Department of Medical
Microbiology, University of Malaya, who have assisted, encouraged and supported me
throughout my research journey.
vii TABLE OF CONTENTS
Abstract ...... iii
Abstrak ...... v
Acknowledgements ...... vii
Table of Contents ...... viii
List of Figures...... xv
List of Tables ...... xix
List of Symbols and Abbreviations ...... xxii
List of Appendices ...... xxvi CHAPTER 1: INTRODUCTION ...... Malaya 1 CHAPTER 2: LITERATURE REVIEW ...... 5 2.1 Genus Rickettsia ...... of 5 2.2 SFG rickettsiae ...... 9
2.2.1 Rickettsia rickettsii ...... 10
2.2.2 Rickettsia conorii ...... 10
2.2.3 Rickettsia raoultii ...... 11
2.2.4 Rickettsia felis/Rickettsia felis-like organisms ...... 12
2.3 Geographical distribution of SFG rickettsiae ...... 14 University2.3.1 Worldwide distribution of SFG rickettsiae ...... 14 2.3.2 SFG rickettsioses in Southeast Asia ...... 18
2.3.3 SFG rickettsioses in Malaysia ...... 20
2.4 Vectors and reservoirs for rickettsioses ...... 22
2.4.1 Tick-borne rickettsioses ...... 25
2.4.2 Flea-borne rickettsioses ...... 26
viii 2.4.3 Mite-borne rickettsioses ...... 28
2.4.4 Mosquitoes ...... 28
2.5 Clinical manifestations of patients with rickettsioses ...... 29
2.6 Laboratory diagnosis of rickettsioses ...... 30
2.6.1 Culture of rickettsiae ...... 30
2.6.2 Serological diagnosis of rickettsioses ...... 31
2.6.3 Molecular detection of rickettsiae ...... 33
2.6.4 Treatment ...... 36
2.7 Genomic features of rickettsial organisms ...... 36
CHAPTER 3: MATERIALS AND METHODS ...... 42 3.1 Ethical approvals for the conduct of research ...... Malaya 42 3.2 Collection of human samples for detection of rickettsiae ...... 43 3.2.1 Patient samples from UMMCof with pyrexia ...... 43 3.2.2 DNA samples from dengue negative febrile patients ...... 43
3.2.3 Serum samples from blood donors ...... 43
3.2.4 Serum samples from animal farm workers...... 44
3.2.5 Serum samples from indigenous community ...... 44
3.3 Serological analysis of human blood samples for rickettsioses using
immunofluorescence assay (IFA) ...... 45
3.3.1 Febrile patient suspected of rickettsioses from UMMC ...... 45 University3.3.2 Blood donors, indigenous community and farm workers ...... 46 3.4 Statistical analysis ...... 47
3.5 Arthropod collection and identification ...... 47
3.6 Animal blood samples collection ...... 50
3.7 Collection and processing of organ tissue samples from small wildlife...... 50
3.8 DNA extraction ...... 51
ix 3.8.1 Human and animal blood samples ...... 51
3.8.2 Animal organ samples ...... 52
3.8.3 Tick and flea samples ...... 52
3.9 Molecular detection and sequence analysis of rickettsiae ...... 53
3.9.1 Molecular detection of rickettsiae from clinical samples ...... 53
3.9.2 Molecular detection of rickettsiae from arthropod samples ...... 55
3.9.3 Molecular detection of rickettsiae from animal samples ...... 55
3.9.4 Sequence determination and analysis of amplified fragments ...... 56
3.10 Phylogenetic analysis of tick and flea-borne rickettsiae ...... 56
3.11 Isolation and maintenance of rickettsial organism ...... 57
3.11.1 Source of tick and flea samples for infection experiments ...... 57 3.11.2 Regeneration and propagation of C6/36Malaya cell line ...... 57 3.11.3 Infection of C6/36 cell line with tick/flea homogenates ...... 59 3.11.4 Storage of rickettsial stock inof sucrose phosphate glutamate (SPG) buffer ………………………………………………………………………60
3.11.5 Storage of infected C6/36 cells in liquid nitrogen ...... 61
3.12 Study on the growth characteristics of Rickettsia spp. in C6/36 cells ...... 61
3.12.1 Inoculation and culture of Rickettsia spp...... 61
3.12.2 DNA extraction of infected cells ...... 62
3.12.3 Preparation of plasmid standard for quantification of rickettsiae ...... 62
3.12.4 Real-time PCR assay ...... 63
University3.13 Study of the morphological characteristics of Rickettsia spp. in C6/36 cells ...... 65
3.13.1 Giemsa stain ...... 65
3.13.2 Transmission electron microscopy of rickettsia-infected cells ...... 65
3.13.3 Toluidine blue stain on semi-thin section of the rickettsia-infected cells
………………………………………………………………………66
x 3.14 Whole genome sequencing and analysis ...... 66
3.14.1 DNA extraction ...... 66
3.14.2 Whole genome sequencing ...... 67
3.14.3 Genome assembly ...... 68
3.14.4 Gene prediction and annotation ...... 68
3.14.5 Pairwise genome comparison ...... 68
3.14.6 Inference of phylogenetic relationships amongst Rickettsia spp...... 69
3.14.7 Comparative analysis of gene functions with R. felis/RFLO and
pathogenomics ...... 70
CHAPTER 4: RESULTS ...... 71 4.1 Determination of the etiological agents of rickettsiosesMalaya in patients with febrile illness ……………………………………………………………………………71 4.1.1 Patient with pyrexia admittedof to UMMC ...... 71 4.1.2 DNA samples from dengue-negative febrile patients ...... 73
4.2 Determination of the antibody prevalence of rickettsiae in urban blood donors,
animal farm workers, and indigenous community ...... 77
4.3 Determination of the type and distribution of rickettsiae in arthropods ...... 84
4.3.1 Collection and identification of ticks ...... 84
4.3.1.1 Collection and identification of ticks from urban areas ...... 84
4.3.1.2 Collection and identification of ticks from animal farms ...... 88 University4.3.1.3 Collection and identification of ticks from rural villages ...... 91 4.3.1.4 Collection of ticks from a forest reserve...... 93
4.3.1.5 Collection and identification of snake ticks ...... 94
4.3.2 Collection and identification of fleas ...... 95
4.3.2.1 Collection and identification of fleas from urban areas ...... 95
4.3.2.2 Collection and identification of fleas from rural villages ...... 96
xi 4.3.3 Collection of other arthropod samples ...... 97
4.3.4 Identification of rickettsial organisms in ticks ...... 97
4.3.4.1 Determination of the type and distribution of rickettsiae in ticks
collected from urban areas ...... 97
4.3.4.2 Determination of the type and distribution of rickettsiae in ticks
collected from animal farms ...... 101
4.3.4.3 Determination of the type and distribution of rickettsiae in ticks
collected from rural villages ...... 105
4.3.4.4 Determination of the type and distribution of rickettsiae in ticks
collected from a forest reserve ...... 109
4.3.4.5 Determination of the type and distribution of rickettsiae in ticks collected from snakes ...... Malaya 113 4.3.5 Identification of rickettsial organisms in fleas ...... 117 4.3.5.1 Determination of theof type and distribution of rickettsiae in fleas collected from urban areas ...... 117
4.3.5.2 Determination of the type and distribution of rickettsiae in fleas
collected from rural villages ...... 117
4.3.6 Identification of rickettsial organisms in other arthropod samples ...... 120
4.4 PCR detection of rickettsiae in various animal hosts ...... 124
4.4.1 Determination of the type and distribution of rickettsiae in animal blood
(small mammals and ruminants) samples ...... 124
University4.4.2 Determination of the type and distribution of rickettsiae in wildlife samples
……………………………………………………………………..124
4.5 Phylogenetic analysis of tick and flea-borne rickettsiae ...... 130
4.5.1 Phylogenetic analysis based on partial fragments of gltA gene ...... 130
4.5.2 Phylogenetic analysis based on ompB genes ...... 132
xii 4.6 Isolation of R. felis-like organisms from infected fleas ...... 135
4.7 Study on the growth characteristics of Rickettsia sp. TH2014 in C6/36 cells .... 137
4.8 Microscopic study of the morphological characteristics of Rickettsia sp. TH2014 in
C6/36 cells ...... 142
4.8.1 Giemsa stain ...... 142
4.8.2 Transmission electron microscopy (TEM) ...... 143
4.8.3 Toluidine blue staining of semi-thin section of infected cells ...... 150
4.9 Genome properties of Rickettsia sp. TH2014 ...... 151
4.10 Phylogenetic position of Rickettsia sp. TH2014 ...... 156
4.11 Comparative whole-genome single nucleotide polymorphism (SNP) analysis .. 161
4.12 Pairwise genome comparison of Rickettsia sp. TH2014 ...... 163 4.13 Comparative analysis of gene functions with R. Malayafelis and RFLO ...... 167 CHAPTER 5: DISCUSSION ...... of 172 5.1 Molecular investigation of suspected cases of rickettsioses in febrile patients
attending to UMMC ...... 172
5.1.1 Serological and molecular findings ...... 172
5.1.2 Clinical presentations of rickettsial PCR-positive patients ...... 174
5.2 Determination rickettsial seropositivity in urban blood donors, animal farm
workers, and indigenous community ...... 176
5.3 Determination of the type and distribution of rickettsiae in arthropods and animals University …………………………………………………………………………..179 5.3.1 Rickettsiae in ticks from urban areas ...... 179
5.3.2 Rickettsiae in ticks from animal farms...... 180
5.3.3 Rickettsiae in ticks from rural villages...... 182
5.3.4 Rickettsiae in ticks from a forest reserve ...... 183
5.3.5 Rickettsiae in snake ticks ...... 185
xiii 5.3.6 Rickettsiae in fleas ...... 186
5.3.7 Rickettsiae in other arthropod samples ...... 188
5.3.8 Rickettsiae in animal hosts ...... 189
5.3.9 Limitation and future study ...... 193
5.4 Phylogenetic analysis of tick/flea-borne rickettsiae ...... 194
5.5 Growth characteristic and comparative phylogenetic analysis of Rickettsia sp.
TH2014 ...... 195
5.6 Genome properties and comparative pathogenomics of Rickettsia sp. TH2014
…………………………………………………………………………..200
5.6.1 RAST prediction o virulence genes in Rickettsia sp. TH2014 ...... 202
5.6.2 Virulence genes for Rickettsia sp. TH2014 searched against VFDB .... 204 5.6.3 Limitations and future study ...... Malaya 207 CHAPTER 6: CONCLUSION ...... of 209 REFERENCES ...... 213
LIST OF PUBLICATIONS AND PAPERS PRESENTED ...... 261
APPENDICES ...... 263
University
xiv LIST OF FIGURES
Figure 2.1: Taxonomic scheme for classification of rickettsiae at the genus and species levels (Raoult et al., 2005)...... 8
Figure 2.2: World distribution of tick-borne rickettsioses (Raoult, 2012)...... 15
Figure 2.3: SFG rickettsioses reported in Southeast Asia ...... 18
Figure 2.4: Life cycle of tick-borne rickettsiae (Walker & Ismail, 2008)...... 25
Figure 2.5: Transmission routes for the maintenance of flea-borne rickettsiae (R. felis and RFLO) infections in the environment (Brown & Macaluso, 2016)...... 27
Figure 3.1: Geographical location of the studied population in Peninsular Malaysia. ... 45
Figure 4.1: Phylogenetic analysis based on the sequences of the outer membrane protein (ompB) gene of rickettsiae identified in this study...... 76
Figure 4.2: Representative confocal images of R. conorii immunofluorescence assay (Fuller lab)...... Malaya 82 Figure 4.3: Representative confocal images of R. felis immunofluorescence assay (Fuller lab)...... of 83 Figure 4.4: The dorsal views of a male (SF023M) (left) and a female (SF025F) (right) Rh. sanguineus...... 86
Figure 4.5: Ventral views of a female (SF025F) (left) and a male (SF023M) (right) Rh. sanguineus...... 86
Figure 4.6: Phylogenetic placement based on the tick mitochondrial 12S rDNA partial sequences (258 bp) of Rh. sanguineus in this study...... 87
Figure 4.7: The dorsal and ventral views of a male (EKR2162M) Rh. microplus (B. microplus) collected from Farm 3...... 89 UniversityFigure 4.8: Major features of a male Haemaphysalis spp. (UN2-40M) collected from Farm 3...... 90
Figure 4.9: A dorsal view (left) and ventral view (right) of H. wellingtoni (SP016F1) collected from a chicken in Perak...... 92
Figure 4.10: H. hystricis (SP007) collected from a dog in Perak...... 92
Figure 4.11: The dorsal view of A. varanense (S3) with metallic yellowish-green ornamentation on the scutum...... 95
xv Figure 4.12: Female C. felis felis (C006F, left) and male C. felis felis (C006M, right) captured from a dog in a rural village, Kg. Orang Asli Semangar Dalam, Johor ...... 96
Figure 4.13: Female C. felis orientis (SD010F, left) and male C. felis orientis (SD010M, right) captured from a dog in a rural village, Kg. Orang Asli Semangar Dalam, Johor...... 96
Figure 4.14: Agarose gel electrophoretogram of representative gltA fragments ( 490 bp) amplified from Rh. sanguineus DNA samples...... 98
Figure 4.15: Agarose gel electrophoretogram of representative PCR-amplified ompA fragments ( 532 bp) amplified from Rh. sanguineus DNA samples...... 98
Figure 4.16: Agarose gel electrophoretogram of representative PCR-amplified ompB fragments ( 855 bp) amplified from Rh. sanguineus DNA samples...... 99
Figure 4.17: Phylogenetic placement of concatenated sequences (gltA and ompA) of known rickettsial species in Table 4.15...... 116
Figure 4.18: Agarose gel electrophoretogram of representative PCR amplified gltA fragments ( 490 bp) from flea DNA samples...... Malaya 118 Figure 4.19: Agarose gel electrophoretogram of representative PCR amplified ompB fragments ( 855 bp) from flea DNA samples.of ...... 118 Figure 4.20: An overview presentation of the seroprevalence and occurrences of rickettsiae in human and arthropod vectors across several states in Peninsular Malaysia...... 123
Figure 4.21: Phylogenetic placement of rickettsiae based on partial sequences of rickettsial gltA (368bp) gene...... 131
Figure 4.22: Phylogenetic placement of rickettsial based on the partial sequences of R. raoultii (749 bp) ompB gene...... 133
Figure 4.23: Phylogenetic placement of rickettsiae based on the partial sequences of R. Universityfelis and RFLO ompB (749 bp) gene...... 134 Figure 4.24: Standard curve for quantitation of Rickettsia sp. TH2014. The plasmid pCR4-TOPO cloning vector showed linearity in the dilution series, for 1.65 × 106 to 1.65 × 1011 copies (R2=0.9923, Efficiency: 99 %, y=-3.3416x+45.812) of gltA gene in the real- time PCR experiment for time-course experiment 1 (C6/36-1)...... 138
Figure 4.25: Growth of Rickettsia sp. TH2014 in Ae. albopictus C6/36 cells for seven days (time-course experiment 1, C6/36-1) in T75 flask...... 139
xvi Figure 4.26: Standard curve for quantitation of Rickettsia sp. TH2014. The plasmid pCR4-TOPO cloning vector showed linearity in the dilution series, for 1.65 × 106 to 1.65 × 1011 copies of gltA gene (R2=0.99, Efficiency= 99 %, y=-3.3472x+46.553) in the real- time PCR experiment for time-course experiment 2 (C6/36-2 and C6/36-3)...... 139
Figure 4.27: Growth of Rickettsia sp. TH2014 in Ae. albopictus C6/36 cells for seven days (time course experiment 2, C6/36-2 and C6/36-3) in T75 flasks...... 140
Figure 4.28: Standard curve for quantitation of Rickettsia sp. TH2014. The plasmid pCR4-TOPO cloning vector showed linearity in the dilution series, for 1.65 × 106 to 1.65 × 1011 copies of gltA gene (R2=0.99, efficiency= 93 % y=-3.5376x+48.111) in the real- time PCR experiment for time-course experiment 3 (C6/36-4 and C6/36-5)...... 140
Figure 4.29: Growth of Rickettsia sp. TH2014 in Ae. albopictus C6/36 cells for 14 days (time-course experiment 3, C6/36-4 and C6/36-5) in 24 well plates...... 141
Figure 4.30: Uninfected C6/36 cells (left), infected C6/36 cells, 2 dpi (Right)...... 141
Figure 4.31: Giemsa stain of Rickettsia sp. TH2014 (11 dpi) ...... 142
Figure 4.32: Transmission electron micrograph of Rickettsia sp. TH2014 in C6/36 mosquito cells (1 dpi)...... Malaya 144
Figure 4.33: Transmission electron micrograph of Rickettsia sp. TH2014 infected C6/36 cells (4 dpi)...... of 145
Figure 4.34: Close-up of the arrow head area in Figure 4.33...... 146
Figure 4.35: Transmission electron micrograph of Rickettsia sp. TH2014 infected C6/36 cells (4 dpi)...... 147
Figure 4.36: Transmission electron micrograph of Rickettsia sp. TH2014 infected C6/36 cells (7 dpi)...... 148
Figure 4.37: Transmission electron micrograph of uninfected C6/36 cells at day 7. ... 149
Figure 4.38: Light photomicrograph showing toluidine blue stain of the infected cells at University1 dpi (A) and 4 dpi (B) ...... 150 Figure 4.39: Light photomicrograph showing toluidine blue stain of the infected cell at 7 dpi (A) and uninfected cell at day 7 (B)...... 151
Figure 4.40: Schematic circular diagram of Rickettsia sp. TH2014 genome generated using DNA plotter software...... 153
Figure 4.41: Functional categories of the RAST predicted genes of Rickettsia sp. TH2014...... 154
xvii Figure 4.42: Multiple alignment of the 16S rRNA genes (1389 bp) for Rickettsia sp. TH2014, R. felis and RFLOs. Nucleotide variations are marked in red box...... 157
Figure 4.43: Phylogenetic placement of Rickettsia sp. TH2014 based on the concatenated sequences (2668 bp – 2674 bp) for rickettsial gltA (1250 bp) and rickettsial 16S rRNA gene (1421 bp – 1424 bp)...... 160
Figure 4.44: SNP-based phylogenetic tree with TG (R. prowazekii and R. typhi) as outgroup...... 162
Malaya of
University
xviii LIST OF TABLES
Table 2.1: Rickettsia spp. and the associated diseases (Merhej et al., 2014)...... 6
Table 2.2: Recognized or potential vectors for the rickettsiae that are associated with human diseases...... 23
Table 2.3: List of rickettsial genomes available in the NCBI genome database...... 38
Table 3.1: Oligonucleotide primers used in this study...... 54
Table 3.2: Reaction mixture used for real-time PCR assay...... 64
Table 3.3: Thermal cycling conditions...... 64
Table 4.1: Seropositivity rates of rickettsiae in febrile patients ...... 71
Table 4.2: The demographic, hematology and blood chemistry profiles of patients investigated in this study...... 73 Table 4.3: Demographic and baseline characteristicsMalaya of the urban blood donors, farm workers and indigenous people (n=250)...... 77 Table 4.4: Seropositivity of R. conorii andof R. felis with respect to different category of the participants investigated in this study...... 78
Table 4.5: Pairwise comparison of different study groups investigated in this study, using Games-Howell Post-hoc tests of the SPSS...... 80
Table 4.6: Pairwise comparison of different age groups investigated in this study, using Games-Howell Post-hoc tests of the SPSS...... 81
Table 4.7: Total number of tick samples collected for this study...... 85
Table 4.8: BLAST analyses of the rickettsia-positive Rh. sanguineus dog ticks...... 100
Table 4.9: Detection rates of rickettsiae in animal farms across different states...... 102
UniversityTable 4.10: BLAST analyses of rickettsial gltA, ompA and ompB gene fragments of ticks from animal farms...... 103
Table 4.11: Detection rates of rickettsiae across rural villages in different states...... 106
Table 4.12: BLAST analyses of rickettsial gltA, ompA and ompB gene fragments of ticks from rural villages...... 107
xix Table 4.13: BLAST results for rickettsial gltA, ompA and ompB gene fragments of ticks collected from Kuala Lompat forest reserve...... 110
Table 4.14: Molecular detection of rickettsiae and BLAST analyses of the sequences derived from snake tick samples in this study...... 114
Table 4.15: GenBank accession numbers of the rickettsial gene sequences used for the construction of a concatenated NJ tree...... 115
Table 4.16: Detection rates of rickettsiae from fleas collected in each locality...... 119
Table 4.17: BLAST analyses of the selected rickettsial gltA and ompB gene sequences from fleas from rural villages...... 119
Table 4.18: Geographical distribution of rickettsiae detected in arthropods and their animal hosts in each location...... 121
Table 4.19: BLAST analyses of the representative sequences derived from the organ of wild animals in Kuala Lompat...... 126
Table 4.20: BLAST analyses of the amplified gene fragments of Rickettsia sp. TH2014 isolated from C. felis orientis...... Malaya 136 Table 4.21: Virulence, disease, defense categoryof of Rickettsia sp. TH2014...... 155 Table 4.22: Comparison of genome information for Rickettsia sp. TH2014 with R. felis and RFLOs...... 156
Table 4.23: Accession number for rickettsial species included in whole-genome kSNP analysis and pairwise genome comparison...... 161
Table 4.24: Average Nucleotide Identity of Rickettsia sp. TH2014 genome with other rickettsial genome based on BLAST(ANIb)...... 164
Table 4.25: Average Nucleotide Identity of Rickettsia sp. TH2014 genome with other rickettsial genome based on MUMmer (ANIm)...... 165
Table 4.26: Pairwise comparison of rickettsial species based on tetra nucleotide Universitycomposition...... 166
Table 4.27: Comparison of the gene functions between Rickettsia sp. TH2014 with R. felis and two RFLOs. Functional assignments of all genes were performed using RAST pipeline...... 168
Table 4.28: Virulence factors in Rickettsia sp. TH2014 and similarity against R. felis URRWXCal2...... 169
xx Table 4.29: Comparison of metabolic reconstruction of programmed cell death and TA systems for Rickettsia sp. TH2014 and R. felis URRWXCal2...... 171
Malaya of
University
xxi LIST OF SYMBOLS AND ABBREVIATIONS
% : percent oC : degree Celsius µl : microliter µm : micrometer µM : micromolar bp : base pair A. varanense : Amblyomma varanense A. helvolum : Amblyomma helvolum A. hydrosauri : Aponomma hydrosauri A. testudinarium : Amblyomma testudinarium Ae. albopictus : Aedes albopictus An. gambiae : Anopheles gambiae ANI : Average nucleotide identity ANIb : Average nucleotide identityMalaya based on BLAST ANIm : Average nucleotide identity based on MUMmer BLAST : Basic Local Alignment Search Tool C. canis : Ctenocephalidesof canis C. felis felis : Ctenocephalides felis felis C. felis orientis : Ctenocephalides felis orientis cell/mL : cell per microliter CI : confidence interval Cx. quinquefasciatus : Culex quinquefasciatus D. andersoni : Dermacentor andersoni D. atrosignatus : Dermacentor atrosignatus D. auratus : Dermacentor auratus D. marginatus : Dermacentor marginatus UniversityD. nuttalli : Dermacentor nuttalli D. silvarum : Dermacentor silvarum DBKL : Dewan Bandaraya Kuala Lumpur DNA : deoxyribonucleic acid DDH : DNA-DNA hybridization dNTPs : deoxynucleotide triphosphates dsDNA : double stranded deoxyribonucleic acid
xxii dpi : days post-infection EDTA : ethylenediamine tetraacetic acid et al. : et alia (Latin), and others FBS : foetal bovine serum FTA : Flinders Technology Associates filter paper g : gram gltA : citrate synthase gene H. bispinosa : Haemaphysalis bispinosa H. concinna : Haemaphysalis concinna H. hystricis : Haemaphysalis hystricis H. longicornis : Haemaphysalis longicornis H. obesa : Haemaphysalis obesa H. sulcata : Haemaphysalis sulcata H. wellingtoni : Haemaphysalis wellingtoni i.e. : id est (Latin), that is IFA : Immunofluorescence assayMalaya IgG : Immunoglobulin G IgM : Immunoglobulin M LAMP : loop-mediated isothermalof amplification Mb : megabase ml : milliliter mg : milligram min : minute MSF : Mediterranean spotted fever ng : nanogram ng/µl : nanogram/microliter NJ : neighbour-joining No. : number UniversityompA : Outer membrane protein A gene ompB : Outer membrane protein B gene PCR : polymerase chain reaction PLA2 : Phospholipase A2 P. irritans : Pulex irritans P. vulgaris : Proteus vulgaris R. aeschlimannii : Rickettsia aeschlimannii
xxiii R. africae : Rickettsia africae R. akari : Rickettsia akari R. asemboensis : Rickettsia asemboensis R. australis : Rickettsia australis R. conorii : Rickettsia conorii R. felis : Rickettsia felis R. heilongjiangensis : Rickettsia heilongjiangensis R. honei : Rickettsia honei R. hoogstraalii : Rickettsia hoogstraalii R. hulinensis : Rickettsia hulinensis R. japonica : Rickettsia japonica R. marmionii : Rickettsia marmionii R. massiliae : Rickettsia massiliae R. parkeri : Rickettsia parkeri R. raoultii : Rickettsia raoultii R. rhipicephali : Rickettsia rhipicephali Malaya R. rickettsii : Rickettsia rickettsii R. slovaca : Rickettsia slovaca R. sibirica : Rickettsia sibiricaof R. tamurae : Rickettsia tamurae R. typhi : Rickettsia typhi Rh. microplus : Rhipicephalus microplus Rh. sanguineus : Rhipicephalus sanguineus RFLO : Rickettsia felis-like organism(s) RMSF : Rocky Mountain spotted fever rpm : revolution per minute rRNA : ribosomal ribonucleic acid rvh : Rickettsiales vir homologs UniversitySca1 : cell surface antigen Sca1 Sca2 : cell surface antigen Sca2 Sca4 : cell surface antigen Sca4 SDS-PAGE : sodium dodecyl-polyacrylamide gel electrophoresis SENLAT : scalp eschar and neck lymphadenopathy after a tick bite SFG : spotted fever group sp. : species
xxiv spp. : species SPG : sucrose phosphate glutamate buffer SPSS : Statistical Package for the Social Sciences T4SS : type IV secretion system TA : toxin-antitoxin Taq : Thermus aquaticus TEM : transmission electron microscopy TETRA : tetranucleotide usage patterns TG : typhus group TIBOLA : tick-borne lymphadenopathy TPB : tryptose phosphate broth UMMC : University of Malaya Medical Center UNG : uracil-N-glycosylase UV : ultraviolet VFDB : virulence factors database WBC : white blood cell Malaya x g : times gravity
of
University
xxv LIST OF APPENDICES
APPENDIX A: Ethics approval for this study 263 APPENDIX B: Approval letter for sample collection in rural villages 264 Approval letter for samples collection in animal farms in APPENDIX C: 266 Peninsular Malaysia Approval letter for samples collection in Veterinary APPENDIX D: 267 Research Institute, Ipoh Approval letter for samples collection in Kuala Lompat APPENDIX E: 268 forest reserve, Pahang APPENDIX F: Details of Rh. sanguineus collected in this study 269 APPENDIX G: Details of cattle ticks collected in this study 274 APPENDIX H: Details of ticks collected from rural villages in this study 285 Details of ticks collected from vegetation area in Kuala APPENDIX I: 289 Lompat forest reserve Details of ticks collected from animal captured in Kuala APPENDIX J: 294 Lompat forest reserve APPENDIX K: Details of ticks collected from snakes 296 Details of fleas collected from cats in rural villages across APPENDIX L: Malaya297 Peninsular Malaysia Details of animal blood samples provided by Veterinary APPENDIX M: 305 Research Institute Details of monkey bloodof samples provided by APPENDIX N: 310 PERHILITAN Details of animal organs collected from Kuala Lompat APPENDIX O: 311 forest reserve Details of other wildlife blood sample on FTA cards APPENDIX P: 312 collected in Pulau Tioman provided by PERHILITAN APPENDIX Q: Cell culture media preparation 314 16S rRNA sequence (238-239 bp) alignment of APPENDIX R: Dermacentor andersoni collected from Kuala Lompat forest 317 reserve, Pahang 16S rRNA sequence alignment (222 bp) of Dermacentor APPENDIX S: atrosignatus collected from Kuala Lompat forest reserve, 318 Pahang APPENDIX T: Published papers 319 University
xxvi CHAPTER 1: INTRODUCTION
Rickettsial organisms are obligate intracellular bacteria which are transmitted to
vertebrates by arthropod vectors mainly ticks, fleas and mites (Renvoisé et al., 2009b).
With the growing number of rickettsiae associated with human infections, some of the
spotted fever group (SFG) rickettsiae are now known as emergent human pathogens
(Parola et al., 2013). The pathogenicity of SFG rickettsiae varies between species
(Hechemy et al., 2005). While much has been learnt about the virulence of Rickettsia
rickettsii, the type strain of SFG rickettsiae, the pathogenicity of some SFG rickettsiae
are still unknown (Parola et al., 2013). Rickettsia sibirica, Rickettsia heilongjiangensis,
Rickettsia japonica, Rickettsia conorii, Rickettsia honei and Rickettsia tamurae have been implicated in human infections in Asia-Pacific region Malaya(Parola et al., 2013). The epidemiology and the disease burden of rickettsioses in Southeast Asia including Malaysia has not been thoroughly investigated.of The lack of appropriate diagnostic tools is likely to contribute to the underestimation of the true epidemiology of rickettsioses in
this region (Aung et al., 2014). Previously, spotted fever group rickettsioses (formerly
known as tick typhus) have been reported as the most frequent infection among febrile
hospitalized patients in the rural areas of Peninsular Malaysia (Tay et al., 2000). However,
little is known on the clinical and microbiological aspects of rickettsioses.
Malaysia is a country rich with natural resources such as forestry and minerals. There Universityhas been vast economic development, deforestation and urbanization in this country. In the past 10 to 20 years, many forests have been cleared to make ways for industrial crops
such as oil palm plantation, timber plantation and rubber plantation (Butler, 2013). The
close proximity of agricultural habitats and urban development may increase the exposure
of urban dwellers to various zoonotic agents leading to potential outbreaks of infectious
diseases including rickettsioses (Ogrzewalska et al., 2011; Tay et al., 2003). Recently,
1 deforestation has been linked to the steep-rise in human cases of Plasmodium knowlesi
infections in Malaysia (Fornace et al., 2016). With this in mind, it is important to have
recent updates on the exposure of local populations to rickettsioses. The indigenous
community and animal farm workers are generally regarded as two populations who are
at high risks of acquiring rickettsioses through tick/flea bites due to their working
environment and lifestyle (Heinrich et al., 2015; Zhang et al., 2008). However, there is
little information on the current serological status of these individuals. Evidence
supporting their exposures to rickettsiae-infected ticks/fleas has not been documented.
Patients diagnosed with SFG rickettsioses often present with fevers and rashes. Eschar
may occur in most but not all of spotted fevers (Renvoisé et al., 2009a). Early sign and
symptoms of these infections are non-specific and mimic other common infections in the tropics such as scrub typhus, malaria, dengue, babesiosis,Malaya ehrlichiosis, and anaplasmosis, thus increasing the possibility of the disease being misdiagnosed (Civen & Ngo, 2008;
Rathi & Rathi, 2010). As delayed diagnosisof may complicate the treatment and
management of patients (Azad, 1990; Premaratna et al., 2008), laboratory investigations
using molecular and serological approaches are important to confirm the diagnosis of
rickettsioses. To date, limited information is available on tick and flea-borne rickettsioses
in Malaysia. The clinical presentations of patients diagnosed with rickettsioses have not
been well documented in Malaysia. It is hypothesized that certain unrecognized rickettsial
pathogens are circulating and responsible for human and wildlife infection in Malaysia. UniversitySuch organisms should be identified and characterized to provide information on the transmission dynamics of rickettsial infections in this region.
Isolation of rickettsiae is a prerequisite for characterization of rickettsial species.
Rickettsiae are difficult to be cultured and this has hampered the identification of the
organisms from the vectors and animal reservoirs. Traditional identification methods used
2 in bacteriology were unable to be applied routinely for rickettsial study due to the strictly
intracellular nature of the organisms. So far, the agents of rickettsioses have never been
grown in axenic media. The culturing of the organisms requires living hosts such as cell
cultures, embryonated eggs, or susceptible animals.
R. felis is an emergent pathogen which is found in a variety of arthropods (including
fleas, ticks, booklice, and mosquitoes) throughout the tropical and subtropical regions
(McQuiston, 2015; Socolovschi et al., 2012c). Isolation and establishment of R. felis have
been reported in different cell lines including Xenopus laevis cell line, mosquito cell line
and tick-derived cell line (Horta et al., 2006; Pornwiroon et al., 2006; Raoult et al.,
2001b). Infection caused by R. felis has been identified as a common cause of febrile
illness in Africa (Parola, 2011). The importance of this organism as a cause of human infection in Southeast Asia has not been evaluated. WithinMalaya the last decade, R. felis-like organisms (RFLO) have been reported in different arthropods (Brown & Macaluso,
2016). Recently, two newly described RFLOof (Rickettsia asemboensis and Candidatus
Rickettsia senegalensis) have been isolated from cat fleas in Africa (Luce-Fedrow et al.,
2015b; Mediannikov et al., 2015). With the advancement of whole genome sequencing
technology, it is now possible to examine the nearly complete genomes of rickettsial
isolates for comparative phylogenetic studies, identification of putative virulence factors
and prediction of zoonotic potential. The information obtained from this study will shed
lights on the rickettsial species circulating in this region, potential vectors for rickettsioses Universityand understanding of the biology of RFLO through genome analysis. The objectives of this study are to:
i. determine the etiological agents of rickettsioses in patients with febrile illness
ii. assess the antibody prevalence of indigenous community, animal farm workers
and urban blood donors against Rickettsia conorii and Rickettsia felis
3 iii. determine the type and distribution of rickettsial organisms in animals and
potential arthropod vectors
iv. assemble and annotate whole genome sequence of a rickettsial endosymbiont in
flea (Rickettsia felis-like organism) for comparative phylogenetic analysis, and
identification of putative virulence factors.
Malaya of
University
4 CHAPTER 2: LITERATURE REVIEW
2.1 Genus Rickettsia
Rickettsia spp. are obligate intracellular Gram-negative bacteria which belong to the
class α-Proteobacteria; order Rickettsiales and family Rickettsiaceae. The bacteria are
known as one of the endosymbionts in arthropods with the capability to infect vertebrate
cells (Munderloh et al., 2005). Rickettsiae are small rod-shaped bacteria and the surface
are covered by a glycocalyx or slime that retain basic fuschin upon staining using
Gimenez stain (Gimenez, 1964; Renvoisé et al., 2009b). The organisms can also be
visualized using Giemsa stain (Ammerman et al., 2008). The size of rickettsia ranged
from 0.3 to 0.5 µm by 0.8 to 2.0 µm (Renvoisé et al., 2009b).
The bacteria within the genus Rickettsia are classified based on their biological, antigenicity and genetic relatedness (Parola et al., 2013).Malaya The latest study on rickettsial phylogeny and taxonomy shows that the genus Rickettsia is comprised of 27 recognized
species and several numbers of uncharacterizedof strains as listed in Table 2.1 (Merhej et
al., 2014). In general, Rickettsia spp. are differentiated into typhus group (TG) which
comprises Rickettsia prowazekii and Rickettsia typhi; spotted fever group (SFG), which
is the largest group comprising of approximately 20 species; and the ancestral group,
which comprises Rickettsia bellii and Rickettsia canadensis (Merhej et al., 2009; Wood
& Artsob, 2012).
Some researchers classified Rickettsia felis, Rickettsia akari and Rickettsia australis Universityinto a transitional group (Gillespie et al., 2007) while most of the investigators categorized them as members of SFG rickettsiae (Angelakis et al., 2016; Bouyer et al.,
2001; Luce-Fedrow et al., 2015a; Parola, 2011). The validity of the transitional group has
been debated since then (Fournier & Raoult, 2009; Hun & Troyo, 2012).
5 Table 2.1: Rickettsia spp. and the associated diseases (Merhej et al., 2014).
Group Rickettsia spp. Diseases/known pathogenesis Rickettsia rickettsii str. Sheila Smith Rocky mountain spotted fever Rickettsia rickettsii str. lowa Avirulent Rickettsia rickettsii str. Hlp2 Unknown pathogenesis R. philipii str. 364D Unnamed rickettsiosis Rickettsia peacockii str. Rustic Unknown pathogenesis Rickettsia montanensis str. OSU85- Unknown pathogenesis 930 Rickettsia sibirica str. 246 Siberian tick typhus Rickettsia sibirica subsp. Lymphangitis associated mongoliitimonae rickettsioses Rickettsia africae ESF5 African tick bite fever Rickettsia parkeri str. Portsmouth Rickettsia parkeri rickettsioses Rickettsia conorii str. Malish 7 Mediterranean spotted fever Rickettsia slovaca str. 13-B Tick-borne lymphadenitis Rickettsia slovaca str. D-CWPP Tick-borne lymphadenitis Rickettsia honei Flinders Island spotted fever Rickettsia heilongjiangensis 054 Far-eastern tick-borne rickettsioses Spotted Rickettsia japonica YH Oriental spotted fever fever Candidatus Rickettsia amblyommii Unknown pathogenesis group str. GAT 30V (SFG) Rickettsia massiliae MTU5 Unnamed rickettsiosis Rickettsia massiliae AZT80 MalayaUnknown pathogenesis Rickettsia rhipicephali str. 37 female6 Unknown pathogenesis CWPP Rickettsia aeschlimannii of Spotted fever rickettsiosis Rickettsia raoultii Scalp eschar and neck lymph adenopathy after a tick bite (SENLAT) Rickettsia helvetica C9P9 Unnamed rickettsiosis Rickettsia asiatica Unknown pathogenesis Rickettsia tamurae Spotted fever rickettsiosis Rickettsia endosymbiont of Ixodes Unknown pathogenesis scapularis Rickettsia monacensis IrR/Munich Spotted fever rickettsiosis Rickettsia akari str. Hartford Rickettsialpox Rickettsia australis str. Cutlack Queensland tick typhus Rickettsia felis URRWXCal2 Spotted fever rickettsiosis Rickettsia hoogstraalii Unknown pathogenesis Rickettsia prowazekii str. Madrid E Epidemic typhus Rickettsia prowazekii str. BuV67 Epidemic typhus CWPP UniversityTyphus Rickettsia prowazekii str. Chernikova Epidemic typhus group (TG) Rickettsia typhi str. TH1527 Murine typhus Rickettsia typhi str. Wilmington Murine typhus Rickettsia typhi str. B9991 CWPP Murine typhus Rickettsia Rickettsia canadensis str. CA410 Unknown pathogenesis canadensis Rickettsia canadensis str. Mckiel Unknown pathogenesis Rickettsia Rickettsia bellii OSU85 389 Unknown pathogenesis bellii Rickettsia bellii RML369 C Unknown pathogenesis
6 However, sufficient evidence and characterization to support the designation of R. felis
as a member of the SGF rickettsiae have been reported by Bouyer et al. (2001) and La
Scola et al. (2002) through genetic analysis, phylogenetic analysis and actin
polymerization. Based on robust phylogenetic analysis, Merhej et al. (2011) concluded
that R. felis as a member of SFG rickettsiae. In their analysis, the identifiable gene
ancestors of R. felis are mainly from SFG rickettsiae which belong to the R. conorii
subgroup (43.5 % of 914 robust phylogenies obtained from R. felis genes common to all
or some Rickettsia spp.), the R. akari subgroup (37 %), the TG (1 %), R. bellii (12 %) and
R. canadensis (0.1 %). The result supported the placement of R. felis in the SFG clade,
and suggested that the putative transitional group is inconsistent (Merhej et al., 2011).
Classification of members in the genus Rickettsia was initially based on the mouse serotyping method developed in 1978 (PhilipMalaya et al., 1978). The micro- immunofluorescence method detects specific epitopes of the high-molecular-mass, and
surface cell antigens (rOmpA, rOmpB andof 120-kDa proteins) of rickettsiae. However,
this method is not highly reproducible and laborious (Fournier et al., 2003a). Hence,
sequence-based guidelines for the classification of rickettsia at taxonomic levels have
been set up (Fournier et al., 2003a). Genes encoding citrate synthase (gltA), outer
membrane protein A (ompA), outer membrane protein B (ompB) and cell surface antigen
(sca4) are used to establish gene sequence-based criteria for new rickettsial isolates.
Raoult et al. (2005) published a complete taxonomic scheme for classification of Universityrickettsiae at the genus and species levels. According to the classification, a new bacterium belongs to the member of Rickettsia genus should have at least 98.1 % and 86.5
% of sequence identities in the 16S rRNA (rrs) and gltA, respectively, compared to a
validated Rickettsia species. In order to be identified up to species level, the rickettsia
should exhibit a sequence similarity of ≥ 99.9 % for gltA, ≥ 99.8 % for rrs, ≥ 98.8 % for
7 ompA, ≥ 99.2 % for ompB and ≥ 99.3 % for sca4 with a validated Rickettsia species
(Figure 2.1). The rickettsia belongs to a validated species if the isolate is available in pure
culture. A potential new species without isolate will be named as Candidatus Rickettsia
sp.
Malaya of
Figure 2.1: Taxonomic scheme for classification of rickettsiae at the genus and species Universitylevels. Genes: rrs encodes the 16S rRNA; gltA encodes the citrate synthase; ompA encodes the rOmpA; ompB encodes rOmpB; sca4 (gene D) encodes the PS-120 (Raoult et al., 2005).
8 2.2 SFG rickettsiae
The spotted fever group rickettsiae have been found in every continent except
Antarctica (Mahajan, 2012). The SFG unites a phylogenetically distinct group of
rickettsiae that have a life cycle involving arthropods, particularly ticks (Socolovschi et
al., 2009). Several members of SFG rickettsiae that are considered as emerging pathogens
around the world include Rickettsia japonica, Rickettsia africae, Rickettsia honei, R. felis,
and Rickettsia slovaca (Luce-Fedrow et al., 2015a). In the past ten years, different groups
of rickettsiologist (clinicians, scientists, veterinarians, field investigators, and laboratory
professions) from different parts of the world have contributed to the accumulation of
knowledge in this research field (Parola et al., 2013).
Recent seroepidemiological data and clinical studies have shown increased prevalences of SFG rickettsiae in United States (DrexlerMalaya et al., 2016), Africa (Parola, 2011), Australia (Derne et al., 2015) and Asia (Fang et al., 2015; Parola et al., 2013;
Reller et al., 2012; Sophie et al., 2014). SFGof rickettsioses are classified as neglected
emerging diseases, especially in developing countries (Crump et al., 2013; Maina et al.,
2012; Mediannikov et al., 2010). The increasing incidents of rickettsia transmission by
arthropods to humans are probably attributed to globalization and climate change (Parola
et al., 2008). There is evidence that Rhipicephalus sanguineus, a brown dog tick
transmitting R. conorii, are more aggressive at higher temperature leading to increased
human attacks (Parola et al., 2008).
UniversitySince the late twentieth century, globalization and travelling have contributed to the spreading of infectious diseases (Tatem et al., 2006). Travel-associated rickettsioses have
been published worldwide, with the most prevalent being murine typhus (caused by R.
typhi), Mediterranean spotted fever (caused by R. conorii), and African tick bite fever
(caused by R. africae) (Delord et al., 2014; Jensenius et al., 2004). Rickettsial infections,
9 particularly tick-borne spotted fever, occurred more frequently than dengue among the
travelers returning from sub-Saharan Africa (Freedman et al., 2006). A case of travel-
acquired SFG rickettsioses (reacting to R. conorii antigen) infection has been reported in
a traveler from Sri Lanka in Australia (Stokes & Walters, 2009). Rapid economic
development, deforestation, land rehabilitation and urbanization may increase the
potential exposure to arthropods and the consequent risk of rickettsial transmission
(Merhej et al., 2014).
2.2.1 Rickettsia rickettsii
R. rickettsii is the causative agent for Rocky Mountain spotted fever (RMSF) which is
a life-threatening disease (Dantas-Torres, 2007). The first clinical report of RMSF was
made as early as 1899 by Dr. Edward E. Maxey (Raoult & Roux, 1997; Ricketts, 1991). The geographical distribution of RMSF is restrictedMalaya to countries of the western hemisphere. The disease has been frequently reported in Brazil (Oliveira et al., 2016;
Straily et al., 2016), Costa Rica (Hun et al.of, 2008), Mexico (Alvarez-Hernandez et al.,
2015), and the United States (Nelson, 2015; Tull et al., 2017). Fatal human infection of
RMSF had been reported in Arizona (Regan et al., 2015), Costa Rica (Argüello et al.,
2012), Panama (Tribaldos et al., 2011) and Mexico (Zavala-Castro et al., 2006).
American dog ticks (Dermacentor variabilis) are the primary vector of R. rickettsii in
United States while brown dog ticks (Rh. sanguineus) serve as the primary vector of R.
rickettsii in Mexico (Dantas-Torres, 2007). A recent study reported the identification of UniversityR. rickettsii with 96.3 – 100 % sequence similarity in six small mammals in Taiwan (Kuo et al., 2015).
2.2.2 Rickettsia conorii
R. conorii is the main etiological agent for Mediterranean spotted fever (MSF) which
is also known as ‘boutonneuse fever’ (Brouqui et al., 2007). R. conorii was first described
10 in 1909 in Tunisia, North Africa by Conor and Bruch (as cited in(Raoult & Roux, 1997).
Several subspecies of R. conorii have been identified which include R. conorii subsp.
caspia, R. conorii subsp. conorii, R. conorii subsp. indica and R. conorii subsp.
israelensis (Parola et al., 2013). Dogs have been reported to suffer from MSF (Solano-
Gallego et al., 2015; Solano-Gallego et al., 2006). The disease is transmitted by Rh.
sanguineus dog ticks in which the ticks also serve as reservoirs (Socolovschi et al.,
2012b). MSF due to R. conorii is prevalent in central Asia (Rossio et al., 2015), southern
and eastern Europe (Brouqui et al., 2007), North Africa (Mouffok et al., 2009), Portugal
(Crespo et al., 2015). The disease complications and fatal cases have been reported in up
to 32 % of patients with MSF (Oteo & Portillo, 2012). Complications which have been
reported in MSF patients include renal and multiorgan failure, acute myocarditis, atrial fibrillation etc. (Ben Mansour et al., 2014; Colomba etMalaya al., 2008; Oteo & Portillo, 2012). 2.2.3 Rickettsia raoultii
R. raoultii was first detected in Rhipicephalusof pumilio and Dermacentor nuttalli ticks
collected from the former Soviet Union in 1999. The bacterium were initially designated
as Rickettsia genotypes RpA4, DnS14 and DnS28 (Rydkina et al., 1999). Isolates for the
Rickettsia species have then obtained and identified as a novel species based on
polyphasic strategy combining genotypic and phenotypic tests (Mediannikov et al.,
2008). Since then, R. raoultii has been found to predominant in Dermacentor ticks
throughout Europe (Spitalska et al., 2012) and some countries in Asia including Mongolia University(Speck et al., 2012) and China (Liu et al., 2016; Wen et al., 2014). Rickettsia spp. closely related to R. raoultii were reported in two tick species (Amblyomma varanense and
Amblyomma helvolum) infesting snakes and an A. helvolum tick from a Varanus salvator
in Thailand (Doornbos et al., 2013; Sumrandee et al., 2014).
11 R. raoultii DNA was first detected in the blood sample of a patient with tick-borne
lymphadenopathy (TIBOLA) in Spain in 2006, and subsequently from a Dermacentor
marginatus tick collected from the scalp of a patient with TIBOLA in France (Ibarra et
al., 2006; Mediannikov et al., 2008). Additional seven probable cases of R. raoultii
infection were later documented in France (Parola et al., 2009). In China, R. raoultii
infection has been identified in two patients with localized rashes around sites of tick bites
(Jia et al., 2014). These cases further confirmed the role of R. raoultii as a human
pathogen.
2.2.4 Rickettsia felis/Rickettsia felis-like organisms
R. felis was first detected in European cat fleas (Ctenocephalides felis felis) in 1918
and was named as Rickettsia ctenocephali by Hilda Sikora (as cited in(Parola, 2011). The work was overlooked until 1990, when a rickettsia-likeMalaya organism was observed in the cytoplasm of the midgut cells of C. felis fleas (Adams et al., 1990). R. felis was cultured
in Xenopus laevis tissue culture cells (XTC-2)of at a low temperature (28oC) (Raoult et al.,
2001b). R. felis strain Marseille-URRWXCAl2 was then proposed as the type strain for
R. felis and the genome was published in 2005 (La Scola et al., 2002; Ogata et al., 2005a).
R. felis was first described as a human pathogen almost two decades ago (Hun &
Troyo, 2012). Thereafter, within the past 20 years, there are fast-growing reports of the
worldwide detection of R. felis in arthropod samples with the availability of molecular
techniques. In conjunction, the reports of R. felis human infections have also increased Universityrapidly (Perez-Osorio et al., 2008). To date, human infections caused by R. felis have been reported in 18 countries across all the continents (Hun & Troyo, 2012). R. felis has
now been described in more than 40 hematophagous arthropods including ticks, fleas,
mosquitoes and mites from more than 20 countries throughout the world (Brown &
Macaluso, 2016; Parola, 2011). In addition, the presence of this pathogen has been
12 detected in other vertebrate host including cats, dogs, opossums, raccoons, rodents and
monkeys (Brown & Macaluso, 2016).
Within the last decade, several reports identified R. felis-like organisms (RFLO) in
different arthropods, including cat fleas, hard- and soft-bodied ticks, throughout the
world. Several uncultured rickettsiae genetically closely related to R. felis including
Rickettsia sp. RF2125 and Rickettsia sp. RF31 were detected in Ctenocephalides species
near Thailand-Myanmar border and Malaysia (Mokhtar & Tay, 2011; Parola et al.,
2003a). Rickettsia sp. RF2125 genotype has been widely reported in various arthropods
including hedgehog fleas (Archeopsylla erinacei) in Algeria (Bitam et al., 2006), human
fleas (Pulex irritans) in Hungary (Hornok et al., 2010), Echidnophaga gallinacea fleas in
Egypt (Loftis et al., 2006) and etc. Angelakis et al. (2016) suggest that RFLO (Rickettsia sp. RF2125 and R. felis strain Pan) probably representMalaya another, not yet isolated species within the R. felis cluster by considering the genetic criterion of 99.9 % gltA sequence
identity in order to be considered the same species,of
The two recently described RFLO isolated from cat fleas are Rickettsia asemboensis
and Candidatus Rickettsia senegalensis. Rickettsia asemboensis was isolated from
Ctenocephalides canis and C. felis fleas collected in Asembo, Kenya (Luce-Fedrow et
al., 2015b; Maina et al., 2016) while Candidatus Rickettsia senegalensis was isolated
from C. felis fleas collected from Senegal (Mediannikov et al., 2015). The rickettsial 16S
rRNA genes showed 99.5 % and 99.65 % similarity to the 16S rRNA gene of R. felis UniversityURRWXCal2 type strain, respectively (Jiang et al., 2013; Mediannikov et al., 2015). R. hoogstraalii, closely related to R. felis, was isolated from Haemaphysalis sulcata ticks in
Croatia and Carios capensis soft ticks in Georgia (Duh et al., 2010). The 16S rRNA gene
of R. hoogstraalii exhibited 99.7 % to that of R. felis URRWXCal2 type strain (Duh et
al., 2010). Detail studies to look into the ecological, genomic or phenotypic differences
13 between R. felis and RFLOs are needed in order to justify the separation of RFLO into
species (Brown & Macaluso, 2016).
R. felis has been recognized as the etiological agent for flea-borne rickettsioses, also
known as flea-borne spotted fever (Brown & Macaluso, 2016). The cases of flea-borne
spotted fever are likely to be underestimated due to the unspecific clinical signs with other
febrile illness (i.e.: fever, rash, headache, and myalgia) and the limited access to
appropriate diagnostic tests (Perez-Osorio et al., 2008). The first human case of R. felis
infection was misdiagnosed as flea-borne endemic typhus as the serological test at that
time were unable to distinguish the two rickettsial species (Schriefer et al., 1994).
Much of the latest works are focusing on the epidemiology of R. felis in Africa due to the considerable frequency of flea-borne spotted feverMalaya in hospitalized febrile patients. It has been reported to be a common cause of fever amongst hospitalized patients (3-15 %) in sub-Saharan Africa (Mediannikov et al.of, 2013a; 2013b; 2014; Richards et al., 2010; Socolovschi et al., 2010). R. felis has also been reported to cause human infections in
China, South Korea and Taiwan (Lai et al., 2014; Yeon-Joo et al., 2005; Zhang et al.,
2014). Although R. felis is an emerging pathogen, it is still unclear whether the increased
incidence in Africa reflects an overall trend or represents an endemic state previously
unknown for R. felis infections (Brown & Macaluso, 2016).
2.3 Geographical distribution of SFG rickettsiae University2.3.1 Worldwide distribution of SFG rickettsiae Molecular evidences show that SFG rickettsiae have a widespread distribution across
different continents including the Americas, Europe, Africa, Australia and Asia (Wood
& Artsob, 2012) (Figure 2.2). The geographical distribution of rickettsiae is closely
associated to their vectors (Merhej et al., 2014). Arthropods have comparatively low
mobility, and their dispersal are associated with the host migration during vector-borne
14 pathogen infestation. Ticks species are highly dependent on their biotopes, and very few
ticks, including Rh. sanguineus, have a worldwide distribution. On the other hand, fleas,
mites and lice are globally distributed (Merhej et al., 2014).
Malaya Figure 2.2: World distribution of tick-borne rickettsioses (Raoult, 2012).
of
More than 10 different tick-borne rickettsioses apart from RMSF, epidemic typhus and
endemic typhus have been described in both arthropods and clinical cases since the year
of 2000 in America (Hidalgo et al., 2013). Spotted fever was first described in South
America in 1931 in Sao Paulo, Brazil (Schoeler et al., 2005). In South America, other
SFG species identified as human pathogens include R. rickettsii, R. parkeri, R. massiliae,
and Rickettsia sp. strain Atlantic rainforest or strain Bahia. The pathogenic SFG Universityrickettsiae reported in North and Central America include R. rickettsii, R. parkeri, R. massiliae, R. africae and Rickettsia philipii (Rickettsia 364D) (Parola et al., 2013).
MSF caused by R. conorii are known to be prevalent in Europe (Brouqui et al., 2004).
Other autochthonous tick-borne rickettsioses that have been described in Europe include
infections caused by R. aeschlimannii in France (Raoult et al., 2002b), R. massiliae in
15 Sicily (Vitale et al., 2006), Rickettsia mongolotimonae in Southern France (Pierre-
Edouard et al., 2000), R. slovaca throughout Europe (Lakos, 2002; Raoult et al., 2002a),
R. helvetica throughout Europe (Fournier et al., 2000), R. felis throughout Europe
(Brouqui et al., 2007), R. conorii subspecies israelensis in Italy and Portugal (Bacellar et
al., 1999; Giammanco et al., 2005) and R. conorii subspecies caspia in Astrakhan and
Kosovo (Fournier et al., 2003b; Tarasevich et al., 1991). In addition, R. africae has been
frequently reported among travelers returning to Europe (Jensenius et al., 2003; Raoult et
al., 2001a).
Tick-borne rickettsioses have been regularly reported in North and South Africa since
1910, particularly MSF associated with R. conorii. R. africae was first detected and
isolated in a woman in the year of 1992 (as cited in(Mediannikov et al., 2010). Since then, this organism has been widely detected across the continentMalaya include Niger, Mali, Burundi, Chad, Ethiopia and most countries in South Africa (Mediannikov et al., 2010). Other
rickettsial species that have been identifiedof as pathogens in North Africa are R. conorii
subsp. israelensis, R. sibirica subsp. mongolitimonae, R. slovaca, R. raoultii, R.
monacensis, R. massiliae, R. africae, and R. helvetica (Parola et al., 2013). In Sub-
Saharan Africa, R. africae, R. conorii subsp. conorii, R. conorii subsp. caspia, R.
aeschlimannii, R. sibirica subsp. mongolitimonae, and R. massiliae are rickettsial
pathogens that have been reported to cause human infections (Parola et al., 2013). In a
study conducted by Jensenius et al. (2009), a total of 82.5 % of the 280 international Universitytravelers studied acquired SFG rickettsiosis during travelling and 87.6 % of the SFG rickettsiosis incidences were acquired in sub-Saharan Africa (Jensenius et al., 2009).
The first cases of SFG rickettsiosis in Australia were described in 1946 in Queensland
with the isolation of R. australis (Parola et al., 2005a). R. honei, the causative agent of
Flinders Island spotted fever was first discovered to cause human infection in 1991
16 (Parola et al., 2005a). Cases of R. africae are commonly reported in Australia among
travelers returning from sub-Saharan Africa (Kelly et al., 1996).
In Asia, Siberian tick typhus (caused by R. sibirica) has been reported in Russia, China,
Mongolia, and Kazakhstan (Byambaa, 2008; Mediannikov et al., 2007; Wu et al., 2013).
Several serological confirmed cases have also been reported in South Korea (Jang et al.,
2005). Far Eastern spotted fever caused by R. heilongjiangensis was first reported in
Russia and China (Parola et al., 2005a) and the organism was subsequently isolated from
Haemaphysalis concinna and Dermacentor silvarum ticks (Mediannikov et al., 2006;
Shpynov et al., 2006). Human infection caused by R. conorii subsp. conorii has only been
reported in Turkey (Kuloglu et al., 2012; Parola et al., 2013) while R. conorii subsp.
indica was reported in India, Laos and Sri Lanka (Chaudhry et al., 2009; Phongmany et al., 2006; Stokes & Walters, 2009). SFG rickettsiosisMalaya caused by R. honei have been described in Thailand and Nepal (Holly et al., 2011; Sangkasuwan et al., 2007). In 2011,
R. tamurae was reported to cause human infectionof in Japan (Imaoka et al., 2011) and
Laos (Phongmany et al., 2006). Other than that, R. japonica rickettsioses have been
reported in Japan and Korea (Chung et al., 2006; Mahara, 1997, 2006; Uchida et al.,
1992). The other SFG rickettsiae found in China are R. akari, R. conorii, R.
heilongjiangensis, R. raoultii and R. sibirica (Jia et al., 2014; Qi et al., 2013; Wu et al.,
2013). University
17 2.3.2 SFG rickettsioses in Southeast Asia
Figure 2.3: SFG rickettsioses reported in Southeast Asia (Map: http://mapsof.net/asia/map-of-southeast-asia). Malaya The countries in Southeast Asia includeof Brunei, Cambodia, East Timor, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand and Vietnam. The incidence
rates of rickettsioses in Southeast Asia are still largely undetermined (Aung et al., 2014).
According to the systematic review by Acestor et al. (2012), rickettsioses have been
reported as the second most frequently reported infections for non-malaria febrile illness
among residents of Southeast Asia, just after dengue. Most studies on rickettsioses have
been reported in Thailand and there is no epidemiologic data available for Brunei, East
Timor and Myanmar (Figure 2.3) (Aung et al., 2014). Serologically confirmed cases of Universityrickettsioses mainly on murine typhus and scrub typhus have been reported in Singapore and Northern Vietnam (Chen et al., 2001; Hamaguchi et al., 2015; Ong et al., 2001). SFG
infections in human caused by R. felis, R. honei, and R. japonica have been documented
in Thailand (Gaywee et al., 2007; Jiang et al., 2005; Sophie et al., 2014). A study
conducted by Parola et al. (2003a) in Kanchanaburi Province, Thailand (Thai-Myanmar
border) reported that five, two and one patients were infected by R. helvetica, R. conorii
18 strain indica and R. felis, respectively (Parola et al., 2003b). Recently, SFG rickettsiae
seropositivity was reported to be relatively high in patients enrolled with undifferentiated
febrile illness in Chiangrai (33 %) and Mae Sot (27.3 %), Thailand (Blacksell et al.,
2015).
R. helvetica, Rickettsia sp. AT1, R. felis, and R. conorii have been detected in patients
from Laos in several studies (Dittrich et al., 2014a; Mayxay et al., 2013; Phongmany et
al., 2006). In Cambodia, 11.6 % of febrile patients in a study showed IgG seropositivity
towards SFG rickettsiae and 14.4 % of the seropositive samples showed 4-fold rise in titer
or seroconversion (Kasper et al., 2012). Camer et al. (2003) reported 1.3 % seropositivity
rate of a SFG rickettsia (R. conorii) in human febrile patients in the Philippines. A total
of 10.4 % and 20.4 % of the healthy residents in Gag Island, Indonesia, showed seroreactivity to R. rickettsii and R. conorii, respectivelyMalaya (Richards et al., 2003). The main reservoirs and vectors for rickettsiosesof in Southeast Asia were rodents and ticks/fleas, respectively (Aung et al., 2014). Rodents were found to be important
reservoirs of SFG rickettsiae and R. felis in Indonesia (Ibrahim et al., 1999; Widjaja et
al., 2016). Infection of mites, lice and ticks with R. felis and other SFG rickettsiae have
also been reported in Indonesia, which further enhance the possibility of human infections
with R. felis and SFG rickettsiae in the region even though no clinical case has been
reported to date (Barbara et al., 2010; Jiang et al., 2006; Widjaja et al., 2016). The role
of dogs as natural mammalian reservoirs for R. felis had been demonstrated in the semi- Universitydomesticated dogs collected from Cambodia (Inpankaew et al., 2016). Rickettsial surveillance in ectoparasites collected from villages in Laos show high detection rates of
R. felis in fleas (76.6 %) and cattle tick (Boophilus spp.) (2.1 %) (Kernif et al., 2012).
Studies in Thailand demonstrated that Bandicota species and Rattus argentiventer may
be the main rodent reservoir for transmission of SFG rickettsiae particularly R. japonica
19 and R. honei (Okabayashi et al., 1996). Studies in Thailand reported the detection of SFG
rickettsiae in A. helvolum and A. varanense (Doornbos et al., 2013; Sumrandee et al.,
2014). Another study by Hirunkanokpun et al. (2003) found three new phylogenetically
distinct SFG rickettsiae in 30 % of Amblyomma testudinarium and 16.8 % of
Haemaphysalis ortnithophila ticks collected from animals and vegetation in Khao Yai
National Park, Thailand. However, the role of new SFG rickettsiae in causing human
diseases and the potential of ticks to feed on humans as a host in Southeast Asia are
unknown (Hirunkanokpun et al., 2003). It is expected that the rates may increase in the
near future in some countries attributed to the improve diagnostic methods (Aung et al.,
2014).
2.3.3 SFG rickettsioses in Malaysia Comprehensive studies on Malaysian ticks speciesMalaya have been carried out by the Institute for Medical Research, Ministry of Health, Malaysia, as early as 1957 (Kohls,
1957). Since then, there are several reportsof on irregular focalized collections of ticks in
different regions of Peninsular Malaysia (Mariana et al., 2008; Nadchatram, 2008). The
tick species found in Malaysia include the Amblyomma, Dermacentor, Haemaphysalis,
Ixodes, and Rhipicephalus (including Rhipicephalus microplus which was previously
known as Boophilus microplus) which may play an important role as potential vectors for
rickettsioses and other arthropod-borne diseases (Nadchatram, 2008; Petney, 1993).
Human tick typhus in Malaya was first detected in 1958 in a patient suspected with Universityscrub typhus (Marchette, 1966). Additional human cases were reported in the following years and the presence of complement-fixing antibodies in jungle small mammals were
also documented (Marchette, 1966). Hemolymph samples of Haemaphysalis conigera
collected in a undisturbed forest in Selangau, Sarawak, were first tested to be positive for
R. conorii using direct immunofluorescence antibody test (Tay et al., 1996).
20 The seropositivity to Rickettsia sp. TT118 (SFG rickettsia) was first reported in 57.3
% of rubber estate workers in Slim River, Perak, Malaysia (Tay et al., 1999). A high
seropositivity of SFG rickettsiae (42.5 %) was also reported among febrile patients from
eight rural areas in close proximity to forest, oil palm and rubber plantations, Peninsular
Malaysia (Tay et al., 2000). Rickettsial infection was also reported among five remote
Orang Ulu villagers in upper Rejang River, Sarawak, East Malaysia. A total of 3.8 % of
the villagers investigated were seropositive to Rickettsia sp. TT118 (Sagin et al., 2000).
On the other hand, a total of 12.9 % of 61, 501 febrile patients’ sera from eight Malaysian
hospitals (Penang, Perak, Johor, Kedah, Terengganu, Kelantan, Sarawak and Sabah)
during 1994-1999 were seropositive to Rickettsia sp. TT118 (Tay & Rohani, 2002).
Antibodies against Rickettsia sp. TT118 was also reported in 1.7 % and 11.6 % of urban blood donors and febrile patients, respectively, from KualaMalaya Lumpur Hospital (Tay et al., 2003). The findings suggested that people from the rural areas and those engaged in the agricultural activities are at higher risk of contractingof rickettsioses in this country.
Mokhtar and Tay (2011) reported the detection of R. felis DNA in 2.9 % of the 209
fleas collected from healthy cats in Kedah and Kuala Lumpur. A higher detection rate of
R. felis (74.4 %, 268/360) was also reported in the fleas collected from Borneo, East
Malaysia (Kernif et al., 2012). Rickettsial DNA was detected in 13.7 % of the wild rats
in wet markets of Kuala Lumpur and Penang (Tay et al., 2014). Sequence analysis showed
the identification of R. honei/R. conorii/R. raoultii based on gltA DNA fragments and UniversityRickettsia TCM1 based on ompA amplicons (Tay et al., 2014). Detection of R. felis in 32.2 % of the fleas collected from Kuala Lumpur and Selangor was also reported in the
same study (Tay et al., 2014). Sequence analysis showed the presence of two genotypes
of R. felis in the cat fleas, with the predominant genotype demonstrated 100 % sequence
similarity to Rickettsia sp. RF2125 (AF516333) and the other genotype representing by
21 only a single sample (HL15c) matched 100 % with Rickettsia sp. Rf31 (AF516331) (Tay
et al., 2014).
With regard to the literature available in this country, data about rickettsial species
circulating in different arthropods and human population are scarce despite of the increase
importance of SFG rickettsioses reported worldwide. Continuous surveillance of
rickettsioses is needed amongst population at high risk to prevent disease outbreak.
Identification of potential vector and host for rickettsiae is essential as a strategy to reduce
disease burden due to rickettsioses.
2.4 Vectors and reservoirs for rickettsioses
Rickettsia spp. are often associated with obligate hematophagous arthropods particularly ticks (Acari: Ixodidae and Argasidae), lice,Malaya fleas and mosquitoes (Merhej et al., 2014). Table 2.2 presents various vectors implicated in the transmission of SFG rickettsioses. Arthropods acquire rickettsiaeof through transovarian transmission or horizontal transmission through co-feeding which occurs when several arthropods feed
on the same host simultaneously (Socolovschi et al., 2009). Many rickettsial species are
considered to be vertically transmitted symbionts of invertebrates, suggesting the role of
arthropod vectors as the reservoirs or amplifiers for the infections (Parola et al., 2013).
Arthropods can serve as bacterial reservoir when rickettsiae are transmitted efficiently,
both transstadially (from larva to nymph and/or nymph to adult) and transovarially (from Universityfemale to progeny) in an arthropod vector (Parola & Raoult, 2001).
22 Table 2.2: Recognized or potential vectors for the rickettsiae that are associated with human diseases.
Rickettsia species Vector Diseases References R. aeschlimannii Hyalomma marginatum, Tick-transmitted Fernandez-Soto Hyalomma rufipes, disease et al. (2003) Haemaphysalis punctata, Rhipicephalus appendiculatus R. africae Amblyomma hebraeum, African tick bite Cazorla et al. Amblyomma lepidum, fever (2008); Portillo Amblyomma variegatum, et al. (2007) Rhipicephalus decoloratus R. akari Liponyssoides sanguineus Rickettsial pox Brouqui and Raoult (2006); Paddock et al. (2006) R. australis Ixodes holocyclus, Queensland tick Sexton et al. Ixodes tasmani, typhus (1991); Graves Ixodes cornuatus et al. (1993) R. conorii subsp. Rh. sanguineus, Mediterranean Raoult and conorii Haemaphysalis leachii spottedMalaya fever Roux (1997) R. conorii subsp. Rh. sanguineus, Israel tick typhus Zhu et al. (2005) israelensis Amblyomma maculatum R. conorii subsp. Rh. sanguineus ofIndian tick typhus Zhu et al. (2005) indica R. felis C. felis, Flea-borne spotted Brown and C. canis, fever Macaluso A. erinacei, (2016); Reif and P. irritans, Macaluso X. cheopis, (2009) An. gambiae, Rh. sanguineus, H. sulcata R. D. silvarum, Far-eastern tick- Fournier et al. heilongjiangensis Haemaphysalis japonica borne rickettsiosis (2003a); douglasi, Mediannikov et H. concinna al. (2006); University (Mediannikov et al., 2009) R. honei Aponomma hydrosauri, Flinders Island Graves and Ixodes granulatus spotted fever Stenos (2003)
23 Table 2.2, continued
Rickettsia species Vector Diseases References R. japonica Haemaphysalis flava, Japanese or Uchida et al. Haemaphysalis Oriental spotted (1992); Fournier longicornis, fever et al. (2002); Dermacentor taiwanensis, Uchiyama and Ixodes ovatus Uchida (1989) R. marmionii Haemaphysalis Australian spotted Stenos et al. novaeguinae, fever (2005); Ixodes holocyclus Unsworth et al. (2007) R. massiliae Rh. sanguineus, Spotted fever Cicuttin et al. Rhipicephalus turanicus (2015); (Wei et al., 2015) R. parkeri A. maculatum, Tick-transmitted Herrick et al. Amblyomma triste disease i.e.: skin (2016) lesions and lymphadenitis R. raoultii D. nuttalli, Tick-borne Mediannikov et D. silvarum, lymphadenopathy, al. (2008); Wen Dermacentor reticulatus, Dermacentor-Malayaet al. (2014); D. marginatus, borne-necrosis- Shpynov et al. Dermacentor nivues erythema- (2001) oflymphadenopathy R. rickettsii Amblyomma aureolatum, Rocky Mountain Minniear and Amblyomma cajennense, spotted fever, Buckingham Dermacentor andersoni, American spotted (2009) Dermacentor variabilis, fever Rh. sanguineus R. slovaca D. marginatus, Tick-borne Ibarra et al. Dermacentor reticulatus lymphadenopathy, (2006); Lakos Dermacentor- (2002); borne-necrosis- Spitalska et al. erythema- (2012) lymphadenopathy R. sibirica subsp. D. nuttalli, Siberian tick Mediannikov et sibirica D. marginatus, D. typhus al. (2007) silvarum, Dermacentor Universitysinicus, D. auratus R. tamurae A. testudinarium Skin inflammation Imaoka et al. (2011)
24 SFG rickettsiae are known to be maintained in nature through transovarial and
transstadial transmission in ticks and horizontal transmission to uninfected ticks that feed
on rickettsemic animals (Figure 2.4). Humans are occasional host for ticks and they are
only rickettsemic for short period of time, and do not serve as reservoirs for tick-borne
rickettsiae. Hence, humans are considered as the dead-end host for rickettsioses which
play no role in maintaining the bacteria in nature (Parola et al., 2013).
Malaya
of
Figure 2.4: Life cycle of tick-borne rickettsiae (Walker & Ismail, 2008).
2.4.1 Tick-borne rickettsioses
Ixodid ticks (hard ticks) are the most important vectors and reservoirs for SFG
rickettsiae in most regions in the world. The distribution of SFG rickettsioses are therefore
determined by the distribution of the ticks. Ixodid ticks have been reported as the vectors
Universityand reservoirs for the disease-causing rickettsiae including R. rickettsii, R. africae, R.
conorii, R. honei, R. sibirica, R. slovaca, R. raoultii, R. parkeri, R. massiliae, R.
aeschlimannii and R. helvetica (Parola et al., 2013; Socolovschi et al., 2013). Other than
that, R. australis, R. heilongjiangensis, R. japonica, R. sibirica mongolitimonae, R.
25 monacensis, and R. philipii have also been reported to have ticks as vectors but their roles
as reservoirs have not been confirmed (Merhej et al., 2014).
Ixodid ticks feed once within each stage for a relatively long period of time, ranging
from few hours to several days. The attachment of the ticks to the host may be unnoticed
due to the painless bite of the ticks which consequently enhance the vector potential of
ticks (Socolovschi et al., 2009).
2.4.2 Flea-borne rickettsioses
Fleas are the vectors of Rickettsia spp. particularly R. typhi and R. felis. R. typhi is the
causative agent for murine typhus and the main vectors are rat flea (X. cheopis) and cat
flea (C. felis) (Portillo et al., 2015). Human cases have been reported in United States, Europe, Taiwan, Thailand, Singapore, Laos, and NepalMalaya (Tsioutis et al., 2017). Primary reservoirs for murine typhus are commensal rodents such as Rattus rattus and Rattus norvegicus (Merhej et al., 2014). Other peri-domesticof and wild animals such as free- ranging cats, dogs, opossums, raccoons and squirrels have also been reported to act as
hosts for murine typhus (Azad et al., 1997; Merhej et al., 2014). Infection in humans can
acquire through different ways including inhalation or self-inoculation of infected flea
feces into skin (Peniche Lara et al., 2012)
R. felis is transmitted vertically and horizontally in cat fleas (C. felis) which supports
the role of fleas as vector and reservoir (Hirunkanokpun et al., 2011). R. felis has also Universitybeen detected in other flea species including Anomiopsyllus nudata, Archaeopyslla eriinacei, Diamanus montanus, C. canis, Ctenocephalides orientis, Ctenophthalmus sp.,
E. gallinacea, Leptopsylla segnis, Polygenis atopus, P. irritans, Spilopsyllus cuniculi,
Tunga penetrans, Xenopsylla brasiliensis, and Xenopsylla cheopis collected from
different animal hosts (dogs, rodents, monkey, opossums, shrew, foxes, hedgehogs, pigs)
and humans (Merhej et al., 2014). Although R. felis has been identified in a large number
26 of blood-feeding arthropods, but only cat fleas, C. felis, are associated with the biological
transmission of this pathogen (Reif & Macaluso, 2009).
The transmission routes for the maintenance of R. felis and RFLO infections in the
fleas has been described by Brown and Macaluso (2016) (Figure 2.5). Flea larvae may
acquire R. felis through infectious adult feaces via vertical non-transovarial transmission
(route A in Figure 2.5). Brown and Macaluso (2016) also determined the adult flea
acquisition of R. felis LSU and LSU-Lb through experimental bioassay (route B in Figure
2.5). Intra-species and inter-species transmission of R. felis can occur through co-feeding
and mating (route C and D in Figure 2.5), as demonstrated using an artificial feeding
system (Brown et al., 2015; Hirunkanokpun et al., 2011). In sustained transmission (route
E in Figure 2.5), co-feeding of R. felis-infected cat fleas continue to spread the bacteria to other uninfected cat fleas in an artificial systemMalaya (Brown et al., 2015; Brown & Macaluso, 2016). of
University
Figure 2.5: Transmission routes for the maintenance of flea-borne rickettsiae (R. felis and RFLO) infections in the environment (Brown & Macaluso, 2016).
27 2.4.3 Mite-borne rickettsioses
R. akari is the etiological agent for mite-borne rickettsioses, also known as
rickettsialpox. It is commonly transmitted by Liponyssoides sanguineus mite which infect
the house mouse, Mus musculus (Denison et al., 2014; Huebner et al., 1947). The
rickettsia is closely related to R. australis and Rickettsia sp. cfland5/ Rickettsia sp. SE313
in the rat mites, Ornithonussus bacoti (Reeves et al., 2007). Rickettsia sp. TwKM02 with
98.4 % gltA sequence similarity to R. australis has been detected in Leptotrombidium
deliense chigger mites in Taiwan (Tsui et al., 2007). Additionally, Rickettsia sp.
TwKM03 with 99.2 % gltA sequence similarity to R. felis URRWXCal2 has also been
reported in L. deliense chigger mite pools and a Mesostigmata mite pool in the same study
(Tsui et al., 2007). R. helvetica and R. monacensis were detected in ectoparasitic mites (Laelapidae and Trombiculidae) infesting rodents in south-westernMalaya Slovakia (Mitkova et al., 2015).
2.4.4 Mosquitoes of
Mosquitoes, the most important vectors of infectious diseases in human, has recently
associated with the transmission of R. felis. The transmission potential of R. felis by
Anopheles gambiae was recently demonstrated in a stimulated model by Dieme et al.
(2015). Molecular detection of R. felis was reported in Aedes albopictus (Asian tiger
mosquitoes) collected from Libreville, Gabon (Socolovschi et al., 2012a) and An.
gambiae collected from Sub-Saharan Africa (Socolovschi et al., 2012c). Other Universityinverbrates that have been associated with Rickettsia species includes beetles (Coleoptera), flies (Diptera), leeches (Hirudinida), non-hematophagous booklice
(Psocoptera), true bugs (Hemiptera), springtails (Collembola), wasps (Hymenoptera), and
amoebae (Mediannikov et al., 2012; Perlman et al., 2006).
28 2.5 Clinical manifestations of patients with rickettsioses
Infections caused by SFG rickettsiae can present with non-specific febrile illness
accompanied by headache, nausea, vomiting, myalgia, anorexia, and diarrhea that usually
appear from two to 14 days after a tick bite (Aung et al., 2014; Mahajan, 2012). In some
cases, eschar may develop at the site of inoculation. Rashes usually appear after three to
five days of onset of fever and begin around the wrists and ankle which subsequently
become maculopapular, petechial or hemorrhagic (Mahajan, 2012; Rathi & Rathi, 2010).
Rashes may absent in some spotted fevers (Rathi & Rathi, 2010). The presence of an
eschar is highly variable in which more significant eschars are usually associated with
mildly pathogenic rickettsiae as compared to highly virulent rickettsiae, such as R.
rickettsii which are not associated with eschars (Wood & Artsob, 2012). Thrombocytopenia, elevated hepatic enzyme levels andMalaya leukocyte count abnormalities are common in patients with rickettsioses (Parola & Raoult, 2001).
The clinical manifestations of murine typhusof usually begin after seven to 14 days of
infection with the common symptoms of fever, musculoskeletal pain, headache and rash.
Complications including pneumonitis, hepatitis, meningoencephalitis and renal failure
may occur but the occurrence usually does not exceed 10 % (Peniche Lara et al., 2012).
Mortality rate reported ranged from 1 % (with use of antibiotic) to 4 % (without use of
antibiotics) (Civen & Ngo, 2008). Serious complications that have been reported included
splenic rupture, pneumonia, meningoencephalitis, renal failure, shock, myocarditis, and Universityendocarditis (Aung et al., 2014). The clinical manifestations of patients with SFG rickettsioses are non-specific and
difficult to be differentiated from other febrile diseases such as malaria, dengue,
leptospirosis and etc. (Civen & Ngo, 2008; Rathi & Rathi, 2010). Untreated rickettsial
infection may result in severe illness or even death. About 30-35 % of fatality rates with
29 multiple organ dysfunction has been reported due to delayed diagnosis and lack of
appropriate treatment (Batra, 2007). Rickettsial infections may developed severe life
threatening manifestation and take a fulminant course especially in patient with glucose-
6-phosphate dehydrogenase (G6PD) deficiency (Rathi & Rathi, 2010). Life threatening
manifestations of rickettsial infection include interstitial pneumonitis,
meningoencephalitic syndrome, acute renal failure, hepatic failure, gangrene and
myocarditis (Rathi & Rathi, 2010).
2.6 Laboratory diagnosis of rickettsioses
2.6.1 Culture of rickettsiae
The diagnosis of rickettsial infections are challenging owing to the non-specific nature
of the clinical manifestations in patients. Conventional identification methods used in bacteriology cannot be applied routinely to studyMalaya rickettsiae due to the strictly intracellular nature of the organisms. Culture techniques are known to be sensitive and
important for confirmation of the clinical diagnosisof of rickettsial disease. Rickettsiae are
cultured in embryonated eggs, tissue culture (Vero cells, L929 cells, C6/36 cells, S2 cells
and etc.) and laboratory animals (La Scola & Raoult, 1997). Isolation of rickettsia from
human samples can be achieved by using buffy coats, whole blood, plasma, and tissue
biopsies as inoculums (Luce-Fedrow et al., 2015a). Triturated arthropod such as ticks and
fleas can also be used as inoculums for rickettsial isolation (Luce-Fedrow et al., 2015a).
The drawback of this method is the time taken for rickettsia to grow and propagate in Universityvitro. In some cases, rickettsial organisms can be detected as early as 48-72 hours post inoculation (Parola et al., 2013) while some rickettsial organisms may require up to
several weeks (Portillo et al., 2017).
Shell vial cell culture technique with slow centrifugation is one of the most commonly
used methods for rickettsial isolation (Portillo et al., 2017). This method has been
30 previously used for culturing of cytomegalovirus and was successfully applied for the
culture of R. conorii from human blood samples (Marrero & Raoult, 1989). The isolation
procedure involves the use of small tubes containing coverslips on which a monolayer of
cells is grown. The small surface area of the cells enhances the ratio of the number of
rickettsia to the number of cells and therefore, increases the chance of isolation. The slow
centrifugation step after the inoculation of the sample increases the rickettsial adhesion
and entry into the host cells. Centrifugation step has proven to be the key point for
successful isolation of the rickettsiae (Kelly et al., 1991).
The bacterial stains that are commonly used to monitor the growth of rickettsiae in cell
culture are Giemsa, Diff-Quik, Gimenez, Macchiavello, Castaneda and acridine orange
stain (Luce-Fedrow et al., 2015a). Giemsa stain is a mixture of methylene blue and eosin that binds to phosphate groups of DNA which containMalaya high amounts of adenine-thymine (A-T) bonding. Rickettsial DNA is very rich in A-T bonding; therefore, Giemsa stain can
be used to distinguish rickettsiae from the hostof cell material (Ammerman et al., 2008).
2.6.2 Serological diagnosis of rickettsioses
Major diagnostic challenges for rickettsioses are the high degree of genetic relatedness
and serological cross-reactivity among rickettsial species (Merhej et al., 2014). Weil-
Felix test is one of the oldest assays which has been widely used around the world,
particularly in resource-limited areas, to diagnose rickettsioses. This is because it is
inexpensive, easy to perform and readily accessible although it is lack of sensitivity and Universityspecificity (Luce-Fedrow et al., 2015a). The Weil-Felix test is based on the detection of antibodies against various Proteus species which contained antigenic epitopes that cross-
react with antigens from members of the genus Rickettsia. The whole cells of Proteus
vulgaris OX-2 react with SFG rickettsiae (except R. rickettsii) while the whole cells of P.
31 vulgaris OX-19 react with TG rickettsiae as well as R. rickettsii (La Scola & Raoult,
1997).
Serodiagnosis using indirect immunofluorescence assay (IFA), remains as the gold
standard for confirmation of infections caused by SFG and TG rickettsiae by the
determination of seroconversion and four-fold increase in the antibody titers (Luce-
Fedrow et al., 2015a). IFA has been used for more than 50 years for the diagnosis of
rickettsial infections around the world (Goldwasser & Shepard, 1959) and can detect both
IgM and IgG antibodies in acute and convalescent human sera. Antibodies usually can be
detected by using IFA at 7-15 days following the onset of symptoms for SFG rickettsioses
(Brouqui et al., 2004). R. rickettsii has been used as antigen for routine serological assays
to detect antibodies for all members of the SFG rickettsiae owing to the cross-reactivity among the group members (Wood & Artsob, 2012). Malaya Due to the extensive cross-reactivity amongof rickettsial species, IFA was unable to identify the causative agent to the species level. To circumvent this problem, monoclonal
antibodies have replaced polyclonal antibodies for the identification of rickettsial isolates
using Western blotting and cross-adsorption techniques (La Scola & Raoult, 1997).
Besides, protein analysis utilizing sodium dodecyl-polyacrylamide gel electrophoresis
(SDS-PAGE) targeting on the outer membrane protein of rickettsiae allows the
identification of the rickettsia up to species level (Beati et al., 1992; Merhej et al., 2014;
Paddock et al., 2006). However, this method is laborious and time consuming (Philippe University& Didier, 2000).
Indirect immunoperoxidase (IIP) assay is another serological test that works similar to
IFA by utilizing peroxidase instead of a fluorescently labeled antibody in which the test
can be interpreted directly under a light microscope. This assay is useful for laboratories
32 in resource-limited countries and has been used for the diagnosis of human infections
(Ando et al., 2010; Tay & Rohani, 2002).
ELISA is another serological assay that can detect rickettsial group-specific IgM, IgG
or both antibodies. Proteins and recombinant proteins have been developed as antigens
for ELISA assays and are now available commercially. For SFG rickettsiae, recombinant
ompA and ompB proteins have been developed by Do et al. (2009). Kowalczewska et al.
(2012) cloned and expressed 10 proteins of R. prowazekii and R. rickettsii, respectively.
Their findings demonstrate that no individual protein is sufficient to be used to diagnose
SFG rickettsiae, except for the three targets (groEL, adr2 and EF-Tu) which is already in
use for diagnosis of both murine typhus and MSF. The advantages of using ELISA assay
include the ability to screen a large number of samples at once, low cost, the technical ease of performing the protocol, and the reproducibilityMalaya of the results (Luce-Fedrow et al., 2015a). However, ELISA is unable to distinguish rickettsial infections to the species
level (Luce-Fedrow et al., 2015a). of
2.6.3 Molecular detection of rickettsiae
Over the decades, the development of molecular detection methods has enhanced the
detection of rickettsial organisms from clinical samples up to species level. Molecular
diagnostic methods produce the most reliable diagnosis in determining the rickettsial
species responsible for an infection. However, this requires a great effort in collecting
appropriate samples which include cutaneous skin swab from the eschar, skin or organ Universitybiopsies of the eschar/rash (Wood & Artsob, 2012). Other types of specimens that can be used for molecular diagnosis of rickettsioses include whole blood, buffy coat,
cerebrospinal fluid (CSF), or pleural fluid. Molecular detection can also be performed on
plasma, serum, formalin fixed paraffin-embedded (FFPE) tissues or fixed slide specimens
if no other options are available (Denison et al., 2014). Polymerase chain reaction (PCR)
33 methods have been reported to be most useful for detection of rickettsiae from eschars
and biopsies samples as compared to acute blood samples as only low numbers of
rickettsiae are circulating in the blood (Portillo et al., 2017).
The molecular diagnostic assays commonly used for detection of rickettsiae include
standard PCR, nested PCR, quantitative real-time PCR and loop-mediated isothermal
amplification (LAMP) assays (Luce-Fedrow et al., 2015a). A variety of gene targets have
been published but none is substantially more effective than others (Paris & Dumler,
2016). The common gene candidates for standard PCR include 16S rRNA (rrs), citrate
synthase (gltA), 17-kDa lipoprotein, outer membrane protein A (ompA), outer membrane
protein B (ompB) and other conserved genes (Paris & Dumler, 2016; Portillo et al., 2017).
Amplification of gltA and ompA genes from different samples is routinely used in well- established rickettsial research laboratories (FenollarMalaya & Raoult, 2004). PCR assays targeting on gltA, ompA and ompB appear to be useful tools for molecular detection of
rickettsial DNA in clinical samples and arthropodsof samples (Santibanez et al., 2013).
Schattner et al. (1992) and Lamas et al. (2008) demonstrated the importance of PCR
technique (targeting on a unspecified 434 bp DNA fragment and ompA gene, respectively)
for detection of rickettsial DNA from blood samples in fatal cases of seronegative
rickettsial infection.
Nested PCR assay is used to enhance the analytical sensitivity of PCR assays
especially for sample with low rickettsiae load. This technique involves two sets of Universityprimers and the second set of primers is used to amplify a secondary target from the first PCR product (Luce-Fedrow et al., 2015a). Nested PCR assays targeting ompB gene and
23S-5S intergenic spacer of Rickettsia sp. have been developed for the detection of
rickettsiae in human and tick samples (Choi et al., 2005b; Kakumanu et al., 2016).The
sensitivity of the PCR can be increased down to the level of detecting 1-10 genomic
34 equivalents per reaction. However, the need to open the first PCR tube in order to retrieve
the template for second PCR reaction also increases the risk of amplicon contamination
(Richards, 2012). This problem can be overcome by replacing nested PCR assays with
real-time PCR technique.
To date, real-time PCR assay has been recognized as an ideal method for detection of
rickettsial organisms as it offers the advantages of rapid detection, high specificity,
reproducibility, quantitative capability and a low risk of contamination (Stenos et al.,
2005; Wölfel et al., 2008). The earliest use of real-time PCR assay for the detection of
SFG rickettsiae was developed by Stenos et al. (2005). Targeting the gltA gene, the assay
is able to detect R. akari, R. australis, R. conorii, R. honei, R. marmionii, R. sibirica, and
R. rickettsii as well as TG rickettsiae (R. typhi and R. prowazekii). Subsequently, a number of genus-specific and species-specific assays targetingMalaya different genes have been developed and widely used for diagnosis (Luce-Fedrow et al., 2015a). The WHO
Collaborative Center for Rickettsioses and ofOther Arthropod-borne Bacterial Diseases in
France reported two years of experience in using real-time PCR assays (Renvoisé et al.,
2012) for diagnosis of rickettsial diseases. New sets of primers and probes have been
designed by comparing all Rickettsia genomes available. The rapidity and sensitivity of
the assays are able to reduce delay in the diagnosis of rickettsial infection and improve
the efficiency of the management of patients suspected with rickettsioses (Renvoisé et
al., 2012).
UniversityLAMP assays have been developed for detection of Rickettsia spp. (both TG and SFG) based on Sca1 gene and rickettsial orthologous genes (Dittrich et al., 2014b; Hanaoka et
al., 2016). LAMP assay targeting on SFG rickettsial ompB gene has also been reported
by Pan et al. (2012). The LAMP assays are useful in economically-restrained areas
because of the single tube procedure and no requirement for thermocycler. The assays
35 provide simple readable endpoints, and comparable accuracy as compared to
conventional PCR but lower sensitivity comparing to nested PCR and real-time PCR
assays. However, the assay has limited usefulness when the rickettsemia levels of the
samples are below the LAMP detectable levels (approximately 40 DNA copies/LAMP
reaction) (Dittrich et al., 2014b; Luce-Fedrow et al., 2015a).
2.6.4 Treatment
To date, doxycycline (100mg, twice a day for 5-7 days) remains the standard treatment
for SFG rickettsioses (Aung et al., 2014; Parola et al., 2013). However, this regime was
contra-indicated in pregnant lady or allergies patients (Botelho-Nevers et al., 2012).
Alternative regimes such as fluoroquinolones, notably ciprofloxacin, have been
presented; nevertheless, fluoroquinolones are associated with increased MSF severity in patients (Botelho-Nevers et al., 2011). FluoroquinolonesMalaya are also contra-indicated in pregnant women (Botelho-Nevers et al., 2012), thus are not suitable for the treatment
MSF (Parola et al., 2013). Macrolide compoundsof (such as josamycin, clarithromycin and
azithromycin) may be used to treat pregnant women under strict follow-up, and represent
safe alternatives for the treatment of rickettsioses (Bella et al., 1990; Cascio et al., 2002;
Cascio et al., 2001). Telithromycin was reported to be active in vitro, but the data are
lacking for in vivo study and thus, requires further evaluation (Botelho-Nevers et al.,
2012).
2.7 Genomic features of rickettsial organisms UniversityIn this genomics-driven era, next generation sequencing technology has been used to enhance our understanding towards the biology, cellular functions, pathogenesis as well
as to assess evolutionary relationship among rickettsial species (Merhej & Raoult, 2011).
Rickettsia spp. are among the bacterial species that are sequenced most rapidly
(Georgiades et al., 2011a). With the advancement of molecular tools and ‘multiomics’, it
36 is now possible to analyze rickettsial genomes with numerous new analytical tools
(Georgiades et al., 2011a). SOAPdenovo, AbySS, Velvet, SSAKE are the assemblers that
are able to exploit paired-end sequencing information to reduce gaps from assembled
contigs (Zhang et al., 2011). In order to understand and harness the full potential of the
genome sequence, a bacterial genome needs to be annotated with biologically relevant
information such as gene models and functional information (Ekblom & Wolf, 2014).
Rapid annotation using subsystems technology (RAST) pipeline is an automatic
annotation server for microbial genomes. It is built based on the framework provided by
the SEED system which made annotation easy for researchers (Overbeek et al., 2014).
RAST pipeline has been widely used for the annotation and analysis of rickettsial
genomes, for instance, R. conorii (Narra et al., 2016), R. felis (Gillespie et al., 2015b) and a rickettsial species associated with endosymbiont ofMalaya the seal louse, Proechinophthirus fluctus (Boyd et al., 2016). Gillespie et al. (2012) have also reported on the annotation of Rickettsia endosymbiont of Ixodes scapularisof and genome-based phylogeny estimation for 46 Rickettsiales genomes by using RAST pipeline.
As of April 2017, a total of 35 known rickettsial genomes are available in the genome
project database of National Center for Biotechnology Information (NCBI,
https://www.ncbi.nlm.nih.gov/genome/browse/), (Table 2.3). The availability of the
complete genomes has enabled comparative genomic study for identification of
differences and commonalities among members in the species (Merhej & Raoult, 2011). UniversityRickettsial genomes exhibit significant intragenus variation in size (1.1 to 2.1 Mb) and gene content (approximately 829-2117 genes) (Duan et al., 2014). The small genome size
of rickettsial species could be attributed to reductive evolution, parasitic adaptation of the
bacteria to the host and pathogenic lifestyle may cause certain gene functions no longer
required, consequently, the bacteria become completely dependent on the hosts and are
unable to survive outside of the host cells (Moran, 1996; Ogata & Renesto, 2007).
37 Table 2.3: List of rickettsial genomes available in the NCBI genome database.
Rickettsia BioProject No. Size G+C Genes Proteins Number of species (Mb) (%) Assemblies (presentative genome) Candidatus Rickettsia PRJNA218069 1.35 32.2 1559 1212 1 gravesii R. aeschlimannii PRJEB6087 1.31 32.2 1560 1125 1 R. africae PRJNA18269 1.29 32.4 1568 1271 1 R. akari PRJNA12953 1.23 32.3 1202 980 1 R. amblyommatis PRJNA212472 1.44 32.8 1715 1115 5 str. Darkwater R. argasii PRJNA232538 1.44 32.3 1715 1243 1 R. asemboensis RJNA271102 1.38 32.3 1604 1235 1 R. australis str. PRJNA75037 1.32 32.3 1475 1185 2 Cutlack R. bellii PRJNA13996 1.52 31.6 1537 1398 5 RML369-C R. buchneri PRJNA238339 1.66 32.5 1846 1388 1 R. canadensis PRJNA12952 1.16 31.1 1142 909 2 str. McKiel Malaya R. conorii str. PRJNA42 1.27 32.4 1539 1289 4 Malish 7 Rickettsia of endosymbiont of PRJNA232537 1.57 32.2 1785 1281 1 Ixodes pacificus R. endosymbiont of PRJNA33979 2.10 33.3 2356 1782 1 Ixodes scapularis R. endosymbiont of PRJNA238474 1.26 32.4 1510 1114 1 Proechinophthir us fluctus R. felis PRJNA13884 1.59 32.5 1704 1363 4 URRWXCal2 R. heilongjiangensi PRJNA66907 1.27 32.3 1525 1210 1 s 054 UniversityR. helvetica PRJNA82855 1.42 32.2 1633 1235 1 C9P9 R. honei RB PRJNA158665 1.29 32.4 1554 1209 1 R. hoogstraalii PRJEB7296 2.30 32.4 1660 1291 2 R. japonica YH PRJDA38487 1.28 32.4 1525 1206 2 R. massiliae str. PRJNA75039 1.28 32.6 1520 1125 2 AZT80 R. monacensis PRJEB4369 1.35 32.4 1447 1164 1 R. montanensis PRJNA75045 1.28 32.6 1457 1161 1
38 Table 2.3, continued
Rickettsia BioProject No. Size G+C Genes Proteins Number of species (Mb) (%) Assemblies (presentative genome) R. parkeri str. PRJNA75031 1.31 32.4 1549 1273 4 Portsmouth R. peacockii PRJNA31309 1.32 32.6 1566 1250 1 R. philipii str. PRJNA75027 1.29 32.5 1537 1263 1 364D R. prowazekii PRJNA61565, 1.11 29.0 921 852 13 str. Brienl PRJNA43 R. raoultii str. PRJNA276402 1.48 32.6 1591 1235 2 Khabarovsk R. rhipicephali str. 3-7-female6- PRJNA75041 1.31 32.4 1556 1142 3 CWPP R. rickettsii str. PRJNA9636 1.27 32.5 1520 1246 12 Sheila Smith R. sibirica 246 PRJNA1414 1.25 32.5 1513 1273 3 R. slovaca str. PRJNA75033 1.28 32.5 1551 1313 2 D-CWPP R. tamurae PRJEB6744 1.45 32.5 Malaya 1704 1297 1 R. typhi str. PRJNA10679 1.11 28.9 881 825 3 Wilmington of
Despite of the reduced genome size, rickettsial genomes contain high fractions of gene
families, DNA repeats, mobile insertion elements which are involved in host-cell
interaction process, and lateral gene transfer (Merhej & Raoult, 2011). Rickettsial
genomes contain palindromic repeats, modules encoding type IV secretion systems
(T4SS), tetratricopeptide and ankyrin repeat motifs, toxin-antitoxin (TA) modules and
paralogous gene families (sca, spoT, tlc, proP and ampG) (Merhej & Raoult, 2011). UniversityMobile genetic elements, including plasmids, have been identified in 11 SFG rickettsial genomes, suggesting the possibility of lateral gene transfer in these intracellular bacteria
(El Karkouri et al., 2016).
T4SS are large protein complexes that traverse the envelope which promote the genetic
exchange with other bacteria and the translocation of effector proteins to eukaryotic target
39 cells (Cascales & Christie, 2003). T4SSs play important roles in the pathogenesis and
genome plasticity of some Gram-negative pathogens as they allow delivery of virulence
factors from bacterial and eukaryotic host membranes to the cytoplasm of the host cell
(Burns, 2003; Christie & Vogel, 2000). There are two specialized type IV secretion
systems that are known in Rickettsia spp., i.e., P-T4SS and F-T4SS. P-T4SS is
Rickettsiales vir homolog (rvh) T4SS which is responsible for protein secretion encoded
solely on chromosome across all genomes in rickettsial families, i.e., Rickettsiaceae,
Anasplasmataceae, and Midichloriaceae (Gillespie et al., 2010; 2015a). Conversely, F-
T4SS, responsible for dissemination of the integrative conjugative element, is known only
from Rickettsiaceae and is present on both the chromosomes and plasmids of some
Rickettsia spp. (Gillespie et al., 2012; 2015a).
Toxin-antitoxin (TA) systems are known to playMalaya an important role in controlling protein expression during nutrient starvation (Buts et al., 2005). Rickettsial TA systems
are presumed to work as addiction systemsof in which the elimination of TA-containing
rickettsia from host cells could lead to the release of the toxin into the cytoplasm of host
and consequently result in cell death (Audoly et al., 2011; Maté et al., 2012). The toxin
protein in the TA system is toxic to the cell and stable, meanwhile its corresponding
antitoxin is labile and requires continuous transcription to inhibit the toxin. If the plasmid
is not correctly segregated during cell division, one of the daughter cells will not carry
the plasmid but will still have a number of copy of the TA complex. Eventually, the Universityantitoxin will be degraded by proteases while the stable toxin will persist and kill the plasmid-free cells and prevents the proliferation of plasmid-free cells in growing bacterial
cultures and therefore, TA systems have been described as ‘addiction modules’ (Maté et
al., 2012).
40 More rickettsial genomes are now available for analysis as the cost and time associated
with whole genome sequencing continue to reduce. Comparative analyses of these
genomes enable the creation of phylogenies based on core gene sequences and
subsequently resolved the conflicting data from MLST analyses, creating a more robust
and accurate phylogeny for rickettsial species (Luce-Fedrow et al., 2015a). K-mer based
methods have been used to estimate genome similarity, identify homologous genomic
sequence and SNP (Treangen et al., 2014). The core-genome SNP-based phylogenetic
comparison is currently the standard method for rapid reconstructing large phylogenies
or closely related microbes (Robinson et al., 2013). kSNP software finds SNPs and build
phylogenies for large numbers of complete, draft and unassembled genomes (Gardner &
Hall, 2013). Additionally, genomic relatedness between microbial strains and confirmation of the identity of microorganisms can Malaya also be determined using average nucleotide identity (ANI) and tetranucleotide usage patterns (TETRA) analyses (Kim et al., 2014; Teeling et al., 2004). The ANIof which is determined based on pairwise comparisons of sequences shared between two strains, has been used for the classification
of bacterial species (Han et al., 2016). Additionally, comparative analysis of full genome
data can be used for the development of new diagnostic tools targeting at features and
regions that are unique to a species or a subset of rickettsial species (Ellison et al., 2008;
Matsutani et al., 2013).
University
41 CHAPTER 3: MATERIALS AND METHODS
3.1 Ethical approvals for the conduct of research
An ethical approval has been obtained from the University of Malaya Medical Center
(UMMC) (Ethics committee reference number: 944.20) (Appendix A) for serological
assessment of febrile patients, blood donors, indigenous community and animal farm
workers. Prior to the commencement of the sample collection, permission was obtained
from the Department of Orang Asli Development (JAKOA, indigenous community)
(Appendix B) for the collection of human blood samples and ectoparasites from
indigenous community; Department of Veterinary Services Ministry of Agriculture and
Agro-based Industry (DVS), Malaysia (Reference number: JPV/PSTT/100-8/1)
(Appendix C) for the collection of human blood samples from animal farm workers, animal blood samples and ectoparasites. For the investigationMalaya of human subjects, an oral briefing on the objective and methodology of the study was given to the participants. Once the participants have voluntarily agreedof to participate, their consents were taken either in written form or verbally followed by thumb prints (for those who were illiterate).
Parents or guardian of child under age of 18 provided informed consent on their behalf.
All data from the studied populations were strictly anonymized.
Written approvals were obtained from the Veterinary Research Institute (VRI)
[Reference no.: JPV: VRI/197/PA/141 Jid.11 (48)] (Appendix D) and Department of
Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia University(PERHILITAN) [reference no.: JPHL&TN (IP):80-4/2 Jilid 15 (51)] (Appendix E) prior to sample collection for screening of rickettsiae in animals and ectoparasites. Approval
was also obtained from Kuala Lumpur City Council, SPCA and Second Chance animal
shelters for the collection of ticks and fleas from cats and dogs.
42 3.2 Collection of human samples for detection of rickettsiae
3.2.1 Patient samples from UMMC with pyrexia
Whole blood and serum samples were obtained from 41 patients admitted to UMMC
with pyrexia of unknown origins, from March 2013 to April 2015. The blood samples
were collected in BD Vacutainer (both plain tube and EDTA tubes) by medical staffs.
According to Petersdorf and Beeson (1961), pyrexia with unknown origin are
unexplained fevers (≥ 38.3 oC) that are persisting for at least three weeks for which no
cause can be identified after three days of investigation in hospital or after three or more
outpatient visits. DNA was immediately extracted from the whole blood samples after
collection. Serum samples were extracted from the plain tubes by centrifugation at 3100
rpm (1000 xg) for 10 min to remove blood clots 30 min to one hour at room temperature after blood collection. The serum samples were subjectedMalaya to serological analysis using an indirect immunofluorescence assay (IFA), as described in section 3.3.
3.2.2 DNA samples from dengue negativeof febrile patients
The DNA extracts obtained from 62 serum samples of patients clinically suspected of
dengue fever were included for molecular detection of rickettsiae (Kho et al., 2016). The
samples were collected from January to June 2010 from the Diagnostic Microbiology
Laboratory, UMMC. The samples were confirmed to be negative for dengue by the
microbiology laboratory. However, serum samples were not available for rickettsial
serological test.
University3.2.3 Serum samples from blood donors The serum samples from 61 healthy blood donors residing in Kuala Lumpur (the
capital city of Malaysia) and Selangor were collected from the blood bank of UMMC for
serological analysis using IFA, as described in section 3.3.
43 3.2.4 Serum samples from animal farm workers
Selection of animal farms was based on the recommendation by the Department of
Veterinary Services (DVS), Ministry of Agriculture and Agro-based Industry, Malaysia,
and the availability of logistics to carry out the study. A total of 11 cattle and goat farms
in Peninsular Malaysia were identified for this study. However, only eight farms agreed
to participate in the study. The eight farms [designated as Farm 1-8] located at six states
of Peninsular Malaysia included a cattle and a goat farm in Negeri Sembilan (Farm 1 and
7) (n=24 workers), two cattle farms in Pahang (Farm 2 and 3) (n=18 workers), one sheep
farm in Kedah (Farm 4 ) (n=7 workers), one cattle farm each in Kelantan (Farm 5) (n=14
workers), Terengganu (Farm 6) (n=7 workers) and Johore (Farm 8) (n=17 workers),
respectively (Figure 3.1). Serum samples were collected from 87 farm workers from February to September, 2013. All the blood samples wereMalaya collected in plain tube (3-5 ml) and processed for serum as described in section 3.2.1. Aliquoted sera were kept at 4 oC before transporting back to laboratory. of
3.2.5 Serum samples from indigenous community
As there is no prior information about rickettsial seropositivity in the indigenous
community, serum samples from 102 individuals residing at six rural villages in Johore
(n=20), Pahang (n=33) and Kelantan (n=49) (Figure 3.1) who participated in a cross
sectional study (October 2012 to February 2013) to determine risk factors associated with
dengue fever (Chandren et al., 2015) were used in this study. The rural villages were Universitymostly located at forest fringe areas and in close proximity with rubber or oil palm estates. All the blood samples were collected in plain tube (3-5 ml) and processed for serum
samples as described in section 3.2.1. Aliquoted sera were kept at 4 oC before transported
back to laboratory.
44 Figure 3.1 shows the locations of the rural villages and animal farm investigated in
this study.
Malaya of
Figure 3.1: Geographical location of the studied population in Peninsular Malaysia.
( Animal farms; rural villages). Farm 1 and 7: Negeri Sembilan; Farm 2 and 3: Pahang; Farm 4: Kedah; Farm 5: Kelantan; Farm 6: Terengganu and Farm 8: Johore. Rural villages: Kedah, Perak, Kelantan, Pahang, Negeri Sembilan, and Johor.
University3.3 Serological analysis of human blood samples for rickettsioses using immunofluorescence assay (IFA)
3.3.1 Febrile patient suspected of rickettsioses from UMMC
Screening for IgG and IgM antibodies against spotted fever group rickettsiae (using R.
rickettsii as antigen) and typhus group (TG) rickettsiae (using R. typhi as antigen) in 41
45 febrile patients were performed using an indirect immunofluorescence assay (IFA) (Focus
diagnostic, Cypress, California, USA) according to the manufacturer’s instructions.
Briefly, serum samples were first diluted (1:64) in phosphate-buffered saline (Focus
diagnostic, Cypress, California, USA) and 25 µL of each diluted serum sample was added
to an antigen well on the IFA slide. After incubation at 37 oC for 30 min in a humidified
chamber, the wells were rinsed with PBS and submerged in PBS for 10 min. The slides
were quick dipped into distilled water prior to incubation with IgG conjugate for 30 min.
The slides were examined under a fluorescence microscope (DM4000B LED, Leica,
Germany) with 400X magnification. Representative confocal images were taken by using
Leica TCS SP5 II laser scanning confocal spectral microscope (Germany) using the 40x oil immersion objective lens. Malaya The test was interpreted according to Lai et al. (2014) whereby an acute infection is suspected when there is a positive detectionof of IgM (≥ 1:64) specific for a rickettsial species or a fourfold or greater increase of IgG titers in paired sera. A past infection is
indicated whenever there is an IgG titer of ≥ 1:64 without a fourfold or greater increase
of titers, and negative IgM. Positive sera were further tested at 1:128, to eliminate
potential false-positivity caused by background seroprevalence in the patients of this
study.
3.3.2 Blood donors, indigenous community and farm workers UniversityThe sera were analyzed for lgG antibody against R. conorii and R. felis by using an indirect immunofluorescence assay (IFA) kit (Fuller Laboratories, USA) in accordance
to the manufacturer’s instructions.
Briefly, serum samples were first diluted (1:64) in phosphate-buffered saline (PBS,
Fuller Laboratories, USA) and 10 µL of each diluted serum sample was added to an
46 antigen well on the IFA slide. After incubation at 37 oC for 30 min in a humidified
chamber, the wells were washed with PBS and distilled water prior to incubation with
IgG conjugate for 30 min. The slides were examined under a fluorescence microscope
(DM4000B LED, Leica, Germany) with 400X magnification. Samples were regarded as
positive when bright apple-green fluorescence of rickettsial antigens was observed.
3.4 Statistical analysis
For comparison of seropositivity rates amongst different study groups, statistical
analysis was performed using SPSS (Statistical Package for the Social Sciences) software
program, version 22 (SPSS Inc., Chicago, IL). Initial data entry was cross-checked in
order to ensure that data was entered correctly. Chi-square and Kruskal-Wallis rank test
were used to determine statistical significance between age, gender and study groups (indigenous population, animal farm workers and bloodMalaya donors). The level of statistical significance was determined at p ≤ 0.05 and 95 % confidence interval (CI). Pairwise
comparisons within the study and age groupsof were performed using Games-Howell post
hoc tests of the SPSS software. A p value of ≤ 0.05 was considered as statistically
significant.
3.5 Arthropod collection and identification
Ticks and fleas were collected with the help of the research students participating in
the Tick-borne Emerging Infectious Diseases Research Program funded by University of
Malaya Research Grant (RP013-2013A), and the assistance provided by staffs from UniversityJAKOA, DVS and PERHILITAN. Ticks were collected by using tweezers from the ear, eyes, flank, abdomen, tail and perineal regions of animals. Fleas were collected using
combing method. The ticks and fleas collected were stored in cryovials and transported
back to laboratory in liquid nitrogen using a cryoshipper (MVE, USA).
47 Ticks were collected from various locations, including urban areas, farms, rural
villages and forest areas. In the urban areas, ticks were collected from strayed dogs in
animal shelters managed by the Society for the Prevention of Cruelty to animals (SPCA)
and Second Chance Animal Shelter in Selangor in March 2013 and an animal shelter at
the Dewan Bandaraya Kuala Lumpur (DBKL) in February 2014 (Appendix F). Ticks
were collected from cattle and sheep during visits to animal farms (as described in section
3.2.5), whenever possible (Appendix G). Ticks were collected from peri-domestic
animals including cat, chicken, dog, goat and cattle in rural villages in Johore, Kedah,
Kelantan, Negeri Sembilan, Perak and Pahang (Figure 3.1) from October 2012 to
February 2013 (Appendix H). Questing ticks were collected under plant leaves along the
trails at Kuala Lompat forest reserve in Pahang from 17 June to 22 June 2013 (Appendix I). Ticks feeding on small mammals (bats, rodents, Malaya skink and squirrel) trapped at the Kuala Lompat forest reserve were also collected for screening of rickettsiae (Appendix J). Amblyomma ticks collected from several ofsnakes ( Python molurus) in Sepang, Selangor (October 2012) (Appendix K) were kindly provided by Mr. Chai Koh Shin, an
experienced researcher on wildlife.
Fleas were collected from cats and dogs in both urban and rural areas. In urban areas,
fleas were collected from cats in DBKL animal shelter and Titiwangsa housing area. Fleas
were also collected from rural villages in Johore, Kedah, Kelantan, Negeri Sembilan,
Perak and Pahang (Figure 3.1) from October 2012 to February 2013 (Appendix L).
UniversityAll the arthropod samples were preserved at -80 oC prior to DNA extraction and further investigation. Individual ticks were examined under a stereomicroscope (Olympus
SZX16, Japan) for sex, growth stage and genus level, by referring to previously described
morphological taxonomic keys (Brahma et al., 2014; Geevarghese & Mishra, 2011;
Walker et al., 2003). Briefly, examination of the gnathosoma of the hard dorsally and
48 ventrally was useful for differentiation of ticks between genera. According to Walker et
al. (2003), the sex of the hard ticks was identified based on the size of scutum covering
the dorsum of the idiosoma. The size of scutum is relatively small in female ticks whereas
it covers the entire dorsum of the male idiosoma. Ticks in larval stage can be
differentiated from nymph and adult based on the number of legs (6 legs in larvae and 8
legs for nymph and adult). The genital apertures are present in adult ticks but are not
distinct in the nymphal stage of ticks, Molecular identification of tick species was
performed by using primers targeting the tick 16S rRNA gene region for all ixodid ticks
(Black & Piesman, 1994). Amplification and sequence analysis of the 12S rRNA gene
was also performed for R. sanguineus (Beati & Keirans, 2001). The sequence obtained
were compared to known sequences in the GenBank database.
All the fleas were identified as C. felis felis andMalaya C. felis orientis on the basis of morphometric characteristics (Lawrence et al., 2015b; Menier & Beaucournu, 1998). The
flea species were differentiated based on theof shape of their head. The head of C. felis felis
appears to be ‘pointier’ than C. canis. The dorsal incrassation of C. felis felis is long and
narrow and while it is short and stout for C. canis. The presence of a single notch bearing
stout setae between postmedian and apical setae on the dorso-posterior margin of the hind
tibia is one of the important characteristics to distinguish C. felis felis (Lawrence et al.,
2015b). Fleas were also subjected to molecular identification method, by amplification of
the cytochrome oxidase I (CoxI) and cytochrome oxidase II (CoxII) gene regions University(Whiting, 2002) (this part of work was performed by Mdm. Lailatul Insyirah for her postgraduate study).
Other than ticks and fleas, DNA extracts for mosquitoes and buffalo flies kindly
provided by Prof. Datin Dr. Indra Vythilingam from Department of Parasitology and Dr.
49 Low Van Lun from Institute of Biological Science, University Malaya were also included
for screening of rickettsial DNA in this study.
3.6 Animal blood samples collection
Blood samples were collected from cattle, sheep and goats during farm survey as stated
in section 3.2.4. Blood samples were collected in both plain tube and EDTA tube by using
vacutainer with the help from veterinarians. Blood and serum samples were kept at 4 oC
before transporting back to our laboratory. In addition, cattle blood samples collected
from Serdang cattle farm were provided by Prof. Dr. Ong Bee Lee and her team from
University Putra Malaysia. Other mammalian (cattle, horse, buffalo, deer, pangolin, goat
and rodent) blood samples collected from February 2013- July 2013 were kindly provided
by Dr. Chandrawathani Panchadcharam from VRI (Appendix M). Dr. Ummukulthum Lawal Hassan provided DNA extracts of cat blood samplesMalaya collected in UPM veterinary clinics (February 2014 – April 2014) for rickettsial DNA screening. Additionally, blood
collected from 50 cynomolgus monkeys caughtof by PERHILITAN staff at 12 residential
areas in Peninsular Malaysia during a population management and wildlife disease
surveillance program (January 2012- December 2013) were also investigated. The
monkeys were active and healthy during blood collection and most monkeys (14 females
and 36 males) were subadult and adult (Appendix N). Wildlife animal blood samples in
Pulau Tioman collected on FTA were provided by PERHILITAN for the screening of
rickettsiae (Appendix O).
University3.7 Collection and processing of organ tissue samples from small wildlife Organ tissue samples were collected from rodents, bats and squirrel in Kuala Lompat
forest reserve (Appendix P). Liver, kidney and spleen samples were collected from the
animals and transported to our laboratory in liquid nitrogen using a cryoshipper (MVE,
USA).
50 3.8 DNA extraction
3.8.1 Human and animal blood samples
Whole blood samples from each febrile patient were used for DNA extraction using a
QIAamp DNA mini kit (Qiagen, Hilden, Germany) in accordance with the instructions
of the manufacturer. Briefly, 200 µl of blood samples were added to the microcentrifuge
tube containing 20 µl Qiagen proteinase K. Then, 200 µl of buffer AL were added to the
samples and mixed by pulse-vortexing (15 s) followed by incubation at 56 oC for 10 min.
A total of 200 µl of ethanol were added to the sample before pipetting the mixture into
the QIAamp spin column and centrifuged at 8, 000 rpm for 1 min. After that, 500 µl of
AW1 were added to the spin column followed by centrifugation at 8, 000 rpm for 1 min.
Five hundred microliters of AW2 were then added to the spin column and centrifuged at 14, 000 rpm for 3 min. Lastly, a total of 100 µl of DNAMalaya were eluted from each blood sample.
For blood samples collected from animals,of two hundred microliters of the whole blood
samples from animals were used for DNA extraction by using the QIAamp DNA mini kit
(Qiagen, Hilden, Germany). A total of 100 µl of DNA were eluted from each blood
sample. Animal blood samples collected on FTA cards were subjected for DNA
extraction using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) in accordance with
the instructions of the manufacturer. Briefly, a 3 mm diameter of dried blood spot was
punched from FTA cards into a 1.5 ml microcentrifuge tube and added with 280 µl of Universitybuffer ATL and 20 µl of proteinase K. The mixture was incubated at 56 oC for an hour. A total of 300 µl AL buffer was then added before incubating the mixture at 70 oC for 10
min. A total of 150 µl of ethanol were added to the sample before pipetting the mixture
into the QIAamp spin column and centrifuged at 8, 000 rpm for 1 min. After that, 500 µl
of AW1 were added to the spin column followed by centrifugation at 8, 000 rpm for 1
min. Five hundred microliters of AW2 were then added to the spin column and
51 centrifuged at 14, 000 rpm for 3 min. Lastly, a total of 100 µl of DNA were eluted from
each blood sample.
3.8.2 Animal organ samples
The organ samples were first removed from the storage in liquid nitrogen and thawed.
A total of approximately 10 mg of spleen and 25 mg of liver and kidney were cut into
small pieces using scalpel blades (Pioneer Surgical, India) and placed in separate 1.5 mL
microcentrifuge tube. The sample was added with 180 µl ATL buffer and 20 µl proteinase
K provided in the QIAamp DNA mini kit (Qiagen, Hilden, Germany). The DNA
extraction was then carried out in accordance with the manufacturer’s protocol as
described in section 3.8.1. A total of 200 µl of DNA solution were eluted for each sample.
3.8.3 Tick and flea samples Malaya The ticks and fleas were surface-sterilized according to Duh et al. (2010) with slight modifications. Briefly, a single or pooled ofsample of ticks and fleas were first thawed, immersed in 5 % sodium hypochlorite and 70 % ethanol (v/v) before washing three times
with sterile distilled water. The ticks and fleas were then triturated by using surgical
blades (Pioneer Surgical, India) and DNA was extracted using QIAamp DNA mini kits
(Qiagen, Hilden, Germany) in accordance to the manufacturer’s instruction. Ticks DNA
was extracted in accordance to the Qiagen supplementary protocol for purification of total
DNA from ticks for detection of Borrelia DNA (www.qiagen.com). Briefly, triturated
tick/flea sample was placed in a 1.5 ml microcentrifuge tube with 180 µl buffer ATL. UniversityTwenty microliters of proteinase K solution was then added to the mixture and the sample was incubated at 56 oC until the tissue lysed completely (30-60 min). Then, 200 µl of
buffer AL was added to the sample and incubated at 70 oC in a heating block for 10 min.
The mixture was added with 230 µl of ethanol and transferred into a QIAamp spin column
for centrifugation at 8, 000 rpm for 1 min. After that, 500 µl of buffer AW1 were added
52 to the spin column followed by centrifugation at 8, 000 rpm for 1 min. Five hundred
microliters of buffer AW2 were then added to the spin column and centrifuged at 14, 000
rpm for 3 min. Lastly, a total of 60 to 80 µl of DNA solution were eluted from each
sample. Two hundred microliters of C6/36 mosquito cell line suspension was included in
the DNA extraction step as a negative control to ensure there was no contamination during
the entire process.
3.9 Molecular detection and sequence analysis of rickettsiae
3.9.1 Molecular detection of rickettsiae from clinical samples
The DNA samples were first screened for rickettsial DNA using PCR primers
CS78/CS323 (gltA) or CS-239/CS-1069 (gltA-1) targeting 410 bp and 830 bp of the
rickettsial citrate synthase gene, respectively (Labruna et al., 2004) (Table 3.1). Positive samples were then subjected to amplification usingMalaya primers Rr190.70p/Rr190.602n, targeting a 532 bp fragment of the 190-kDa outer membrane protein gene (ompA)
(Regnery et al., 1991) and primers 120-M59/120-807,of targeting a 866 bp fragment of 135-
kDa outer membrane protein gene (ompB) (Roux & Raoult, 2000) (Table 3.1). All PCR
assays were performed in a final volume of 20 µL containing 2 µL of DNA template, 1
X ExPrime Taq DNA polymerase (GENET BIO, Daejeon, South Korea) and 0.2 µM of
each primer, in a Veriti thermal cycler (Applied Biosystems, Foster City, California,
USA). DNA extracted from R. conorii antigen slides (Fuller Laboratories, Fullerton,
California, USA) was used as a positive control for the PCR assay. Sterile distilled water Universitywas used as the negative control in each PCR reaction. The presence of PCR-inhibitors in the human and clinical samples were ruled out by amplifying the housekeeping gene
(human β-globin), using primers GH20/PC04 (Gauduchon et al., 2003) (Table 3.1). PCR
products (5 µl) were separated on a 1.5 % (w/v) agarose gel (FirstBase Laboratories,
Malaysia) at 100 V for 45 min and visualized using a UV transilluminator (G-Box,
Syngene, UK).
53 Table 3.1: Oligonucleotide primers used in this study.
Annealing Expected Target Samples Primers Primer sequence (5’-3’) temperature amplicon Reference gene (oC) size (bp) TTG GGC AAG AAG 16 S+2 Black and 16S ACC CTA TGA A Ixodidae 55 300 Piesman rRNA CCG GTC TGA 16 S-1 (1994) ACTCAGATCAAGT AAA CTA GGA TTA T1B Beati and Rh. 12S GAT ACC CT 51 and 53 360 Keirans sanguineus rDNA AAT GAG AGC GAC T2A (2001) GGG CGA TGT AGA GTT TGA TCC fD1 Weisburg 16S TGG CTC AG Eubacteria 58 1500 et al. rDNA ACG GCT ACC TTG rP2 (1991) TTA CGA CTT CAA CTT CAT CCA PC04 Gauducho Beta CGT TCA CC Human 55 268 n et al. globin GAA GAG CCA AGG GH20 (2003) ACA GGT AC CCA TCC AAC ATC CytB-F TCA GCA TGA TGA Oshaghi et Vertebrate Cyt b AA 58 358 Malayaal. (2006) CCC CTCA GAA TGA CytB-R TAT TTG TCC TCA GCA AGT ATC GGT CS 78 GAG GAT GTA AT of Labruna et Rickettsiae gltA 55 401 GCT TCC TTA AAA al. (2004) CS 323 TTC AAT AAA TCA GGA T GCT CTT CTC ATC CS 239 CTA TGG CTA TTA T Labruna et Rickettsiae gltA-1 48 830 CS106 CAG GGT CTT CGT al. (2004) 9 GCA TTT CTT Rr ATG GCG AAT ATT 190.70 190- TCT CCA AAA p KDa Regnery et Rickettsiae 58 532 Rr antigen al. (1991) AGT GCA GCA TTC 190.60 (ompA) GCT CCC CCT 2n 120- CCG CAG GGT TGG M59 TAA CTG C Roux and Rickettsiae ompB 50 855 Raoult 120- CCT TTT AGA TTA (2000) University807 CCG CCT AA ATG AGT AAA GAC D1f GGT AAC CT Sekeyova Rickettsiae Sca4 50 928 et al. AAG CTA TTG CGT D928r (2001) CAT CTC CG
54 3.9.2 Molecular detection of rickettsiae from arthropod samples
Primers targeting three rickettsial-specific genes were used for amplification of
rickettsial DNA from arthropod samples (ticks, fleas, mosquitoes and buffalo flies), i.e.,
citrate synthase gene (gltA) (Labruna et al., 2004), 190-kDa outer membrane protein gene
(ompA) (Regnery et al., 1991) and 135-kDa outer membrane protein gene (ompB) (Roux
& Raoult, 2000) (Table 3.1). However, due to the large sample sizes of fleas collected
from both urban areas and rural villages as well as ticks collected from animal farms, the
DNA samples were first screened using a PCR assay targeting gltA. Any positive samples
were then subjected to further amplification by using primers targeting ompA and ompB.
For the rest of the samples, i.e., ticks collected from rural villages and urban areas, all the
three rickettsial genes were screened simultaneously.
All PCR assays were performed in a final volume Malayaof 20 µL containing 2 µL of DNA template, 1X ExPrime Taq DNA polymerase (GENET BIO, Daejeon, South Korea) and
0.2 µM of each primer, in a Veriti thermalof cycler (Applied Biosystems, Foster City,
California, USA). DNA extracted from R. conorii antigen slides (Fuller Laboratories,
Fullerton, California, USA) was used as a positive control for all the PCR assays. Sterile
distilled water was used as a negative control in each PCR reaction. The presence of PCR-
inhibitors in tick and flea samples was ruled out by amplifying the 16S rRNA gene of the
ticks (Black & Piesman, 1994) and CoxII gene of the fleas (Slapeta et al., 2011) (Table
3.1). PCR products (5 µl) were separated on a 1.5 % (w/v) agarose gel (FirstBase UniversityLaboratories, Malaysia) at 100 V for 45 min and visualized using a UV transilluminator (G-Box, Syngene, UK).
3.9.3 Molecular detection of rickettsiae from animal samples
The animal blood and organ samples were first screened for rickettsial gltA (primers:
CS78/CS323) (Labruna et al., 2004) and ompA (Regnery et al., 1991) (Table 3.1). All the
55 positive samples were further screened for ompB (primers: 120-M59/120-807) (Roux &
Raoult, 2000). All PCR assays were performed in a final volume of 20 µL containing 2
µL of DNA template, 1 X ExPrime Taq DNA polymerase (GENET BIO, Daejeon, South
Korea) and 0.2 µM of each primer, in a Veriti thermal cycler (Applied Biosystems, Foster
City, California, USA).
DNA extracted from R. conorii antigen slides (Fuller Laboratories, Fullerton,
California, USA) was used as a positive control for the PCR assay. Sterile distilled water
was used as a DNA blank in each PCR reaction. The presence of PCR-inhibitors in animal
samples were ruled out by amplifying the mitochondrial DNA cytochrome B gene
(Oshaghi et al., 2006) (Table 3.1). PCR products (5 µl) were separated on a 1.5 % (w/v)
agarose gel (FirstBase Laboratories, Malaysia) at 100V for 45 min and visualized using a UV transilluminator (G-Box, Syngene, UK). Malaya 3.9.4 Sequence determination and analysisof of amplified fragments PCR products were purified using a GeneAll ExpinTM Combo GP kit (GeneAll
Biotechnology, Seoul, South Korea). The purified DNA was then subjected to sequencing
using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, Foster City, California,
USA), with both forward and reverse primers of each PCR assay. The sequences obtained
were subjected to BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for
homologous sequences in the GenBank database (Altschul et al., 1990).
University3.10 Phylogenetic analysis of tick and flea-borne rickettsiae To determine the phylogenetic position of the rickettsiae identified in this study, a
dendrogram was constructed based on gltA sequences (368 nucleotides) by using the
neighbour-joining method of MEGA 7.0 software (Kumar et al., 2016). Reference
sequences were retrieved from the GenBank database, i.e.: R. raoultii (DQ365804),
Rickettsia sp. Rf31 (AF516331), Rickettsia sp. RF2125 (AF516333), Rickettsia
56 asemboensis (JN315974), Candidatus Rickettsia senegalensis (KF666472), Rickettsia sp.
California 2 (AF210692), Rickettsia felis URRWXCal2 (CP000053), R. tamurae
(AF394896), Rickettsia sp. kagoshima6 (JQ697956), R. honei (AF022817), R. conorii
(HM050292), R. heilongjiangensis (AB473994), R. japonica (U59724), Rickettsia sp.
LON-13 (AB516964), Rickettsia sp. TCM1 (AB359458) and R. belli (DQ865204).
Rickettsiae reported in previous Malaysian studies, i.e.: Rickettsia sp. clone HL15c
(KF963603) (Tay et al., 2014), derived from cat fleas were also included in the
phylogenetic analysis. Dendrograms were also constructed based on R. raoultii ompB and
R. felis ompB by using the neighbour-joining method of MEGA 7.0 software (Kumar et
al., 2016).
3.11 Isolation and maintenance of rickettsial organism 3.11.1 Source of tick and flea samples for infectionMalaya experiments Ticks and fleas which were positive in the PCR assays were selected for culturing in
this study. Repeat samplings were also conductedof in rural villages with high rickettsial
detection rates in fleas (R. felis and RFLO) in Perak and Pahang during January 2016.
Efforts were taken to ensure that the fleas collected were alive until the time they were
delivered to the laboratory. Identification of flea was performed as described in section
3.5.
3.11.2 Regeneration and propagation of C6/36 cell line
Ae. albopictus clone C6/36 (ATCC CRL-1660) was kindly provided by Assoc. Prof. UniversityDr. Chan Yoke Fun from the Department of Medical Microbiology, Faculty of Medicine, University of Malaya. The passage level of the cell line used in this study was between
50-85. The cell line was stored in cell culture cryogenic tubes (Nalge Nunc, USA) and
kept in a liquid nitrogen tank. The cell line was retrieved from the liquid nitrogen tank
storage and immediately thawed in a 37 oC water bath. The cells were then transferred
57 into a 15 mL conical centrifuge tube containing Leibovitz-15 medium (with L-glutamine
but no sodium bicarbonate) (GIBCO, New York) supplemented with 10 % heat-
inactivated foetal bovine serum (FBS) (Hyclone, United States) and 10 % tryptose
phosphate broth (TPB) (complete medium) (Appendix Q). The cell suspensions were
centrifuged at 1200 rpm (150 x g) for 5 min at room temperature (Sigma 3-18K, Sartorius
Stedim Biotech, Germany). The supernatant was discarded and the pellet was re-
suspended in 1 ml of complete L-15 medium and transferred into either 25 cm2 or 75 cm2
flasks (SPL Life Science, South Korea). The cells were then cultivated at 28 oC.
Antibiotics were not used for the maintenance of the cell line.
Adherent cell cultures were routinely sub-passaged once they became confluent in 25
cm2 or 75 cm2 flasks (SPL Life Science, South Korea). Old medium was replaced by 5 mL of PBS (pH7.3, Oxoid, England) and the cells wereMalaya harvested by using a cell scrapper (SPL Life Science, South Korea). The harvested cells were centrifuged at 1200 rpm (150
x g) for 5 min following which the pellet wasof re-suspended in 1 mL of complete L-15
medium. A volume of 10 µL of the cell suspension was subjected to cell count using a
hemocytometer (Assistent, Germany). If there were too many cells (>200), 1:10 or 1:100
dilution was made in the L-15 medium. The cell suspension was mixed with an equal
volume of trypan blue solution (0.4 %, sterile-filtered) (Sigma-Aldrich, United States)
and 10 µL was pipetted onto a hemocytometer with a coverslip. The cells were viewed
under an inverted phase contrast microscope (Motic, Hong Kong) with 20X Universitymagnification. The numbers of viable cells (live cells) were calculated using the following equation:
Number of live cells counted Viable cell count = X 2 X dilution factor X 104 Number of square counted
Approximately 1.5 X 105 cells/ml were seeded into the shell vials (Diagnostic Hybrids,
USA) to obtain confluent layer of cells prior to the inoculation of tick/flea homogenates.
58 For 25 cm2 and 75 cm2 flasks, the cells were seeded between 2.5 X 105 cells to 5.0 X 105
cells per flask.
3.11.3 Infection of C6/36 cell line with tick/flea homogenates
All of the works involved rickettsia were performed in Biosafety Cabinet class II, type
A2 (Nuaire, USA). The isolation of rickettsia in C6/36 cell line was carried out according
to Hun et al. (2011) with some modification. Individual or pool of tick(s) and flea(s) from
the same host were identified and disinfected prior to trituration. The samples were
surface-sterilized by a quick rinse in 5 % sodium hypochlorite, washing in 70 % ethanol
for two min, followed by washing twice in sterile distilled water for two min. The
sterilized ticks and fleas were air-dried in a biosafety cabinet and collected into 1.5 mL
centrifuge tubes containing brain heart infusion broth. The fleas were manually triturated and transferred into a shell vial containing monolayerMalaya of C6/36 cells by using a syringe filter (0.45 µm membrane). From each triturated sample, 900 µL of triturate was used for
inoculation and 100 µL of triturate was usedof for DNA extraction(s) using QIAamp DNA
mini kit (Qiagen, Hilden, Germany). The shell vials were centrifuged for one hour at 2594
rpm (700 x g) at 22 oC after inoculation. The monolayer was then washed and fed with 1
mL of L-15 medium (GIBCO, New York) with 1 % amphotericin B (Hyclone, United
States), 1 % penicillin-streptomycin (Hyclone, United States), 100 µg/mL gentamicin
(Duchefa Biochemie, Netherlands), 2.5 % heat-inactivated FBS (Hyclone, United States)
and 2.5 % TPB (Sigma-Aldrich, United States). This was followed by three-day Universityincubation at 28 oC, after which, the medium was changed to antibiotic-free medium. The inoculated cultures were examined under an inverted microscope daily to check
for contamination until 14 days post-infection (dpi). The cultures were replenished with
fresh medium (2.5 % FBS and TPB, respectively) every 3 days. DNA was extracted from
the aspirated medium at 1, 6 and 12 dpi. The aspirated medium was centrifuged at 13,
59 000 rpm for 10 min, and the pellet was subjected to DNA extraction by using a G-spinTM
Total DNA extraction kit (iNtRON Biotechnology, Korea) according to manufacturer’s
protocols. All the extracted DNA from tick(s)/fleas(s) and cell cultures were subjected to
gltA-PCR screening assays for detection of rickettsial DNA as described in section 3.9.
After 14 dpi, infected cells were harvested and transferred to uninfected monolayers
in a 25 cm2 flask and subsequently to a 75 cm2 flask. An isolate was considered
established in the laboratory after three passages through 75 cm2 flasks (Labruna et al.,
2004).
The rickettsial growth was monitored under an inverted phase contrast microscope
(Motic, Hong Kong) daily. The rickettsia was continually sub-passaged every week into new flasks of C6/36 cells to maintain an ongoing sourceMalaya of live and actively growing rickettsia. The infected cells were dislodged from the flask and transferred into a 15 mL conical tube. The cell suspension was centrifugedof at 3100 rpm (1, 000 x g) for 5 min at 4 oC. Centrifugation of the cell suspension at a low speed enabled the host cells and heavy
cellular debris to be separated in the pellet leaving behind the rickettsia and small cellular
debris in the supernatant (Ammerman et al., 2008). The supernatant was then transferred
to a sterile 15 mL high-speed conical tube and the pellet was discarded. The supernatant
(2 mL to 5 mL) containing rickettsiae were used to infect a new monolayer of C6/36 cells
which was maintained in fresh L-15 medium supplemented with 2.5 % heat-inactivated
FBS and 2.5 % TPB. The remaining supernatant containing rickettsiae were stored in UniversitySPG buffer as stated in section 3.11.4.
3.11.4 Storage of rickettsial stock in sucrose phosphate glutamate (SPG) buffer
The remaining supernatant (3-5 mL) in a 15 mL high-speed conical tube collected
from section 3.11.3 were again spun at 12786 rpm (17, 000 x g) for 10 min at 4 oC. The
rickettsial organism was harvested from the high speed centrifugation with some small
60 cellular debris (Ammerman et al., 2008). The supernatant was removed with extra care
not to dislodge the rickettsial pellet. The pellet was re-suspended in 500 µL SPG and
transferred into separate cryovials. The rickettsial stock cultures were aliquoted and kept
at -80 oC.
3.11.5 Storage of infected C6/36 cells in liquid nitrogen
The rickettsia-infected cells were kept in liquid nitrogen for long-term storage. The
medium was removed from an infected culture flask when distinct cell morphological
changes were observed (3 to 7 days depending on the number of rickettsia used in the
infection). The cells were gently scraped with a sterile cell scraper and washed with L-15
medium supplemented with 10 % FBS and 10 % TPB. The cells were centrifuged at
12786 rpm (17, 000 x g) for 10 min at 4 oC. The supernatant was discarded and the pellet was resuspended in a total volume of 5-10 mL L-15 mediumMalaya (Gibco, USA) supplemented with 10 % FBS and 10 % TPB. A total of 950 µL of the cell suspension were aliquoted
into each cryovial containing 50 µL DMSO ofand mixed well. The cells were slowly frozen
to -80 oC by using a Mr. FrostyTM freezing container (Nalgene, Thermo Fisher Scientific,
USA) before transferring a to liquid nitrogen tank.
3.12 Study on the growth characteristics of Rickettsia spp. in C6/36 cells
3.12.1 Inoculation and culture of Rickettsia spp.
For the time-course experiment 1, a fifth-passage culture (5 dpi) was used to infect
confluent C6/36 cells in T75 cell culture flasks (C6/36-1). For the time-course experiment University2, a 18th-passage culture (19 dpi) was used to infect confluent C6/36 cells in T75 cell culture flasks. The infections in time-course experiment 2 were performed in duplicates
(C6/36-2 and C6/36-3). In both time-course experiment 1 and 2, 1 mL of the infected
C6/36 cells was inoculated into each flask containing 9 mL of L-15 medium
supplemented with 2.5 % FBS and 2.5 % TPB. The cells were harvested at 1, 2, 4 and 7
61 dpi. The growth medium was not replaced during the cultivation period and the cultures
were checked daily for bacterial contamination.
For the time-course experiment 3, 13th-passage cultures (10 dpi) were used to infect
confluent C6/36 cells in 24-well plates (SPL Life Science, Pocheon-Si, South Korea) for
14 days. In two parallel series of infection, 100 µL of the infected C6/36 cells was
inoculated into each well of uninfected C6/36 monolayers in a 24-well tissue culture plate
containing 900 µL of L-15 medium supplemented with 2.5 % FBS and 2.5 % TPB. The
infections were carried out in duplicates (C6/36-4 and C6/36-5) and the cells were
harvested on 2, 4, 6, 8, 10, 12 and 14 dpi. The growth medium was not replaced during
the cultivation period and the cultures were checked daily for bacterial contamination. The harvested cells for all the experiments were frozenMalaya at -80 oC and later the DNA was extracted for quantification by using a real-time PCR as described in section 3.12.4.
3.12.2 DNA extraction of infected cells of
DNA was extracted from 100 µL of the infected cells used to initiate infections
(represent the total number of DNA copies inoculated into the cultures), 1 mL of the
harvested infected and uninfected (control) cells. The cells were centrifuged at 14,000
rpm (Hermle AG, Germany) for 30 min at 4 oC. The supernatant was discarded and the
pellet was resuspended with remnant supernatant by tapping or vigorous vortexing. DNA
was then extracted using a G-spinTM Total DNA extraction kit (iNtRON Biotechnology, UniversityKorea) according to the manufacturer’s protocol. Fifty microliters of DNA were eluted for real-time PCR assays.
3.12.3 Preparation of plasmid standard for quantification of rickettsiae
A standard curve was generated from a plasmid harbouring the cloned gltA fragment
(168 bp) amplified from Rickettsia sp. TH2014. The gltA fragment was amplified with a
62 primer pairs as described by Socolovschi et al. (2010) with slight modification. The
primer pairs were consisted of the forward primer: 5’-TAG-TGA-ATG-AAA-GAT-
TAC-ACT-ATT-TAT-TTC-AAA-C-3’, and the reverse primer: 5’-GTA-TCT-TAG-
CAA-TCA-TTC-TAA-TAG-CGG-TAA-3’. Then, the gltA fragment was cloned into
pCRTM4-TOPO cloning vector using a TOPO TA cloning kit (Invitrogen, USA)
according to the manufacturer’s protocol. The plasmid was extracted using a Exprep
Plasmid SV kit (GeneAll, Korea). The concentration of the plasmid was measured using
a NanoDropTM 2000c (Thermo Scientific, USA).
Serially-diluted (tenfold dilution) plasmid extract containing the cloned gltA fragment
(168 bp) of Rickettsia sp. TH2014 was used to construct standard curve for quantification
of the rickettsial organism using real-time PCR assay. The copy number of the constructed plasmid was determined by using a formulaMalaya converting nanograms to copy number, as illustrated on the Integrated DNA Technologies, Inc. website
(https://www.idtdna.com/pages/decoded/decoded-articles/pipet-of
tips/decoded/2013/10/21/calculations-converting-from-nanograms-to-copy-number):
where:
X = amount of amplicon (ng)
N= length of dsDNA amplicon
University660 g/mole = average mass of 1 bp dsDNA
3.12.4 Real-time PCR assay
A real-time PCR assay (RKND03 system) as described by Socolovschi et al. (2010)
with slight modifications on the primers and probe targeting the gltA gene was performed
63 for quantification of rickettsial DNA in the infected cells. The 168 bp long fragment of
the gltA gene was amplified using the forward primer, 5’-TAG-TGA-ATG-AAA-GAT-
TAC-ACT-ATT-TAT-TTC-AAA-C-3’, the reverse primer, 5’-GTA-TCT-TAG-CAA-
TCA-TTC-TAA-TAG-CGG-TAA-3’ and the probe FAM-TAT-TAT-GCT-TGC-GGC-
T-C-NFQ. The appropriate handling and DNA extraction procedure was monitored by
amplification of the Eukaryotic 18S rRNA endogenous control (4319143E, Applied
Biosystems, USA) designed by Applied Biosystems. The PCR reaction mix and thermal
cycling conditions are shown in Table 3.2 and Table 3.3. The real-time PCR assays were
performed using ABI StepOnePlusTM system (Applied Biosystems, USA) and the results
were analyzed using StepOneTM Software v2.2.1.
Table 3.2: Reaction mixture used for real-time PCR assay.
Component Volume (µL) for 1 reactionMalaya Final concentration TaqMan Environmental 15.0 1 X Master Mix 2.0 (2X) TaqMan gene expression of 1.5 1 X assay (20 X) DNA template (10ng/µl) 3.0 - Ultrapure water 10.5 - Total volume per reaction 30.0 -
Table 3.3: Thermal cycling conditions.
Thermal-cycling profile Temperature (oC) Time (mm:ss) Cycles Hold (UNG incubation) 50.0 2:00 1 Hold (Polymerase 95.0 10:00 1 Universityactivation) Denature 95.0 00:15 40 Anneal/extend 60.0 01:00
64 3.13 Study of the morphological characteristics of Rickettsia spp. in C6/36 cells
3.13.1 Giemsa stain
Giemsa stain was used to examine the morphology of rickettsia in the infected C6-36
cells. An aliquot of the sample prepared in section 3.11.3 was used for standard Giemsa
stain. Briefly, 20 µL of the infected cell culture were pipetted and smeared on a glass
slide and air-dried in a biosafety cabinet. The slide was subsequently fixed in ice-cold
methanol (Merck, Germany) for 20 min in a coupling jar and air-dried in a biosafety
cabinet. Modified Giemsa stain (GS500 Sigma-Aldrich, United States) was diluted at
1:20 ratio in ultrapure water. The slide was stained with the diluted Giemsa solution for
60 min, rinsed in ultrapure water and air-dried for viewing under a light microscope
(Nikon, Japan).
3.13.2 Transmission electron microscopy of rickettsia-infectedMalaya cells Infected C6/36 cells (1, 4 and 7 dpi) and uninfected C6/36 cells harvested from time-
course experiment 1 in section 3.12.1 forof transmission electron microscopy (TEM)
analysis. The infected cells were removed from the tissue culture flasks by scraping with
a cell scrapper and then transferred to a 15 mL conical tube for centrifugation at 1, 200
rpm for 5 min (Luce-Fedrow et al., 2015b). The supernatant was discarded and the cell
pellet was fixed in approximately 5 mL of 4 % glutaraldehyde (Agar Scientific, UK).
Each sample was then washed twice in approximately 1 mL of cacodylate buffer, post-
fixed in approximately 1 mL of osmium tetraoxide: cacodylate buffer (1:1) (Agar UniversityScientific, UK) for 2 hours at 4 oC and subsequently incubated in approximately 1 mL of cacodylate buffer (Agar Scientific, UK) for overnight. The pellet was dehydrated in
approximately 1 mL of ethanol and embedded in Epon (Appendix Q) for overnight and
incubated at 37 oC for 5 hours, then 60 oC for overnight. The ultrathin sections were
placed in formvar carbon-coated grids, stained with 8 % uranyl acetate (Agar Scientific,
UK) and subsequently counterstained with lead citrate (Agar Scientific, UK). The images
65 were captured by using a LEO-Libra 120 transmission electron microscope (Carl Zeiss,
Germany).
3.13.3 Toluidine blue stain on semi-thin section of the rickettsia-infected cells
Semi-thin section (1 µm) of the infected cells embedded in Epon (from section 3.13.2)
was transferred onto a drop of distilled water on a glass slide. The section on the glass
slide was dried on a slide warmer. A few drops of toluidine blue staining solution (Agar
Scientific, UK) were dropped on the completely dried section for 1 to 2 min. The excess
stain was rinsed off gently with distilled water and air dried. The stained section was
viewed under a bright field microscope (Leica, Germany).
3.14 Whole genome sequencing and analysis 3.14.1 DNA extraction Malaya DNA was extracted from a 15 dpi rickettsial culture using a QIAamp® DNA Microbiome kit (Qiagen, Hilden, Germany)of according to the manufacturer’s protocols (QIAamp DNA Microbiome Handbook, 05/2014) with slight modifications. The
extraction procedure involved lysis of host cells, degradation of host nucleic acids,
chemical and mechanical disruption of bacterial cells, binding of the DNA to the QIAamp
UCP Mini membrane, removal of contaminants and lastly elution of pure bacterial nucleic
acid. All of the reagents used in the extraction were provided in the kit.
Briefly, the infected cells were harvested from a 75 cm2 tissue culture flask and Universitycentrifuged at 3100 rpm (1, 000 x g) for 5 min to remove the host cells. The supernatant was then centrifuged at 12786 rpm (17, 000 x g) for 10 min to harvest all the bacteria.
The pellet was re-suspended in 1 mL PBS and added with 500 µL AHL buffer prior to 30
min of incubation at room temperature with an end to end rotator (Grant Instrument,
England). The tube was centrifuged at 9806 rpm (10, 000 x g) for 10 min and the pellet
was re-suspended in 190 µL buffer RDD and 2.5 µL Benzonase. The mixture was
66 incubated at 37 oC for 30 min at 300 rpm in a shaking incubator. During the incubation,
the host nucleic acid was degraded by using Benzonase in which the bacterial cells were
kept intact. The host lysis was continued by adding 20 µL proteinase K solution and
incubated at 56 oC for 30 min at 300 rpm. Two hundred microliters of buffer ATL
(containing Reagent DX) was added and the mixture was transferred to pathogen lysis
tube L for bacterial cell lysis. Pathogen lysis tube L was vortexed for 10 min at maximum
speed. The supernatant was then transferred to a new microcentrifuge tube and 40 µL of
proteinase K solution was added. The mixture was incubated at 56 oC for 30 min at 300
rpm. Buffer APL2 (200 µL) was then added and the mixture was incubated at 70 oC for
10 min. Ethanol (200 µL) was added to the lysate and the mixture was transferred into a
QIAamp UCP mini column. This was then followed by several washing steps using buffers AW1 and AW2. The bacterial DNA was Malaya eluted in 50 µL of buffer AVE (containing RNAse free water with 0.04 % sodium azide). The extracted bacterial DNA was loaded onto a 1.0 % agarose gel (FirstBaseof Laboratories, Malaysia) at 80 V for 1 hour and visualized using a UV transilluminator (G-Box, Syngene, UK). The DNA
concentration was measured using a NanoDropTM 2000c (Thermo Scientific, USA).
3.14.2 Whole genome sequencing
Library preparation and genome sequencing were performed using the service
provided by a genome sequencing facility (Beijing Genomics Institute, China). Quality
control (QC) was performed in every step of the procedures to ensure data reliability. The UniversityDNA sample passed the QC criteria, i.e., DNA purity (OD260/OD280<1.8), no DNA degradation and potential contamination (agarose gel electrophoresis) and DNA
concentration of >7 µg (Qubit 2.0). For library construction, the genomic DNA was first
sheared into smaller fragments with a desired size using Covaris S/E 210 or Bioruptor.
The overhangs resulting from fragmentation were then converted into blunt ends by using
T4 DNA polymerase (Klenow Fragment and T4 polynucleotide kinase). Adapters were
67 ligated onto both ends of DNA fragments after adding an ‘A’ base to the 3’ end of the
blunt phosphorylated DNA fragments. The desired fragments were purified though gel-
electrophoresis, then selectively enriched and amplified by PCR. Library quality test was
carried out and only the qualified libraries were sequenced using Illumina high-
throughput Illumina HiSeq 4000 with a 150 Paired-End (PE) strategy.
3.14.3 Genome assembly
The raw sequencing reads obtained from the sequence provider were pre-processed by
removing poor quality reads (Phred score ≤ 20) and ambiguous N bases. The clean reads
were used for de novo genome assembly and scaffolding using three different well-
established software, i.e.: SPAdes (Bankevich et al., 2012), ABySS (Simpson et al.,
2009) and Velvet (Zerbino & Birney, 2008). The assemblies generated were compared and the best assembly in terms of N50 size, contig numberMalaya and also genome size was chosen for downstream analyses. of 3.14.4 Gene prediction and annotation
For consistency and easier comparison, the genome sequence was annotated (i.e. gene
prediction) by using RAST (Rapid Annotation using Subsystem Technology) annotation
pipeline (Aziz et al., 2008). Reference genome sequences were obtained from NCBI
genome database (www.ncbi.nlm.nih.gov/genome). Schematic circular diagram was
generated by using DNAPlotter (Carver et al., 2009).
University3.14.5 Pairwise genome comparison Pairwise genome comparison of Rickettsia sp. TH2014 with other rickettsial genomes
were carried out using JSpeciesWS (http://jspecies.ribohost.com/jspeciesws) to
determine the average nucleotide identity (ANI) values based on BLAST (ANIb) and
based on MUMmer (ANIm) (Richter et al., 2016). MUMmer is a system for rapidly
aligning entire genomes, whether in complete or draft form (Kurtz et al., 2004). ANI
68 values are based on pairwise alignment of genome stretches. JSpeciesWS also enables
the calculation of correlation indexes of tetra-nucleotide signature frequency correlation
coefficient (TETRA) based on tetra nucleotide composition in the genome (Richter et al.,
2016; Teeling et al., 2004). Closely related genomes may show very high ANI and
TETRA correlation values. For most intraspecific results, the ANIm values are above
96% identity and usually corresponded to very high TETRA correlation coefficients
(>0.99). In general observation, TETRA values >0.99 may support the species
circumscription based on the ANI range >95-96 %, but both values should agree (Richter
& Rossello-Mora, 2009).
3.14.6 Inference of phylogenetic relationships amongst Rickettsia spp.
The Rickettsia sp. TH2014 together with the completed and draft sequences downloaded from NCBI genome database were subjectedMalaya to a series of pre-processing to generate a FASTA input file compatible with the analysis software. The kchooser script
was used to select an optimum value of k-merof size for the data set. Jellyfish in the kSNP
package was used to enumerate a list of k-mers at optimum k-value for each genome in
the data set. The sequence variation of the data set was evaluated and the fraction of core-
k-mers at optimum k-value was identified. kSNP was used to search for putative SNP
loci in the data set and the identified SNP alleles were concatenated into a string. Multiple
sequence alignment was produced for each nucleotide in the SNP matrix using MUSCLE
(version 3.8.31) (Edgar, 2004). The best fit nucleotides substitution model was selected Universityusing jModelTest and trees of different models were assessed using Akaike Information Criterion framework (AIC). PhyML 3.0 (Guindon et al., 2010) was used to generate
maximum likelihood tree with 1000 bootstrap replicates using the jModelTest suggested
model.
69 3.14.7 Comparative analysis of gene functions with R. felis/RFLO and
pathogenomics
Comparative gene function analysis of Rickettsia sp. TH2014, R. felis URRWXCal2
(Accession: SAMN02603143) and two RFLOs, i.e., Rickettsia hoogstraalii (Accession:
SAMEA2771239), and Rickettsia asemboensis (SAMN03272870) retrieved from the
GenBank database was performed. For pathogenomic study, RAST-predicted genes of
Rickettsia sp. TH2014 were searched against Virulence Factors Database (VFDB,
www.mgc.ac.cn/VFs/) (Chen et al., 2005) for prediction of putative virulence genes.
Malaya of
University
70 CHAPTER 4: RESULTS
4.1 Determination of the etiological agents of rickettsioses in patients with
febrile illness
4.1.1 Patient with pyrexia admitted to UMMC
Serum and whole blood samples were obtained from 41 febrile patients admitted to
UMMC for serological and molecular testing. The patients aged between 14 to 76 years,
of which 28 (68.3 %) were male and 13 (31.7 %) were female. The seropositivity against
rickettsial antigens, i.e., R. rickettsii and R. typhi were determined (Table 4.1). IgM titers
of ≥ 1:64 against R. typhi and R. rickettsii were detected from seven (17.1 %) and four
(9.8 %) out of 41 patients, respectively. IgG titers of ≥ 1:64 against R. typhi and R.
rickettsii were detected from three (7.3 %) and four (9.8 %) patients, respectively. Further testing of the positive sera at IgM titer of ≥ 1:128 showedMalaya that only three (7.3 %) patients were positive for R. typhi, and none of them were positive against R. rickettsii. IgG titers of ≥ 1:128 against R. typhi and R. rickettsii wereof only detected from two (4.9 %) patients, respectively. Of five patients with paired sera, only one patient (referred patient A)
showed a rise in IgM titer against R. typhi (1:256 to ≥ 2048). A fourfold or greater increase
of IgG titers against R. typhi (1:256 to ≥ 2048) and R. rickettsii (1:256 to ≥ 1024) in paired
sera was observed in the same patient. None of the patients revealed any history of
exposure to arthropod vectors. However, history of preceding travel is not available for
all the patients. UniversityTable 4.1: Seropositivity rates of rickettsiae in febrile patients Rickettsia spp. IgM IgG titer ≥ 1:64 R. typhi 7 (17.1 %) 3 (7.3 %) R. rickettsii 4 (9.8 %) 4 (9.8 %) titer ≥ 1:128 R. typhi 3 (7.3 %) 2 (4.9 %) R. rickettsii 0 (0) 2 (4.9 %)
71 A rise in IgM titers against R. typhi in patient A suggesting a recent infection of R.
typhi or its antigenically closely related species, including R. felis (Schriefer et al., 1994).
High IgG titers (≥ 2048) against R. typhi were noted in both acute and convalescent
samples of this patient. Since the IgG titers for R. rickettsii were lower than that of R.
typhi in both acute and convalescent samples, the results are suggestive of cross-reactivity
amongst rickettsial antigens.
Using conventional PCR assays, rickettsial gltA and ompB gene fragments were
amplified from only one (2.4 %) blood sample of the 41 patients. OmpA gene was not
amplified from any of the samples. The positive blood sample was derived from a 15-
year-old boy (patient A) who experienced fever, myalgia, arthralgia, mild headache and
loss of appetite for the past one week. He had conjunctival suffusion and the presence of petechiae was noted in the limbs. His white blood cellMalaya (WBC) count, hemoglobin, urea, creatinine and bilirubin values were within normal limits (Table 4.2). Elevated levels of
alanine aminotransferase and aspartate aminotransferaseof associated with a low white
blood cell count and albumin were noted. Laboratory tests including blood cultures and
serological tests for dengue, malaria, and hepatitis C were negative. His fever persisted
despite treatment with ceftriaxone. He was then started on doxycycline and the
temperature subsided within 24 hours. Upon review one week later, he was well and his
platelet count and liver enzymes were normal. The sequences of the gltA [deposited in
GenBank, accession no.: KU255716, 399/402 bp, 99.3 %] and ompB (deposited in UniversityGenBank, accession no.: KU255717, 772/772 bp, 100 %) gene fragments amplified from the blood DNA of patient A had the closest match with those of Rickettsia sp. RF2125
(GenBank accession no.: AF516333 and JX183538), and next with those of R. felis type
strain URRWXCal2 (GenBank accession no.: CP000053).
72 4.1.2 DNA samples from dengue-negative febrile patients
All the DNA samples obtained from 62 dengue-negative febrile patients were included
for molecular detection of rickettsia. The serum samples obtained from three patients (B,
C and D) were positive for rickettsial DNA in the PCR assays. One sample (Patient B)
was positive for R. typhi based on the amplification and sequence analyses of gltA-1
(deposited in GenBank, accession no.: KU255718, 726/726 bp) and ompB (deposited in
GenBank, accession no.: KU255719, 767/767 bp) gene fragments, with both fragments
demonstrated 100 % sequence similarity to those of R. typhi str. Wilmington (GenBank
accession no.: U59714).
Table 4.2: The demographic, hematology and blood chemistry profiles of patients investigated in this study.
Patient A Patient B Patient C Patient D Reference Malayarange Demographic data Age (years) 15 72 33 42 Gender Male Femaleof Female Male Occupation student farmer - - Blood profiles Hemoglobin (g/L) 12.6 13.3 - - 12-15 White blood cells 4.8 7.6 1.9 3.5 4-10 (109/L) Platelets (109/L) 78 50 115 126 150-400 Urea (mmol/L) 4.8 13.8 - - 2.5-6.4 Creatinine (mol/L) 105 196 - - 62-115 Bilirubin (mol/L) 11 15 - - 3-17 Albumin level (g/L) 32 28 - - 35-50 Alkaline phosphatase 129 122 - - 50-139 (U/L) Alanine 111 67 - - 12-78 aminotransferase (U/L) UniversityAspartate 141 126 - - 15-37 aminotransferase (U/L) Tick bite history No No No No Rashes/eschars Yes (limb) No No No Molecular detection of Rickettsia Rickettsia Rickettsia Rickettsia rickettsia sp. typhi closely closely RF2125 related to related to R. raoultii R. raoultii -, data not available.
73 Patient B was a 73-year-old lady living in a farm at the East coast of Malaysia. She
was admitted to UMMC with febrile illness and underlying interstitial pulmonary
fibrosis. During admission, she presented with the complaints of backache, anorexia,
diarrhea, abdominal pain, reduced effort tolerance, and productive cough. She developed
atrial fibrillation and was hypotensive. Chest examination revealed bilateral lung
crepitation. All her other body systems were grossly normal and there was no rash or
eschar. The patient was thrombocytopenic with raised plasma levels of urea, creatinine,
and aspartate aminotransferase (Table 4.2). Hypoalbuminemia was also noted. The
thorax computerized tomography (CT) scan showed extensive patchy consolidation,
bilateral pleural effusion and mediastinal lymphadenopathy, suggesting severe
pneumonia. Septic work up including blood and stool cultures did not reveal any bacterial growth. Blood smear for malaria parasites and Malaya serological tests for leptospira, mycoplasma, and legionella were all negative. She was treated for septic shock and was started on intravenous amoxyl-clavulanic acidof and azithromycin. However, due to poor response and symptom worsening, her antibiotics were changed to piperacillin-
tazobactam and doxycycline on day four of admission. Her clinical condition improved
gradually and the fever subsided.
Rickettsial gltA-1 and ompB gene fragments were amplified from the serum DNA
sample of two patients (C and D) with mild fever. Patient C (33 years old, female)
presented with five days of fever, headache and presumed upper respiratory tract Universityinfection. She had nausea but no jaundice or rash. Blood profile investigations showed low WBC and platelet counts. She was advised on hydration and discharged without
further treatment when her WBC count reverted back to normal range. Patient D (42
years old, male) presented with a history of fever for seven days. He had arthralgia,
myalgia, rhinorrhea and cough with yellowish sputum but no hemoptysis or dyspnea. No
rash or eschar was noted. All his body systems were normal, except for lower WBC and
74 platelet counts. BLAST analyses of the gltA-1 amplicons from both patients showed the
closest match (deposited in GenBank, accession no.: KU255720 and KU255721, 711/722
bp, 98.5 %) to a rickettsia closely related to R. raoultii (GenBank accession no.:
JQ697956) detected from Haemaphysalis hystricis ticks in Japan. The amplified ompB
gene fragments showed the highest sequence similarity (deposited in GenBank, accession
no.: KU2557122 and KU255723, 679/691 bp, 98.3 %) to R. raoultii strain Khabarovsk
(GenBank accession no.: DQ365798). Based on the sequence analyses, the rickettsia
detected from the serum DNA sample of the patients were identified as a rickettsia closely
related to R. raoultii.
Figure 4.1 is a dendrogram constructed based on the rickettsial ompB gene sequences
(746-764 bp) obtained in this study and those available in the GenBank database. The dendrogram confirms the closest genetic relatednessMalaya of the rickettsia from the blood sample of patient A with Rickettsia sp. RF2125 which had been reported from C. canis
dog flea in Thailand (GenBank accession no.:of JX183538, Parola et al., unpublished), and
RFLO detected from monkey (section 4.4.2) (Rickettsia sp. 0095, GenBank accession
no.: KP126804,(Tay et al., 2015). The rickettsia was next closest to R. felis URRWXCal2
type strain (GenBank accession no.: CP000053) and a Malaysian cat flea (HL15c,
GenBank accession no.: KF963608) (Tay et al., 2014). The rickettsia identified from
patient B (diagnosed with murine typhus) was clustered with R. typhi str. Wilmington
strain (GenBank accession no.: NC006142), while the rickettsia identified from the serum UniversityDNA samples of patient C and D were clustered with several R. raoultii type strains and an uncultured R. raoultii strain (GenBank accession no.: KJ769652) identified from
Malaysian Amblyomma snake tick (Section 4.3.4.5) (Kho et al., 2015).
75
Malaya of
Figure 4.1: Phylogenetic analysis based on the sequences of the outer membrane protein (ompB) gene of rickettsiae identified in this study. * denotes rickettsia detected in the patients in this study.
Bootstrap analysis was performed with 1000 replications. Scale bar indicates the nucleotide substitutions per site. Rickettsiae identified from monkey, cat flea and Amblyomma tick (GenBank accession nos.: KP126804, KF963608, and KJ769652) in this study were also included in the dendrogram analysis, for comparison with the reference strains. University
76 4.2 Determination of the antibody prevalence of rickettsiae in urban blood
donors, animal farm workers, and indigenous community
Serum samples of healthy individuals were collected from 61 urban blood donors, 87
farm workers and 102 indigenous people across several states in Peninsular Malaysia
(section 3.2.3-3.2.5). The median ages of 87 farm workers, 61 blood donors and 102
indigenous people were 38 years (range, 23-59 years), 31 years (range, 19-54 years) and
27 years (range, 8-78 years), respectively (Table 4.3). The male to female ratio was 1.34
(143:107).
The majority of the animal farm workers and urban blood donors were of the Malay
ethnic groups, which is the largest ethnic group in Malaysia, followed by Chinese and
Indians. The indigenous people comprised different tribes, including Temiar, Semoq Beri, Semai, Temuan, Jakun, Jah Hut, Kensui and others.Malaya
Table 4.3: Demographic and baseline characteristics of the urban blood donors, farm workers and indigenousof people (n=250).
Study Blood donors Farm workers Indigenous people populations (n=61) (n=87) (n=102) Variable No. (%) No. (%) No. (%) Age Range 19-54 23-59 8-78 32.93 39.92 30.24 Mean (± SD) (±9.19) (±11.14) (±16.29) Age groups (years) 8 - 20 2 3.3 0 0 35 34.3 21 - 30 28 45.9 22 25.3 25 24.5 31 - 40 16 26.2 25 28.7 19 18.6 41 - 50 12 19.7 18 20.7 9 8.8 51 and above 3 4.9 22 25.3 14 13.7 UniversityGender Female 29 47.5 15 17.2 63 61.8 Male 32 52.5 72 82.8 39 38.2 Race group Malay 36 59.0 81 93.1 0 0 Non-Malay 25 41.0 5 5.7 0 0 (Chinese, Indian) Indigenous people 0 0.0 1 1.1 102 100
77 Table 4.4 presents the rickettsial seropositivity rates of different study groups based
on gender and age. The indigenous people had the highest seropositivity rates to R.
conorii (50.0 %, 95 % CI: 40.1-59.9 %) and R. felis (22.5 %, 95 % CI: 14.3-30.8%). A
total of 13.8 % (95 % CI: 6.4-21.2 %) and 16.1 % (95 % CI: 8.2 %-24.0 %) of the farm
workers were seropositive for R. conorii and R. felis, respectively. The seropositivity rates
for rickettsiae were the lowest amongst the urban blood donors, given that only 3.3 % (95
% CI: 0.0-7.9 %) were seropositive for R. conorii and none was seropositive for R. felis.
Table 4.4: Seropositivity of R. conorii and R. felis with respect to different category of the participants investigated in this study.
R. conorii R. felis Categories No. (%) p value 95 % CI No. (%) p value 95 % CI Malaya Study group Blood donors 2 (3.3) # 0.0 – 7.9 0 (0.0) # 0.0-0.0 Farm 12 (13.8) # 6.4of – 21.2 14 (16.1) 8.2 – 24.0 workers < 0.001 < 0.001 Indigenous 51 (50.0) 40.1 – 59.9 23 (22.5) 14.3 – 30.8 people
Gender Male 31 (21.7) 14.8 – 28.5 23 (16.1) 10.0 – 22.2 0.072 0.509 Female 34 (31.8) 22.8 – 40.7 14 (13.1) 6.6 – 19.6
Age group
(years) ≤20 10 (27.0) 12.0 – 42.0 2 (5.4) * 0.0 – 13.0 21-30 19 (25.3) 15.3 – 35.4 11 (14.7) 6.5 – 22.9 31-40 16 (26.7) 0.011 15.1 – 38.2 3 (5.0) * 0.001 0.0 – 10.7 41-50 3 (7.7) * 0.0 – 16.4 8 (20.5) 7.3 – 33.8 ≥51 17 (43.6) 27.3 – 59.9 13 (33.3) 17.9 – 48.8 University CI: confidence level. # Significant difference in the rickettsial seropositivity rate when compared to the indigenous people (Games-Howell post hoc test, refer to Table 4.4). * Significant difference in the rickettsial seropositivity rate when compared to those ≥51 years of age (Games-Howell post hoc test, refer to Table 4.5).
78 Pairwise comparison within the study groups (using Games-Howell post-hoc test)
demonstrated a significantly higher R. conorii-seropositivity rate (50.0±50.2 %) in the
indigenous people, as compared with the animal farm workers (13.8±34.7 %, p<0.001),
and the urban blood donors (3.3±18.0 %, p<0.001) (Table 4.5). The R. felis-seropositivity
rate of the indigenous people (22.5±42.0 %) was also significantly higher as compared
with urban blood donors (0.0 %, p<0.001), but not animal farm workers (16.1±37.0 %,
p=0.500). Seropositivity against both R. conorii and R. felis was detected in 23 individuals
[0 (0.0 %) in urban blood donors, including 5 (5.7 %) for farm workers and 18 (17.6 %)
for the indigenous community] in this study. Representative confocal images were taken
for R. conorii and R. felis immunofluorescence assays as depicted in Figure 4.2 and Figure
4.3, respectively.
No significant differences in the seropositivity ratesMalaya for R. conorii and R. felis were noted in any of the study groups based on gender (p=0.072 and p=0.509 for R. conorii
and R. felis, respectively) (Table 4.4). of Significant differences were noted in the
seropositivity rates of R. conorii (p=0.011) and R. felis (p=0.001) within different age
groups. The participants in the age group of ≥51 years old demonstrated the highest
seropositivity rates for both R. conorii and R. felis (Table 4.6). Games Howell post-hoc
tests revealed significantly higher R. conorii-seropositivity rate amongst participants over
50 years old (43.6±50.2 %) compared with those 41-50 years old (7.7±27.0 %, p=0.002).
R. felis-seropositivity rate was also significantly higher amongst participants over 50 Universityyears old of age (33.3±47.8 %), compared with those ≤20 years old (5.4±22.9 %, p=0.015) and 31 to 40 years old (5.0±22.0 %, p=0.009) (Table 4.6). Comparison of
rickettsial seropositivity rates between ethnic groups was not possible as each study group
was composed of different ethnic groups.
79 Table 4.5: Pairwise comparison of different study groups investigated in this study, using Games-Howell Post-hoc tests of the SPSS.
95 % Study Study Mean Confidence Group Group Difference Seropositivity Sig. Interval Lower Upper (I) (J) (I-J) Bound Bound Farm -10.5* 0.046 -20.9 -0.2 Blood workers donors Indigenous -46.7* <0.001 -59.7 -33.7 people
Blood 10.5* 0.046 0.2 20.9 Farm donors R. conorii workers Indigenous -36.2* <0.001 -50.9 -21.5 people
Blood 46.7* <0.001 33.7 59.7 Indigenous donors people Farm 36.2* <0.001 21.5 50.9 workers Malaya
Farm -16.1* <0.001 -25.5 -6.6 Blood workersof donors Indigenous -22.6* <0.001 -32.4 -12.7 people
Blood 16.1* <0.001 6.6 25.5 Farm donors R. felis workers Indigenous -6.5 0.5 -20.0 7.1 people
Blood 22.6* <0.001 12.7 32.4 Indigenous donors people Farm 6.5 0.5 -7.1 20.0 workers University* The mean difference is significant at the 0.05 level.
80 Table 4.6: Pairwise comparison of different age groups investigated in this study, using Games-Howell Post-hoc tests of the SPSS.
Age Mean 95 % Confidence Age group Difference Interval Seropositivity Sig. group (I) Lower Upper (J) (I-J) Bound Bound 21-30 1.7 1.000 -23.4 26.8 31-40 0.4 1.000 -25.8 26.6 ≤ 20 41-50 19.3 0.174 -4.8 43.5 ≥ 51 -16.6 0.556 -47.1 14.0 ≤20 -1.7 1.000 -26.8 23.4 31-40 -1.3 1.000 -22.5 19.9 21-30 41-50 17.6 0.068 -0.8 36.1 ≥ 51 -18.3 0.316 -44.9 8.4 ≤20 -0.4 1.000 -26.6 25.8 21-30 1.3 1.000 -19.9 22.5 R. conorii 31-40 41-50 19.0 0.072 -1.0 39.0 ≥ 51 -16.9 0.434 -44.6 10.7 ≤20 -19.3 0.174 -43.5 4.8 21-30 -17.6 0.068 -36.1 0.8 41-50 31-40 -19.0 0.072 -39.0 1.0 ≥ 51 -35.9* Malaya 0.002 -61.6 -10.2 ≤20 16.6 0.556 -14.0 47.1 21-30 18.3 0.316 -8.4 44.9 ≥ 51 31-40 of 16.9 0.434 -10.7 44.6 41-50 35.9* 0.002 10.2 61.6 21-30 -9.3 0.463 -24.8 6.2 31-40 0.4 1.000 -12.8 13.6 ≤ 20 41-50 -15.1 0.279 -36.4 6.1 ≥ 51 -27.9* 0.015 -52.0 -3.9 ≤20 9.3 0.463 -6.2 24.8 31-40 9.7 0.305 -4.2 23.5 21-30 41-50 -5.8 0.942 -27.5 15.8 ≥ 51 -18.7 0.213 -43.1 5.7 ≤20 -0.4 1.000 -13.6 12.8 21-30 -9.7 0.305 -23.5 4.2 R. felis 31-40 41-50 -15.5 0.206 -35.7 4.7 ≥ 51 -28.3* 0.009 -51.4 -5.2 University≤20 15.1 0.279 -6.1 36.4 21-30 5.8 0.942 -15.8 27.5 41-50 31-40 15.5 0.206 -4.7 35.7 ≥ 51 -12.8 0.708 -41.0 15.3 ≤20 -27.9* 0.015 3.9 52.0 21-30 18.7 0.213 -5.7 43.1 ≥ 51 31-40 -28.3* 0.009 5.2 51.4 41-50 12.8 0.708 -15.3 41.0 * The mean difference is significant at the 0.05 level.
81
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UniversityFigure 4.2: Representative confocal images of R. conorii immunofluorescence assay (Fuller lab).
A: Negative control (human serum provided in the kit), B: Positive control (human serum provided in the kit), C: a positive serum sample from a farm worker (UL02), D: a positive serum sample from a rural villager (PS139), E: a positive serum sample from a farm worker (AH12) and F: a positive serum sample from a rural villager (PI006). Analysis was performed by using a Leica TCS SP5 II laser scanning confocal spectral microscope with 40x oil immersion objective lens. Scale bar: 50 µm.
82
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UniversityFigure 4.3: Representative confocal images of R. felis immunofluorescence assay (Fuller lab).
A: Negative control (human serum provided in the kit), B: Positive control (human serum provided in the kit), C: a positive serum sample from a farm worker (G013), and D: a positive serum sample from a rural villager (PS095) E: a positive serum sample from a farm worker (TT02) and F: a positive serum sample from a rural villager (SM042). Analysis was performed by using a Leica TCS SP5 II laser scanning confocal spectral microscope with 40x oil immersion objective lens. Scale bar: 50 µm. Scale bar: 50 µm.
83 4.3 Determination of the type and distribution of rickettsiae in arthropods
4.3.1 Collection and identification of ticks
4.3.1.1 Collection and identification of ticks from urban areas
A total of 129 individual ticks were collected at three animal shelters in Kuala Lumpur
(Table 4.7 , Appendix F). Thirty-three ticks identified as Rh. sanguineus (brown dog
ticks) were collected from 13 dogs in SPCA and Second Chance Animal Shelter during
the sampling trips from March to May 2013. Following several visits to DBKL animal
shelter in August 2013, January 2014 and February 2014, an additional 48 ticks were
collected from seven dogs and 48 unfed ticks were collected from the surrounding of the
animal shelter i.e., the wall, floor, corner of the cages.
Rh. sanguineus can be recognized by its red brown color, elongated body shape, and hexagonal basis capituli (Figure 4.4 and Figure 4.5). TheMalaya identification of Rh. sanguineus was confirmed by sequence analysis of the tick 12S ribosomal DNA sequences. The tick
mitochondrial 12S rDNA sequences of sevenof representative tick samples showed 100 %
(367/367 bp) identity to Rh. sanguineus (GenBank accession no.: DQ002997). The 12S
rDNA sequences for Rh. sanguineus collected from urban areas and rural villages in this
study are homogenous. A dendrogram constructed based on the 12S rDNA sequences of
the Rh. sanguineus in this study (including the Rh. sanguineus collected from rural
villages in Negeri Sembilan and Perak, respectively; section 4.3.1.3) shows that the
species of Malaysian dog ticks are closely related to those in Taiwan, Thailand, Brazil Universityand Iran (Figure 4.6).
84 Table 4.7: Total number of tick samples collected for this study. Animal host examined Localities Ticks species (n) (n)/source Urban areas SPCA and second Dog (13) Rh. sanguineus (33 individuals) chance animal shelters DBKL Unfed tick, Dog (20) Rh. sanguineus (96 individuals) Total 129 individual ticks Farms Negeri Sembilan (Farm H. bispinosa (41) and Rh. Cattle (30) 1) microplus (6) Pahang (Farm 2) Cattle (39) Rh. microplus (14) Haemaphysalis bispinosa (57) Pahang (Farm 3) Cattle (40) and Rh. microplus (37) Kedah (Farm 4) Sheep (40) H. bispinosa (44) Kelantan (Farm 5) Cattle (40) Not determined H. bispinosa (58) and Rh. Terengganu (Farm 6) Cattle (40) microplus (13) Negeri Sembilan (Farm Goat (40) 0 7) Johore (Farm 8) Dairy cattle (41) 0
Total 270Malaya individual ticks Rural area Cat (12), chicken (1), Rh. sanguineus (3 individuals), Negeri Sembilan dog (40), goat (8)of Haemaphysalis spp. (4 pools) Cat (18), chicken (5), Pahang Haemaphysalis spp. (7 pools) dog (21), Cat (17), chicken (1), Kedah Rh. microplus (3 individuals) cattle (3), dog (16) Cat (27), chicken (9), Haemaphysalis spp. (10 Kelantan dog (4) individuals and one pool) Cat (23), chicken (2), Haemaphysalis spp. (1 Johore dog (15), individual and 8 pools) Haemaphysalis spp. (13 Cat (6), chicken (3), individuals and 11 pools), Rh. Perak dog (9) sanguineus (1 individual), Dermacentor spp. (2 individual) Total 33 individuals and 31 pools Forest area Bats (18), rat (15), Haemaphysalis spp. (21), Kuala Lompat, Pahang Universitysquirrel (2), Skink (1) Dermacentor spp. (1) Amblyomma spp. (3), Vegetation areas Haemaphysalis spp. (35), Dermacentor spp. (61) Others A. varanense (11) and A. Sepang Snakes (7) helvolum (10) Johore Snake (1) A. helvolum (1 pool)
85
Figure 4.4: The dorsal views of a male (SF023M) (left) and a female (SF025F) (right) Rh. sanguineus. Hexagonal basis capituli is an identifying character for Rh. sanguineus. Scale bar: 5000 µm; magnification: 10X, zoom: 2X.
Malaya of
Figure 4.5: Ventral views of a female (SF025F) (left) and a male (SF023M) (right) Rh. sanguineus. Scale bar: 5000 µm; magnification: 10X, zoom: 2X. University
86 Rhipicephalus sanguineus (Taiwan DQ002997) D1001 (SPCA Kuala Lumpur) D1005 T1B (SPCA Kuala Lumpur) D7001 (SPCA Kuala Lumpur) D7002 (SPCA Kuala Lumpur) SC03f (second chance shelter Hulu Langat) 65 SC03h (second chance shelter Hulu Langat) SC06b (second chance shelter Hulu Langat) DK014 (Dusun Kubur Negeri Sembilan rural villages) SP009F2 (Sungai Perah Perak rural villages) 95 Rhipicephalus sanguineus (Thailand AY987377) Rhipicephalus sanguineus (Brazil AY559842) Rhipicephalus sanguineus strain 32 (NA DQ003004) 94 Rhipicephalus sp. voucher No RML (Iran FJ536563) Rhipicephalus sanguineus AF (Reunion JQ425164) 96 Rhipicephalus turanicus isolate 6K Turan SP (Livingstone Zambia DQ849232) 39 92 Rhipicephalus turanicus strain Zim (Zimbabwe AF150017) Rhipicephalus camicasi voucher USNTC-RML 107410 (Ethiopia FJ536556) 57 Rhipicephalus turanicus isolate T090960 (Sicily Italy HM014442) 36 Rhipicephalus turanicus strain Israel-63 (Israel AF150013) Rhipicephalus sulcatus voucher USNTC-RML 116669 (Zambia FJ536564) 51 Rhipicephalus sanguineus isolate OK (Oklahoma USA HM138900) 60 42 Rhipicephalus sanguineus group KM-2005 (Corsica France AY947467) 52 Rhipicephalus turanicus isolate 575-tot (NA AF483264) 49 Rhipicephalus turanicus strain Fra (France AF150018) Rhipicephalus turanicus isolate 127-tot (NA AF483244) Rhipicephalus sanguineus (Argentina AY559841) 67 Malaya 49 Rhipicephalus sp. voucher CAS35 (South Africa FJ536555) Rhipicephalus sanguineus (Uruguay AY559843) 96 Rhipicephalus sanguineus IS (Israel HM138901) Rhipicephalus sanguineus (California USA: HM014443) 86 of Rhipicephalus sanguineus isolate T6 AZ2004 (Arizona USAHM138903) 24 27 Rhipicephalus sanguineus voucher V4 (Portugal FJ536553) Rhipicephalus pravus (Tanzania AJ150025) Rhipicephalus zumpti (South Africa AF150016) Rhipicephalus muhsamae voucher USNTC-RML 116980b (Zambi FJ536559) 85 Rhipicephalus compositus (NA AF031860) 48 55 Rhipicephalus simus (Tanzania AF150025)
0.01
Figure 4.6: Phylogenetic placement based on the tick mitochondrial 12S rDNA partial sequences (258 bp) of Rh. sanguineus in this study.
Phylogenetic analysis was performed by using neighbour-joining method (Kimura 2- Universityparameter model). Rhipicephalus camicasi , Rhipicephalus compositus, Rhipicephalus muhsamae, Rhipicephalus simus, and Rhipicephalus zumpti were included as out-group. Bootstrap analysis was performed with 1000 replications. Scale bar indicates the nucleotide substitutions per site. Rh. sanguineus ticks in this study are marked with .
87 4.3.1.2 Collection and identification of ticks from animal farms
A total of 270 ticks (70 Rhipicephalus microplus and 200 Haemaphysalis bispinosa)
collected from five animal farms (Farm 1, 2, 3, 4, and 6) were included in this study
(Appendix G). No ticks were collected from Farms 5, 7 and 8. Table 4.7 summarizes the
total number of ticks collected in animal farms for this study.
i) Rhipicephalus (Boophilus) microplus
The Rhipicephalus (Boophilus) genus ticks were identified based on their small palpal
segments, hexagonal basis capituli, indistinct eyes, coxa I with small paired spur, and
coxa II to IV without spurs (Brahma et al., 2014). The male of the Rhipicephalus genus
has extra characteristics with the absence of festoons and the presence of adanal and accessory adanal ventral plates, as shown in Figure 4.7.Malaya The caudal appendage in the male is the most conspicuous features to differentiate between Rh. microplus (present) and Rh. annulatus (absent) (Brahma et al., 2014). Ofof the 70 ticks morphologically identified as Rh. microplus, 11 representative samples (three males and eight females) were sequenced
for 16S rRNA gene and they are all belongs to the same sequence type. The sequences
analyses showed 100 % (276/276 bp) similarity with those mitochondrial 16S rRNA gene
sequence of Rh. microplus from Thailand (GenBank accession no.: KT428015-
KT428016), Taiwan (GenBank accession no.: AY974242 and AY974232) and
Mozambique (GenBank accession no.: EU9188187).
University
88
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University Figure 4.7: The dorsal and ventral views of a male (EKR2162M) Rh. microplus (B. microplus) collected from Farm 3. Scale bar: 200 µm; magnification: 10X, zoom: 3.2X.
89 ii) Haemaphysalis bispinosa
Haemaphysalis ticks were identified based on the characteristic reported in previous
report (Brahma et al., 2014), i.e.: the presence of small palpal segments, lateral extension
of second palpal segment, basis capitulum rectangular (straight lateral margins), absence
of eyes, presence of festoons. Ventral plates are absent in males. Most of these features
were observed in the ticks collected from animal farms (Figure 4.8).
Malaya of
Figure 4.8: Major features of a male Haemaphysalis spp. (UN2-40M) collected from Farm 3. Scale bar: 100 µm; magnification: 10X, zoom: 11.5X.
A total of 13 representatives of morphologically identified Haemaphysalis spp. were Universitysubjected to sequence analysis of the 16S rRNA gene. All of the sequences revealed 100 % identity (287 bp) with that of H. bispinosa reported in India (GenBank accession no.:
KC853418- KC853420) (Brahma et al., 2014).
90 4.3.1.3 Collection and identification of ticks from rural villages
A total of 186 ticks collected from 47 peri-domestic-animals (i.e., cattle, cats,
chickens, dogs, and goat) from the rural villages were segregated into 64 pools (one to
ten individuals) (Table 4.7) prior to DNA extraction, in accordance with the size of the
ticks (Appendix H). Majority of the ticks (56 pools) collected from cats, chicken and
goats were morphologically identified as Haemaphysalis spp. Other than Haemaphysalis
spp., four Rh. sanguineus, three Rh. microplus, and two Dermacentor spp. were collected
from three dogs, two cattle, and a dog, respectively.
Of the 25 pools of Haemaphysalis spp. selected for sequence analyses using the tick
mitochondrial 16S rRNA gene, three different species of Haemaphysalis spp. were
identified based on BLAST analyses: H. bispinosa (GenBank accession no.: KC853419), Haemaphysalis wellingtoni (GenBank accession no.: MalayaAB819221) (Figure 4.9, Appendix H) and H. hystricis (GenBank accession no.: KC170733) (Figure 4.10, Appendix H). Two
sequence types of H. wellingtoni were observedof in 14 ticks [13 and one ticks showing
100 % (228/228 bp) and 99 % (227/228 bp) identity, respectively]. For H. hystricis, three
sequence types were observed for six ticks, showing 97 % (237/246 bp and 238/246 bp)
and 99 % (232/233 bp), respectively to H. hystricis (GenBank accession no.: KC170733).
Five H. bispinosa collected shared 100 % sequence similarity (287/287 bp) with that of
H. bispinosa reported in India (GenBank accession no.: KC853418- KC853420) and H.
bispinosa collected from animal farms (section 4.3.1.2).
UniversityAdditionally, two ticks collected from a dog in a rural village in Perak were identified as Dermacentor auratus. The tick mitochondrial 16S rRNA gene sequences demonstrated
the highest sequence similarity (99 %, 290/292 bp) to that of Dermacentor auratus
(GenBank accession no.: KC170746) reported in Thailand.
91
Figure 4.9: A dorsal view (left) and ventral view (right) of H. wellingtoni (SP016F1) collected from a chicken in Perak.
Dorsal view: scale bar: 500 µm; magnification: 10X, zoom: 2.5X. Ventral view: Scale bar: 200 µm; magnification: 10X, zoom: 3.2X.
Malaya of
Figure 4.10: H. hystricis (SP007) collected from a dog in Perak. UniversityScale bar: 1000 µm; magnification: 10X, zoom: 1X.
92 4.3.1.4 Collection of ticks from a forest reserve
A total of 100 ticks (61 Dermacentor spp., 35 Haemaphysalis spp. and four
Amblyomma spp.) were collected from the vegetation in Kuala Lompat forest reserve,
Pahang. DNA were extracted from 76 ticks (48 Dermacentor spp., 26 Haemaphysalis
spp., and two A. testudinarium spp.) (Appendix I) while the remaining ticks were served
as voucher specimens. In the vegetation, the ticks were usually found to be residing on
the tips and behind the leaves along the jungle trail. Small mammals were also captured
and a total of 22 ticks (15 Haemaphysalis spp., six Dermacentor spp. and one A.
helvolum) were collected from ten rats, a bat, a squirrel and a skink.
A total of 30 Dermacentor ticks were identified based on BLAST analyses of the 16S
rRNA gene sequences. The sequences for 19 Dermacentor ticks collected from vegetation demonstrated the highest sequence similarityMalaya of 92-93 % (218/233 bp, 218/233 bp, 220/234 bp, 221/234 bp) to that of Dermacentor andersoni (GenBank accession no.:
AF309032). The 16S rRNA gene sequencesof of another nine Dermacentor ticks (six ticks
collected from vegetation and three ticks collected from rodents) showed the highest
sequence similarity of 99 % (218/239 bp, 230/231 bp) to 100 % (229/229 bp, 239/239
bp) to that of Dermacentor atrosignatus (GenBank accession no.: KC170745). The 16S
rRNA gene of two Dermacentor ticks collected from vegetation showed the highest
sequence similarity of 93 % (210/227 bp and 222/239 bp, respectively) to that of D.
nuttalli (Appendix I). In total, there were five 16S rRNA sequence types identified for Universityboth D. andersoni and D. atrosignatus, respectively (Appendix R and appendix S). For Haemaphysalis ticks, a total of 16 ticks were randomly selected for tick 16S rRNA
sequence determination. BLAST analyses showed one tick with the highest sequence
similarity of 93 % (220/236 bp) to that of Haemaphysalis qinghaiensis (GenBank
accession no.: KJ609201); two ticks with the highest sequence similarity of 99 %
93 (237/239 bp) to that of H. hystricis (GenBank accession no.: KC170733); and ten ticks
with the highest sequence similarity of 94 % (222/234 bp, 222-223/236 bp) to that of
Haemaphysalis obesa (GenBank accession no.: KC170732). Hence, four sequence types
were identified based on the 16S rRNA gene sequences of H. obesa. Two Haemaphysalis
ticks collected from a bat and a rodent showed the highest sequence similarity of 99 % to
100 % (230/231 bp; 233/233 bp) to that of H. bispinosa (GenBank accession no.:
KC853419). The 16S rRNA gene sequence of one tick collected from a rodent exhibits
the highest sequence similarity [93 % (223/239 bp)] to that of Haemaphysalis asiatica
(GenBank accession no.: KC170734).
The 16S rRNA gene sequences of two Amblyomma ticks collected from vegetation
had the highest sequence similarity [97% (232/239 bp)] to that of A. testudinarium (GenBank accession no.: KC170737). A. helvolum wereMalaya identified from a skink with 100 % (236/236 bp) identity. The lists of the ticks collected from vegetation and animals are
available in Appendix I and J. of
4.3.1.5 Collection and identification of snake ticks
Twenty-one adult ticks (11 A. varanense and 10 A. helvolum) were collected from
seven Python molurus snakes at Sepang (2°49′10.862″N, 101°44′1.262″E) from 1st to 8th
September 2012. Of the 21 ticks collected, 11 were identified morphologically as A.
varanense (three females, eight males) whereas 10 female ticks were identified as A.
helvolum based on a previous publication (Chao et al., 2013). The A. varanense ticks had Universitybrown scutum with metallic yellowish-green ornamentation (Figure 4.11). Besides, a pool of six ticks collected from a wild snake (Spitting cobra) in a rural village in Johor
on 29 November 2012 were identified as A. helvolum.
94
Figure 4.11: The dorsal view of A. varanense (S3) with metallic yellowish-green ornamentation on the scutum.
The ventral view was not shown as the legs were damaged. Scale bar: 500 µm; magnification: 10X, zoom: 2.5X. Malaya 4.3.2 Collection and identification of fleas 4.3.2.1 Collection and identification of fleasof from urban areas A total of 162 and 48 fleas collected from 18 strayed cats in Dewan Bandaraya Kuala
Lumpur (DBKL) animal shelter and Titiwangsa housing area in Kuala Lumpur,
respectively, were included in this study. Out of 162 fleas collected in DBKL animal
shelter, there were 121 female fleas and 41 male fleas. Meanwhile, 46 female fleas and
two male fleas were identified in the fleas collected from Titiwangsa. All of the fleas were
morphologically identified as C. felis felis. Briefly, C. felis felis is characterized by a long, Universityacutely angled frons (Figure 4.12) while C. felis orientis is characterized by a short, rounded frons (Figure 4.13).
95
Figure 4.12: Female C. felis felis (C006F, left) and male C. felis felis (C006M, right) captured from a dog in a rural village, Kg. Orang Asli Semangar Dalam, Johor. Scale bar: 500 µm; magnification: 10X, zoom: 2.5X.
Malaya
of Figure 4.13: Female C. felis orientis (SD010F, left) and male C. felis orientis (SD010M, right) captured from a dog in a rural village, Kg. Orang Asli Semangar Dalam, Johor. Scale bar: 500 µm; magnification: 10X, zoom: 2X.
4.3.2.2 Collection and identification of fleas from rural villages
A total of 201 fleas were collected from 77 pet animals (cats and dogs) in nine rural
villages located in seven states (Selangor, Negeri Sembilan, Pahang, Kedah, Kelantan, UniversityJohore, and Perak) (Appendix L). These fleas were identified as C. felis felis (n=52) and Ctenocephalides felis orientis (n=159). The identity of the fleas in both urban and rural
areas were confirmed based on sequence analyses of the CoxI and CoxII genes performed
by Mdm. Lailatul Insyirah as part of her postgraduate study.
96 4.3.3 Collection of other arthropod samples
(i) Haematobia exigua
The DNA extracts of 53 H. exigua (buffalo flies) collected from animal farms (Farm
2, 3, 6 and 8) were included in this study. The species of the buffalo flies were identified
by Dr. Low Van Lun. Another 22 H. exigua collected from an animal farm in Penang
were also included in this study.
(ii) Mosquitoes
The DNA extracted from 165 Ae. albopictus collected around Selangor and Kuala
Lumpur were subjected for rickettsial screening. Another 70 DNA extracts from Culex
quinquefasciatus collected from various locations (Kuala Terengganu, Melaka, Johor, Selangor, Pahang, Penang, Kelantan, Kuala Lumpur,Malaya Negeri Sembilan, Kedah, Perlis, Perak, Sarawak, Sabah) were also included ofin this study. 4.3.4 Identification of rickettsial organisms in ticks
4.3.4.1 Determination of the type and distribution of rickettsiae in ticks collected
from urban areas
The overall rickettsial detection rate in R. sanguineus collected from urban areas was
16.3 % (21/129). Rickettsial DNA (either gltA, ompA or ompB gene fragments) was
amplified from 13 (39.4 %) of 33 Rh. sanguineus dog ticks in two animal shelters (SPCA
and Second Chance) collected from March to May 2013 (Figure 4.14, Figure 4.15, and
UniversityFigure 4.16). Of the six randomly selected rickettsial-positive Rh. sanguineus samples,
three gltA, four ompA and five ompB were obtained for sequence analyses. BLAST results
revealed the highest sequence similarity (98 %, 369/375 bp) similarity of gltA sequences
to that of Rickettsia sp. RF2125 (GenBank accession no.: AF516333) from one tick, and
97 99 % (370/375 bp) similarity to those of R. conorii type strain/R. raoultii strain
Khabarovsk (GenBank accession no.: DQ365804) from two ticks.
Figure 4.14: Agarose gel electrophoretogram of representative gltA fragments ( 490 bp) amplified from Rh. sanguineus DNA samples.
Lane M: 100 bp DNA ladder (Solis Biodyne, Estonia);Malaya Lane 1: sample SP02a; Lane 2: sample SP02b; Lane 3: sample SP05a; Lane N: DNA blank (distilled water); Lane P: positive control (R. conorii DNA extracted offrom a commercial IFA slide).
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Figure 4.15: Agarose gel electrophoretogram of representative PCR-amplified ompA fragments ( 532 bp) amplified from Rh. sanguineus DNA samples.
Lane M: 100 bp DNA ladder (Solis Biodyne, Estonia); Lane 1: sample SP02a; Lane 2: sample SP02b; Lane 3: sample SP05b; Lane 4: sample SC01a; Lane 5: sample SC03d; Lane N: DNA blank (distilled water); Lane P: positive control (R. conorii DNA extracted from a commercial IFA slide).
98
Figure 4.16: Agarose gel electrophoretogram of representative PCR-amplified ompB fragments ( 855 bp) amplified from Rh. sanguineus DNA samples.
Lane M: 100 bp DNA ladder (Solis Biodyne, Estonia); Lane 1: sample SP01b; Lane 2: sample SP02a; Lane 3: sample SP02b; Lane 4: sample SP04a; Lane 5: sample SP04b; Lane 6: sample SP05b; Lane 7: sample SP06a; Lane 8: sample SC01a; Lane N: DNA blank (distilled water); Lane P: positive control (R. conorii DNA extracted from a commercial IFA slide). Malaya of Sequence analyses of the partial ompA gene fragments from four ticks revealed highest
sequence similarity (99 %, 512/518 bp) to that of R. heilongjiangensis. The amplified
ompB fragments derived from three dog ticks showed the highest sequence similarity (99
%, 790/802 bp) to that of R. raoultii strain Khabarovsk (GenBank accession no.:
DQ365798) and, in two ticks, the sequences had the highest sequence similarity (99 %,
752/756 bp and 100 %, 756/756 bp, respectively) to that of Rickettsia sp. RF2125
(GenBank accession no.: JX183538). Table 4.8 summarizes the BLAST results of the Universityrickettsia-positive Rh. sanguineus dog ticks.
A total of eight Rh. sanguineus ticks (8.3 %) of the 96 Rh. sanguineus ticks collected
from DBKL dogs were positive for rickettsial DNA. None of the tick sample was positive
upon amplification for gltA gene region. Only one Rh. sanguineus tick (D6003) was
positive upon amplification for ompA and the sequence showed the highest sequence
99 similarity of 99 % (470/475 bp) to that of Rickettsia sp. HL-93 strain HL-93 (GenBank
accession no.: AF179364, also known as Rickettsia hulinii). OmpB gene was successfully
amplified from seven Rh. sanguineus dog ticks. OmpB genes of four dog ticks, including
three fed ticks and an unfed tick, were positive for R. conorii strain Malish 7 (GenBank
accession no.: AE006914) with 99 % sequence similarity while OmpB genes for two
unfed dog ticks demonstrated the highest sequence similarity of 99 % to 100 % similarity
to that of Rickettsia sp. RF2125 (GenBank accession no.: JX183538) (Table 4.8).
Table 4.8: BLAST analyses of the rickettsia-positive Rh. sanguineus dog ticks.
Ticks BLAST analyses (closest relative) ID gltA ompA ompB R. raoultii strain SP01b, R. heilongjiangensis Khabarovsk [DQ365 SP04a negative [CP002912; 511/518 798, 790/802 bp (99 (dog) bp (99 %)] Malaya%)] R. raoultii strain Rickettsia sp. RF2125 SP02a Khabarovsk [DQ365 [AF516333, 369/375 bp negative (dog) 798, 790/802 bp (99 (98 %)] of %)] R. heilongjiangensis Rickettsia sp. SP02b negative [CP002912; 511/518 RF2125 [JX183538, (dog) bp (99 %)] 752/756 bp (99 %)] R. conorii type strains/ Rickettsia R. raoultii strain Rickettsia sp. SP05a heilongjiangensis Khabarovsk RF2125 [JX183538, (dog) [CP002912; 511/518 [DQ365804, 369/375 bp 756/756 bp (100%)] bp (99 %)] (98 %)] R. conorii type strains/ SP05b R. raoultii strain negative negative (dog) Khabarovsk [DQ365804, 369/375 bp (98 %)] Rickettsia sp. HL-93 D6003 strain HL-93 negative negative University(dog) [AF179364; 470/475 bp (99 %)] R. conorii strain SF006, Malish 7 SF011 negative negative [AE006914; 800/801 (unfed) bp (99 %)] R. conorii strain D6001 Malish 7 negative negative (dog) [AE006914; 800/801 bp (99 %)]
100 Table 4.8, continued
Ticks BLAST analyses (closest relative) ID gltA ompA ompB R. conorii strain D2007 negative negative Malish 7 [AE006914; (dog) 798/801 bp (99 %)] R. conorii strain D3008 negative negative Malish 7 [AE006914; (dog) 740/741 bp (99 %)] Rickettsia sp. RF2125 SF023 negative negative [JX183538, 749/750 (unfed) bp (99 %)] Rickettsia sp. RF2125 SF034 negative negative [JX183538, 750/750 (unfed) bp (100 %)]
4.3.4.2 Determination of the type and distribution of rickettsiae in ticks collected from animal farms Malaya Rickettsial DNA was detected in 25 (9.3 %) ticks (21 H. bispinosa and four Rh. microplus) of 270 ticks collected from fourof farms (Farms 1, 3, 4, and 6 in Negeri Sembilan, Pahang, Kedah and Terengganu, respectively). The detection rates for
rickettsial DNA in the cattle ticks ranged from 2.1 % to 27.7 % (Table 4.9). A total of 42
rickettsial sequences (20 gltA, 7 ompA and 15 ompB) were successfully obtained from
cattle ticks and analyzed. Sequence analyses of the gltA fragments (375 bp) from 20 ticks
revealed the identification of rickettsiae closely related to R. raoultii (n=15), R.
heilongjiangensis (n=2), Rickettsia sp. RF2125 (n=1), R. tamurae (n=1), and Rickettsia
sp. TCM1 (n=1) with the highest sequence similarity ranging from 97 % to 99 %. There Universityare four gltA sequences types of R. raoultii obtained from this study. BLAST analyses of ompA (518 bp) gene sequences in seven cattle ticks indicate the identification of a
rickettsia closely related to R. heilongjiangensis with 99 % (500/509 bp) sequence
identity. Of the 15 ompB sequences (774 bp - 826 bp) obtained, nine and six matched
those of R. raoultii (93 %, 740/800 bp) and Rickettsia sp. RF2125 (790/790 bp),
101 respectively. Detailed BLAST analyses of rickettsial gltA, ompA and ompB gene
fragments are shown in Table 4.10.
A potentially novel rickettsia (closely related to R. raoultii) was identified from 12 H.
bispinosa from three cattle farms (Negeri Sembilan, Kedah and Terengganu). The gltA
sequences obtained from these ticks demonstrated the highest sequence similarity (98 %)
to that of R. raoultii (also known as Rickettsia sp. Kagoshima6, GenBank accession no.:
JQ 697956) detected from H. hystricis in Japan. The ompB sequences amplified from
most of the ticks showed the highest sequence similarity (93 %, 740/800 bp) similarity to
that of R. raoultii strain Khabarovsk (GenBank accession no.: DQ365798), whereas the
rickettsial ompA gene was not amplifiable. Table 4.9: Detection rates of rickettsiae in animalMalaya farms across different states. Farms Animal host Ticks species (n) No. (%, 95 % CI) of examined (n) rickettsia-positive ticks Negeri Sembilan Cattle (30) H.of bispinosa (41); 13 (27.7, 14.4-40.9 %) (Farm 1) Rh. microplus (6) Pahang (Farm 2) Cattle (39) Rh. microplus (14) 0 (0.0, 0.0-0.0 %) Pahang (Farm 3) Cattle (40) H. bispinosa (57); 2 (2.1, 0.0-5.1 %) Rh. microplus (37) Kedah (Farm 4) Sheep (40) H. bispinosa (44) 7 (15.9, 4.7-27.2 %)
Kelantan (Farm 5) Cattle (40) Not determined - Terengganu (Farm 6) Cattle (40) H. bispinosa (58); 3 (4.2, 0.0-9.0 %) Rh. microplus (13) Negeri Sembilan Goat (40) 0 0 (0.0, 0.0-0.0 %) (Farm 7) Johore (Farm 8) Dairy cattle (41) 0 0 (0.0, 0.0-0.0 %) UniversityTotal 270 individual ticks 25 (9.3, 5.8-12.7 %)
102 Table 4.10: BLAST analyses of rickettsial gltA, ompA and ompB gene fragments of ticks from animal farms.
Tick species positive BLAST analyses (closest relative) Farms for Rickettsia (n; gltA [n] ompA [n] ompB [n] host) R. raoultii strain R. raoultii [JQ697956; 366/375 negative Khabarovsk [DQ365798; bp (98 %)]; [6] 740/800 bp (93 %)]; [5] R. heilongjiangensis type strains [393-373/375 bp (99 negative negative %)]; [1] Malaya H. bispinosa (11; R. heilongjiangensis type R. heilongjiangensis Rickettsia sp. RF2125 cattle) strains [373/375 bp (99 %)]; [CP002912; 500/506 bp [JX183538, 790/790 bp Negeri Sembilan [1] of (99 %)]; [1] (100 %)]; [1] (Farm 1) R. heilongjiangensis Rickettsia sp. RF2125 negative [CP002912; 500/506 bp [JX183538, 790/790 bp (99 %)]; [3] (100 %)]; [2]
R. heilongjiangensis Rickettsia sp. RF2125 Rh. microplus (2; negative [CP002912; 500/506 bp [JX183538, 790/790 bp cattle) (99 %)]; [2] (100 %)]; [2]
R. conorii type strains /R. R. heilongjiangensis Rh. microplus (2; raoultii type strains Pahang (Farm 3) [CP002912; 500/506 bp negative cattle) [370-375/375 bp (99-100 %)]; (99 %)]; [1] [2]
103 103 University
Table 4.10, continued
Tick species positive BLAST analyses (closest relative) Farms for Rickettsia (n; ompA [n] gltA [n] ompB [n] host) negative R. raoultii strain Khabarovsk [DQ365798; R. raoultii [JQ697956; 366/375 740/800 bp (93 %)]; [3] bp (98 %)]; [5] Rickettsia sp. RF2125 [JX183538, 790/790 bp H. bispinosa (7; Malaya (100 %)]; [1] Kedah (Farm 4) sheep) R. tamurae strain AT-1 negative [AF394896; 365/375 bp (9 negative 7%)]; [1] Rickettsia sp. TCM1of negative [B359458; 374/375 bp (99 %)], negative [1] negative R. raoultii strain R. raoultii [JQ697956; 366/375 Khabarovsk [DQ365798; bp (98 %)]; [1] 740/800 bp (93 %)]; [1] Rickettsia sp. RF2125 negative negative H. bispinosa (3; Terengganu (Farm 6) [AF516333, 392/397 bp (99 cattle) %)]; [1] R. conorii type strains /R. negative negative raoultii type strains, [375/375 bp (100 %)]; [1]
104 University
4.3.4.3 Determination of the type and distribution of rickettsiae in ticks collected
from rural villages
Of 186 ticks (64 pools) collected from 47 peri-domestic animals in the rural villages,
rickettsial DNA was amplified from 40.6 % (26/64) of the ticks (23 pools Haemaphysalis
spp., one Rh. sanguineus, and two Rh. microplus). Rickettsia positive ticks were
identified from nine villages in all the six states studied, with the detection rates ranging
from 14.3 % to 66.7 % (Table 4.11). Table 4.12 shows the BLAST results obtained in
this study. A total of 43 sequences (13 gltA, 11 ompA, and 19 ompB) were analyzed. The
gltA fragments amplified from a Haemaphysalis cat tick in a rural village in Johore
showed the highest sequence similarity (98 %, 366/375 bp) to that of R. tamurae (Table
4.12). BLAST analyses of rickettsial gltA and ompB partial fragments from two ticks (a Rh. microplus and a Haemaphysalis sp., from Kedah andMalaya Perak, respectively) showed the identification of a rickettsia closely related to R. felis URRWXCal2 (99 % sequence similarity). The gltA and ompB sequences ofobtained from nine and five Haemaphysalis ticks, respectively, from Kelantan and Johore, were identical to that of Rickettsia sp.
RF2125. OmpB sequences resembling R. raoultii Khabarovsk (GenBank accession no.:
DQ365798) were identified from 11 ticks (one Rh. sanguineus, one Rh. microplus and
nine Haemaphysalis spp.) from four states (Negeri Sembilan, Pahang, Kedah and Perak).
Based on BLAST analyses of ompA gene fragments, a rickettsia closely related to R.
heilongjiangensis was identified from 11 Haemaphysalis ticks in Kelantan and Johore. UniversityAdditionally, a rickettsia identified from a Haemaphysalis cat tick from Kelantan shared 100 % sequence similarity with the gltA sequence of Rickettsia sp. LON-13 [GenBank
accession no.: AB516964, (Fujita, 2008)] whereas the ompB sequence derived from the
tick resembled that of Rickettsia hulinensis (GenBank accession no.: AY260452).
105 Table 4.11: Detection rates of rickettsiae across rural villages in different states.
Rural Animal host Ticks species (n) No. (%, 95 % CI) of villages examined (n) ticks with rickettsia detection Negeri Cat (12), chicken Rh. sanguineus, 4 (57.1, 7.7-100.0 %) Sembilan (1), dog (40), goat Haemaphysalis spp. (3 (8) individuals and 4 pools) Pahang Cat (18), chicken Haemaphysalis spp. (7 1 (14.3, 0.0-49.2 %) (5), dog (21), pools) Kedah Cat (17), chicken Rh. microplus (3 2 (66.7,0.0-100.0 %) (1), cattle (3), dog individuals) (16) Kelantan Cat (27), chicken Haemaphysalis spp. (10 7 (63.6, 29.7-97.5 %) (9), dog (4) individuals and one pool) Johore Cat (23), chicken Haemaphysalis spp. (one 5 (55.6, 15.0-96.1 %) (2), dog (15) individual and 8 pools) Perak Cat (8), chicken Haemaphysalis spp. (16 7 (25.9, 8.3-43.6 %) (2), dog (10) individuals and 11 pools) Total 33 individuals and 31 pools 26 (40.6, 28.3-53.0 %) Malaya
of
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106 Table 4.12: BLAST analyses of rickettsial gltA, ompA and ompB gene fragments of ticks from rural villages.
Tick species positive BLAST analyses (closest relative) Rural area for Rickettsia (n; host) gltA [n] ompA [n] ompB [n] R. raoultii strain Khabarovsk Rh. sanguineus (1; dog) negative negative [DQ365798, 790/802 bp (99 %)]; [1] Negeri Sembilan Haemaphysalis spp. (2; 1 negative negative R. raoultii strain Khabarovsk chicken and dog, [DQ365798, 787-789/802 bp respectively) (98 %)]; [2] negative Malayanegative R. raoultii strain Khabarovsk Haemaphysalis spp. (1; Pahang [DQ365798, 787-787/802 bp chicken) (98 %)]; [1] R. felis URRWXCal2 negative R. felis URRWXCal2 [CP000053, 369/370 bpof (99 [CP000053, 807/808 bp (99 %)]; [1] %)]; [1] Kedah Rh. microplus (2; cattle) negative R. raoultii strain Khabarovsk negative [DQ365798, 790/802 bp (99 %)]; [1] Rickettsia sp. LON-13 negative R. hulinensis [AB516964, 375/375 bp (100 [AY260452, 776/802 bp (97 %)]; [1] %)]; [1] Haemaphysalis spp. (8; 4 Rickettsia sp. RF2125 R. heilongjiangensis Kelantan chickens and 4 cats, [AF516333, 373-374/376 bp [CP002912; 500/506 bp negative respectively) (99 %)]; [6] (99 %)]; [5] R. heilongjiangensis negative [CP002912; 500/506 bp negative (99 %)]; [1]
107 University
107 Table 4.12, continued
Tick species positive BLAST analyses (closest relative) Rural area for Rickettsia (n; host) gltA [n] ompA [n] ompB [n] R. heilongjiangensis Rickettsia sp. RF2125 R. tamurae [AB812551, [CP002912; 500/506 bp (99 [JX183538, 756/756 bp 366/375 bp (98 %)]; [1] %)]; [1] (100 %)]; [1]
R. heilongjiangensis Rickettsia sp. RF2125 Rickettsia sp. RF2125 [CP002912; 500/506 bp (99 Haemaphysalis spp. (5; 2 [AF516333, 367/376 bp (98 [JX183538, 756/756 bp Malaya%)]; [1] Johore dogs, a cat and chicken, %)]; [1] (100 %)]; [1]
respectively) Rickettsia sp. RF2125 R. heilongjiangensis Rickettsia sp. RF2125 [AF516333, 373/376 bp (99 [CP002912; 500/506 bp (99 [JX183538, 756/756 bp %)]; [2] of %)]; [2] (100 %)]; [2] R. heilongjiangensis Rickettsia sp. RF2125 negative [CP002912; 500/506 bp (99 [JX183538, 756/756 bp %)]; [1] (100 %)]; [1] R. felis URRWXCal2 R. felis URRWXCal2 [CP000053, 369/370 bp (99 negative [CP000053, 807/808 bp (99 %)]; [1] %)]; [1] Haemaphysalis spp. (7; 2 Perak R. raoultii strain dogs and 5 cats) Khabarovsk negative negative [DQ365798, 789-790/802 bp (98-99 %)]; [6] N/A- not available
108 University
108 4.3.4.4 Determination of the type and distribution of rickettsiae in ticks collected
from a forest reserve
In the Kuala Lompat forest reserve, 22 feeding ticks collected from 12 small mammals
and a skink were screened for rickettsial DNA. A total of 31.8 % (7/22) of the feeding
ticks were positive upon amplification targeting either one or more genes of gltA, ompA
and ompB. Detailed BLAST results for rickettsia detected in ticks collected from Kuala
Lompat forest reserve are shown in Table 4.13. A gltA sequence amplified from a
Haemaphysalis tick collected from a rodent (R0008) was identical to those of R.
raoultii/R. heilongjiangensis/Rickettsia sp. TCM1 (286/286 bp). The two gltA sequences
amplified from two Dermacentor ticks (R0011-1 and R0011-2) collected from a rodent
showed the highest sequence similarity (99 %) to that of Rickettsia sp. asiatica (282/286 bp, GenBank accession no.: AF394901). Another twoMalaya gltA sequences amplified from a Dermacentor atrosignatus and a Haemaphysalis sp. tick (R002-1 and R002-3) collected from a rodent (R002) exhibited 100 % identityof to Rickettsia sp. RF2125 (286/286 bp, GenBank accession no.: AF216333). The ompB sequence of the Haemaphysalis sp. tick
(R002-3) showed 100 % sequence identity to that of Rickettsia sp. RF2125 (754/754 bp,
GenBank accession no.: JX183538).
The gltA (286/286 bp, 100 %), ompA (459/463 bp, 99 %) and ompB (770/775 bp, 99
%) sequences obtained from a Haemaphysalis bispinosa tick collected from a rodent
(R0019) exhibited the highest sequence similarities to R. heilongjiangensis (GenBank Universityaccession no.: CP002912). The only Amblyomma tick collected from a skink (L001) was positive for a rickettsia closely related to R. raoultii strain Khabarovsk (GenBank
accession no.: CP010969), as its gltA, ompA and ompB gene sequences were 100 %
(286/286 bp), 97.6 % (453/464 bp) and 98.3 % (762/775 bp) similar, respectively, to the
rickettsial species.
109 Table 4.13: BLAST results for rickettsial gltA, ompA and ompB gene fragments of ticks collected from Kuala Lompat forest reserve.
BLAST analyses (closest relative) Sample code/ID (Tick species) gltA ompA ompB Ticks collected from wildlife R0002-1 Rickettsia sp. RF2125 (AF516333, negative negative (D. atrosignatus) 286/286 bp (100 %)) R0002-3 Rickettsia sp. RF2125 (AF516333, Rickettsia sp. RF2125 (JX183538, negative (H. obesa) 286/286 bp (100 %)) 754/754 bp (100 %)) R. raoultii (DQ365803)/R. R0008 heilongjiangensis (CP002912)/ Malaya negative negative (Haemaphysalis sp.) Rickettsia sp. TCM1 (AB359458) (286/286 bp (100 %)) R0011-1 and R0011-2 Rickettsia sp. asiatica (AF394901, of (Dermacentor sp. and D. negative negative 282/286 bp (99 %)) atrosignatus) R0019 R. heilongjiangensis (CP002912, R. heilongjiangensis (CP002912, R. heilongjiangensis (CP002912, (H. bispinosa) 286/286 bp (100 %)) 459/463 bp (99 %)) 770/775 bp, (99 %)) L001 R. raoultii (CP010969, 286/286 bp R. raoultii (CP010969, 453/464 bp R. raoultii (CP010969, 762/775 bp (A. helvolum) (100 %)) (98 %)) (98 %)) Ticks collected from vegetation KLV008 Rickettsia sp. TCM1 (AB359459, negative negative (Dermacentor sp.) 491/491 bp (100 %)) Rickettsia sp. RF2125 (JX183538, Rickettsia sp. RF2125 (AF516333, KLV014 (Haemaphysalis sp.) negative 685/690 bp (99 %)) -low sequence 285/286 bp (99 %)) quality R. raoultii (DQ365803)/R. Rickettsia sp. RF2125 (JX183538, heilongjiangensis (CP002912)/ KLV023 (Haemaphysalis sp.) negative 586/589 bp (99 %)) -low sequence Rickettsia sp. TCM1 (AB359458) quality 110 University(281/286 bp (98 %))
110 Table 4.13, continued
BLAST analyses (closest relative) Sample code/ID gltA ompA ompB Rickettsia sp. TCM1 (AB359459, KLV027 (Haemaphysalis sp.) negative negative 491/491 bp (100 %)) Rickettsia sp. RF2125 (JX183538, KLV032 (H. obesa) negative negative 754/754 bp (100 %)) Rickettsia sp. RF2125 (AF516333, KLV035 (H. obesa) negative negative 281/286 bp (99 %)) MalayaRickettsia sp. RF2125 (JX183538, KLV040 Rickettsia sp. TCM1 (AB359459, negative 586/589 bp (99 %)) -low sequence (D. andersoni) 491/491 bp (100 %)) quality KLV046 Rickettsia sp. TCM1 (AB359459, Rickettsia sp. RF2125 (JX183538, negative of (D. atrosignatus) 490/491 bp (99 %)) 754/754 bp (100 %)) KLV052 Rickettsia sp. RF2125 (JX183538, negative negative (Dermacentor sp.) 536/536 bp (100 %)) KLV053 Rickettsia sp. RF2125 (AF516333, Rickettsia sp. TCM1 (AB359459, negative (D. andersoni) 286/286 bp (100 %)) 491/491 bp (100 %)) KLV060 Rickettsia sp. TCM1 (AB359459, negative negative (D. atrosignatus) 491/491 bp (100 %)) KLV062 Rickettsia sp. RF2125 (JX183538, negative negative (Dermacentor sp.) 754/754 bp (100 %)) KLV064 Rickettsia sp. TCM1 (AB359459, negative negative (D. nuttalli) 490/491 bp (99 %)) KLV070 R. raoultii (CP010969, 282/286 bp negative negative (D. andersoni) (99 %)) KLV074 R. raoultii (CP010969, 767/775 bp negative negative (D. andersoni) (99 %))
111 111 University
111 Table 4.13, continued
BLAST analyses (closest relative) Sample code/ID gltA ompA ompB KLV078 Rickettsia sp. TCM1 (AB359458) Rickettsia sp. TCM1 (AB359459, negative (D. andersoni) (286/286 bp (100 %)) 491/491 bp (100 %)) R. raoultii (CP010969)/ R. conorii KLV091 Rickettsia sp. RF2125 (JX183538, (AE006914)/R. honei (U59726) negative (D. andersoni) 753/753 bp (100 %)) 279/286 bp (99 %)) KLV098 R. tamurae (AF394896, 273/283 bp negative negative (A. testudinarium) (97 %)) Malaya KLV099 R. tamurae (AF394896, 273/283 bp negative negative (A. testudinarium) (97 %)) of
112 University
112 Of the 76 questing ticks (48 Dermacentor spp., 26 Haemaphysalis spp., and two
Amblyomma spp.) collected from the vegetation, 25 ticks (32.9 %) were PCR-positive to
either one or more of the three genes screened in this study (gltA, ompA and ompB). A
total of 19 rickettsia-positive tick samples were sequenced. From the nine gltA amplicons
sequenced, three sequences (KLV014, KLV035 and KLV053) shared 98-100 % identities
to that of Rickettsia sp. RF2125 (GenBank accession no.: AF516333), two sequences
(KLV098 and KLV099) shared 97 % identities to that of R. tamurae (GenBank accession
no.: AF394896) and a sequence (KLV070) shared 99 % identities to R. raoultii (GenBank
accession no.: CP010969). The gltA sequences of three samples showed highest identity
to those of R. raoultii/R. heilongjiangensis and Rickettsia sp. TCM1 (KLV023 and
KLV078) and R. raoultii/ R. conorii/R. honei (KLV091), respectively.
Eight ompA amplicons were sequenced and showedMalaya highest sequence similarity of 99 %- 100 % to Rickettsia sp. TCM1 (GenBank accession no.: AB359459). A total of nine
representative ompB amplicons were sequencedof of which eight sequences showed the
highest sequence similarity (99-100 %) to that of Rickettsia sp. RF2125 and one sequence
showed 99 % sequence similarity to R. raoultii.
4.3.4.5 Determination of the type and distribution of rickettsiae in ticks collected
from snakes
Rickettsial DNA was detected from four (19 %) of 21 Amblyomma ticks (S1-S7)
collected in Sepang. A pool of Amblyomma ticks (P1) collected from Johor was also
Universitypositive in the PCR detection of rickettsial DNA.
Rickettsial gltA gene was amplified from three A. varanense (S5, S4-2 and S7-2) and
an A. helvolum tick (S6-1). The gltA and ompA sequences from the S5 tick was almost
similar (99.0 % and 97.7 %, respectively) with R. tamurae strain AT-1 from A.
113 testudinarium tick in Japan (Fournier et al., 2006). However, the ompB and Sca4 genes
of the rickettsiae were unable to be amplified (Table 4.14).
Table 4.14: Molecular detection of rickettsiae and BLAST analyses of the sequences derived from snake tick samples in this study.
Tick sample BLAST analyses (closest relative) (Species, gltA ompA ompB sca4 location) Candidatus Rickettsia sepangensis R. tamurae strain R. tamurae strain S5 (A. AT-1 No significant AT-1 (AF394896) varanense, (DQ103259) negative similarity in (1033/1043, 99.0 Sepang) (417/427, 97.7 BLAST %) %) Candidatus Rickettsia johorensis P1 (pooled A. helvolum, R. raoultii R. raoultii Johore), R. raoultii strain R. raoultii strain strain strain Khabarovsk Khabarovsk Khabarovsk Khabarovsk S4-2 (A. (DQ365804) (DQ365801) Malaya (DQ365798) (DQ365808) varanense, (1057/1060, 99.7 (418/429, 97.4 (762/775, 98.3 (795/816, 97.4 Sepang), S6-1 %) %) %) %) (A. helvolum, of Sepang) R. raoultii R. raoultii R. raoultii strain R. raoultii strain strain strain S7-2 (A. Khabarovsk Khabarovsk Khabarovsk Khabarovsk varanense, (DQ365804) (DQ365801) (DQ365798) (DQ365808) Sepang) (1057/1060, 99.7 (418/429, 97.4 (762/775, 98.3 (795/816, 97.4 %) %) %) %) The sequences obtained for rickettsiae from S5 and P1 ticks have been deposited in the GenBank database under the accession numbers: [gltA (GenBank: KJ769648, KJ769650), ompA (GenBank: KJ769649, KJ769651), ompB (GenBank: KJ769652), sca4 (GenBank: KM977711)]. UniversityBLAST analyses of the rickettsial gltA sequence from two samples (an individual and a pool) of A. helvolum (S6-1, P1) and two A. varanense (S4-2 and S7-2) ticks
demonstrated the closest match (99.7 %) to R. raoultii strain Khabarovsk (Table 4.14),
which was cultivated from Dermacentor ticks in Russia and France (Mediannikov et al.,
2008). The sequence similarity of the ompA, ompB and sca4 sequences of these ticks with
those of R. raoultii strain Khabarovsk were 97.4 %, 98.3 % and 97.4 %, respectively.
114 Based on the current criteria for speciation of rickettsial species (Raoult et al., 2005),
the rickettsiae are tentatively named as Candidatus Rickettsia sepangensis and
Candidatus Rickettsia johorensis, respectively, in accordance to the location of their first
sample collection. The dendrogram constructed using concatenated sequence of gltA and
ompA gene fragments (Table 4.15,Figure 4.17) confirmed the clustering of Candidatus
Rickettsia sepangensis with the type strain of R. tamurae, and Candidatus Rickettsia
johorensis with the R. raoultii type strains.
Table 4.15: GenBank accession numbers of the rickettsial gene sequences used for the construction of a concatenated NJ tree.
GenBank accession no. for targeted Rickettsia sp. genes MalayagltA ompA R. raoultii strain Elanda-23/95 EU036985 EU036986 R. raoultii strain Khabarovsk DQ365804 DQ365801 R. raoultii strain Marne DQ365803 DQ365799 Rickettsia sp. DnS14 ofAF120028 AF120021 R. aeschlimannii AY259084 AY259083 R. massiliae Mtu 1 U59719 U43799 R. rhipicephali strain HJ5 DQ865206 DQ865208 R. parkeri KF782319 KF782320 R. sibirica 246 U59734 U43807 R. conorii U59730 U43806 R. honei AF018074 AF018075 R. rickettsii R (Bitterroot) U59729 U43804 Rickettsia montana U74756 U43801 R. tamurae strain AT-1 AF394896 DQ103259 Rickettsia sp. IRS3 AF140706 AF141909 Rickettsia sp. IRS4 AF141906 AF141911 UniversityCandidatus Rickettsia tasmanensis GQ223391 GQ223392 R. japonica YM U59724 U43795 R. heilongjiangensis strain CH8-1 AB473812 AB473813 Rickettsia sp. California 2 AF210692 AF210694 R. slovaca N.A. 13-B U59725 U43808 Candidatus Rickettsia sepangensis KJ769648 KJ769649 Candidatus Rickettsia johorensis KJ769650 KJ769651
115 Malaya
Figure 4.17: Phylogenetic placement of concatenated sequences (gltA and ompA) of known rickettsial speciesof in Table 4.15. Bootstraps analysis was performed with 1000 replications. Scale bar indicates the nucleotide substitutions per sites. * denote the rickettsiae identified from Amblyomma snake ticks in this study.
University
116 4.3.5 Identification of rickettsial organisms in fleas
4.3.5.1 Determination of the type and distribution of rickettsiae in fleas collected
from urban areas
From a total of 210 C. felis felis fleas examined, only 17 (8.1 %) of C. felis felis were
positive in the rickettsial gltA PCR assays; however, the sequences were not determined
due to the presence of insufficient amounts of amplified fragments.
4.3.5.2 Determination of the type and distribution of rickettsiae in fleas collected
from rural villages
A total of 66.2 % (133/201) fleas collected from rural villages were positive for
rickettsial DNA using both gltA and ompB PCR assays (Figure 4.18 and Figure 4.19).
Rickettsial-positive ticks were identified from nine villages in the six states studied except Kedah, with the detection rates ranging from 0.0 % toMalaya 96.9 % (Table 4.16). Due to the large number of rickettsia-positiveof flea samples, only 26 amplified gltA and ompB fragments from different hosts and geographical locations were selected for
sequence determination. The sequences were differentiated into two distinct types, of
which one was more closely related to R. felis URRWXCal2 [(99 %, 373/375 bp) for gltA
and 100 % (808/808 bp) for ompB sequences, GenBank accession no.: CP000053)]. The
other was more closely related to Rickettsia sp. RF2125 (99 %, 373/374 bp) for gltA
(GenBank accession no.: AF516333) and 100 % (756/756 bp) for ompB sequences
(GenBank accession no.: JX183538)]. BLAST analyses of the R. felis and RFLO
Universitysequences are summarized in Table 4.17.
117
Figure 4.18: Agarose gel electrophoretogram of representative PCR amplified gltA fragments ( 490 bp) from flea DNA samples.
Lane M: 100 bp DNA ladder (Solis biodyne, Estonia); Lane 1: sample SW002 (M1); Lane 2: sample SW002 (M2); Lane 3: sample SW008 (M1); Lane 4: sample SW009 (M1); Lane 5: sample JJ003 (F1); Lane 6: sample SD001 (F1); Lane 7: sample SD002 (M1); Lane 8: sample PP001 (M1); Lane 9: sample PP001 (F1); Lane 10: sample SP013 (M); Lane N: DNA blank; Lane P: positive control (R. conorii DNA extracted from a commercial IFA slide). Malaya of
Figure 4.19: Agarose gel electrophoretogram of representative PCR amplified ompB fragments ( 855 bp) from flea DNA samples.
Lane M: 100 bp DNA ladder (Solis biodyne, Estonia); Lane 1: sample SW002 (M1); Lane 2: sample SW002 (M2); Lane 3: sample SW008 (M1); Lane 4: sample SW009 University(M1); Lane 5: sample JJ003 (F1); Lane 6: sample SD001 (F1); Lane 7: sample SD002 (M1); Lane 8: sample PP001 (M1); Lane 9: sample PP001 (F1); Lane 10: sample SP013 (M); Lane 11: sample SP013 (F); Lane N: DNA blank; Lane P: positive control (R. conorii DNA extracted from a commercial IFA slide).
118 Table 4.16: Detection rates of rickettsiae from fleas collected in each locality.
No. of No. (%, 95% CI) of Animal host Localities Flea species fleas fleas with rickettsia (n) tested detection Urban areas DBKL Cat (18) C. felis felis 162 17 (10.5, 5.7-15.3 %) Titiwangsa Cat (18) C. felis felis 48 0 (0.0, 0.0-0.0) Total 210 17 (8.1, 4.4-11.9 %) Rural areas Selangor Dog (5) C. felis orientis 48 41 (87.2, 75.1-95.8 %) Negeri Dog (19), C. felis orientis, 36 26 (72.2, 56.9-87.6 %) Sembilan cat (3) C. felis felis Pahang Dog (16) C. felis orientis 26 15 (57.7, 37.3-78.0 %) Kedah Cat (7) C. felis felis 14 0 (0.0, 0.0-0.0) C. felis felis, C. Kelantan Cat (13) 26 7 (26.9, 8.7-45.2 %) felis orientis Johore Dog (9) C. felis orientis 32 31(96.9, 90.5-100.0 %) Dog (9), cat C. felis orientis, Perak Malaya19 13 (68.4, 45.4-91.4 %) (1) C. felis felis Total 201 133 (66.2, 59.6-72.8 %)
of
Table 4.17: BLAST analyses of the selected rickettsial gltA and ompB gene sequences from fleas from rural villages.
Location (No. of No. of BLAST analyses (closest relative) Rickettsia positive samples gltA ompB fleas) sequenced C. felis felis SW, Kelantan (5) 2 R. felis URRWXCal2 R. felis URRWXCal2 (CP000053, (CP000053, JJ, Kelantan (1) 1 373/375(99 %)) 808/808(100 %)) C. felis orientis Gombak, Selangor 4 (41) UniversityJJ, Kelantan (1) 1 SD, Johore (31) 5 Rickettsia sp. Rickettsia sp. PP, Pahang (5) 2 RF2125 (AF516333, RF2125 (JX183538, PS, Pahang (10) 2 373/375 (99 %)) 756/756 (100 %)) TH, Perak (7) 2 SP, Perak (6) 2 Negeri Sembilan 5 (26)
119 4.3.6 Identification of rickettsial organisms in other arthropod samples
(i) Buffalo flies
The DNA of 53 and 22 H. exigua (buffalo flies) collected from animal farms
(Farm 2, 3, 6 and 8) and an animal farm in Penang were screened for rickettsial gltA gene.
The DNA for three (4.0 %) fly samples were positive for rickettsia. The gltA sequences
obtained from two rickettsia-positive buffalo flies (J26 and J18) collected from the Johore
farm (Farm 8) demonstrated 99.7 % (324/325 bp) and 100 % (325/325 bp) similarity,
respectively, to that of R. raoultii strain Khabarovsk (GenBank accession no.: CP010969)
and Candidatus Rickettsia johorensis (GenBank accession no.: KJ769650). The
rickettsial gltA fragment amplified from a buffalo fly in Terengganu (Farm 6) generated
a low-quality sequence. The fly was negative in the amplification for rickettsial ompA gene and further characterization was unable to be carriedMalaya out due to insufficient DNA sample. of (ii) Mosquitoes
A total of 165 Ae. albopictus collected around Selangor and Kuala Lumpur were
screened for rickettsial DNA. Another 70 DNA samples obtained from Cx.
quinquefasciatus from 14 states (Kuala Terengganu, Melaka, Johor, Selangor, Pahang,
Penang, Kelantan, Kuala Lumpur, Negeri Sembilan, Kedah, Perlis, Perak, Sarawak,
Sabah) were also included in this study. All the DNA samples were negative upon Universityscreening for rickettsial gltA gene.
The geographical distribution of rickettsiae detected in arthropods (ticks, fleas and
buffalo flies) in Peninsular Malaysia is summarized in Table 4.18. An overview of the
seroprevalence and occurrence of rickettsia in human and arthropods across several states
of Peninsular Malaysia is presented in Figure 4.20.
120 Table 4.18: Geographical distribution of rickettsiae detected in arthropods and their animal hosts in each location.
Closely related Arthropods Host Location Rickettsia species Rickettsia asiatica Dermacentor spp. rodent Pahang R. conorii Rh. sanguineus dog Kuala Lumpur R. felis C. felis felis cat Kelantan URRWXCal2 Haemaphysalis spp. dog Perak Rh. microplus cattle Kedah Johore, Pahang, Perak, C. felis orientis cat Negeri Sembilan Negeri Sembilan, H. bispinosa cattle Terengganu H. bispinosa sheep Kedah Haemaphysalis spp. cat Kelantan, Johore
R. felis - like Haemaphysalis spp. chicken Kelantan, Johore organisms (RFLO) Haemaphysalis spp. dogMalaya Johore Haemaphysalis spp., vegetation Pahang Dermacentor spp. Rh. microplus of cattle Negeri Sembilan Rh. sanguineus dog Kuala Lumpur Dermacentor spp. rodent Pahang Haemaphysalis spp. rodent Pahang H. bispinosa cattle Negeri Sembilan Haemaphysalis spp. cat Kelantan, Johore Haemaphysalis spp. chicken Kelantan, Johore R. Haemaphysalis spp. dog Johore heilongjiangensis Rh. microplus cattle Negeri Sembilan Rh. microplus cattle Pahang UniversityRh. sanguineus dog Kuala Lumpur Haemaphysalis spp. rodent Pahang Rickettsia Haemaphysalis spp. cat Kelantan hulinensis Rickettsia sp. HL- Rh. sanguineus dog Kuala Lumpur 93
121 Table 4.18, continued
Closely related Arthropods Host Location Rickettsia species Negeri Sembilan, H. bispinosa cattle Terengganu H. bispinosa sheep Kedah Negeri Sembilan, Haemaphysalis spp. chicken Pahang, Perak Haemaphysalis spp. dog Negeri Sembilan, Perak R. raoultii Haemaphysalis spp. cat Perak Rh. microplus cattle Kedah, Pahang Negeri Sembilan, Kuala Rh. sanguineus dog Lumpur Haemaphysalis spp. rodent Pahang Haemaphysalis spp., vegetation Pahang Dermacentor spp. A. helvolum, A. R. raoultii snake Johore (Candidatus varanense Rickettsia Amblyomma sp. skinkMalaya Pahang johorensis) H. exigua cattle Johore
H. bispinosa of cattle Terengganu H. bispinosa sheep Kedah R. tamurae Haemaphysalis spp. cat Johore A. testudinarium vegetation Pahang
R. tamurae (Candidatus A. varanense snake Kuala Lumpur Rickettsia sepangensis) H. bispinosa sheep Kedah Rickettsia sp. TCM1 Haemaphysalis spp. rodent Pahang Haemaphysalis spp., vegetation Pahang UniversityDermacentor spp.
122 Malaya of
Figure 4.20: An overview presentation of the seroprevalence and occurrences of rickettsiae in human and arthropod vectors across several states in 123 University Peninsular Malaysia.
123 4.4 PCR detection of rickettsiae in various animal hosts
4.4.1 Determination of the type and distribution of rickettsiae in animal blood
(small mammals and ruminants) samples
The blood samples collected from four rodents, 19 cattle, 32 horses, 55 buffaloes, 78
deer, 15 pangolins and 74 goats [kindly provided by Dr. Chandrawathani from the
Veterinary Research Institute, Ipoh] were included for investigation in this study
(Appendix M). Only two blood samples from buffaloes and a horse blood sample were
positive upon amplification for rickettsial ompA gene, thus, contributing to an occurrence
of 1.1 % (3/273) in the animal samples. All sequences demonstrated the highest sequence
similarity of 99 % (393/395 bp) to 100 % (395/395 bp) to that of Rickettsia sp. TCM1
(GenBank accession no.: AB359459).
In addition, 30 blood samples collected from a cattleMalaya farm in Serdang, Selangor, were free from rickettsial DNA. None of the 305 blood samples collected from five cattle
farms (Farm 1, 2, 3, 5 and 6), a sheep farm of(Farm 4) and a goat farm (Farm 8) across six
states in Peninsular Malaysia were positive for rickettsial DNA. Additionally, a total of
150 blood samples collected from cats admitted to a veterinary clinic in Serdang,
Selangor, were negative for rickettsial DNA based PCR assays targeting gltA and ompA
genes.
4.4.2 Determination of the type and distribution of rickettsiae in wildlife samples
A total of 50 blood samples from monkeys (Macaca fascicularis) in Perlis and Kedah Universitywere provided by PERHILITAN (Appendix N). The monkeys appeared to be healthy and active during blood samplings. R. felis gltA gene was detected from the blood
samples of 12 (24.0 %) monkeys in this study. BLAST analyses of gltA nucleotides
amplified from the samples demonstrated 100 % (373/373 bp) sequence similarity with
those of Rickettsia sp. RF2125 (GenBank accession no.: AF516333); Rickettsia
124 asemboensis (GenBank accession no.: JN315968) and Rickettsia spp. clone 4G/JP102
(GenBank accession no.: JN982949). The rickettsial gltA sequence showed 99.0 %
similarity (360/373 bp) with R. felis URRWXCal2 (GenBank accession no.: CP000053).
Rickettsial ompB gene was amplified from only five samples and the sequences showed
the closest match 99 % (777/779 bp) to 100% (779/779 bp) with that of Rickettsia sp.
R14 (GenBank accession no.: HM370113); 99 % (776/779 bp; 778/779 bp) with that of
Rickettsia asemboensis (GenBank accession no.: JN315972); and 99 % (752/754 bp) to
100% (754/754 bp) with that of Rickettsia sp. RF2125 (GenBank accession no.:
JX183538) with 96.0 % query coverage. The sequences showed 93 % (706/759 bp;
708/759 bp) with R. felis URRWXCal2 (GenBank accession no.: CP000053). A 210 bp
of the trimmed gltA sequence and 779 bp of ompB sequence amplified from sample 0095 were deposited into GenBank database with the accessionMalaya number KP126803 and KP126804, respectively.
In the Kuala Lompat forest reserve area,of organ samples (liver, kidney and spleen)
were collected from four rats, seven bats and a squirrel (Appendix O). All of the animals
were positive for one or more rickettsial genes (gltA, ompA and ompB) in the PCR assays.
Both kidney and spleen samples of two rodents; and the liver and spleen samples of a
rodent were positive in the screening assay. One rodent was positive for only liver and
only spleen sample, respectively. All of the organs collected in five bats (B0014, B0032,
B0034, B0037, and B0038) and a squirrel (S0040) were positive upon screening for one Universityor more rickettsial genes. The liver and spleen of a bat (B0015) were screened positive for one or more rickettsial genes. The liver and kidney of another bat samples (B0031)
were also positive upon screening using rickettsial PCR assays. BLAST analyses
revealed the detection of Rickettsia closely related to those of R. asiatica, R. raoultii,
Rickettsia sp. RF2125 and Rickettsia TCM1 (Table 4.19).
125 Table 4.19: BLAST analyses of the representative sequences derived from the organ of wild animals in Kuala Lompat.
Animal species BLAST analyses (closest relative) Sample ID (organ examined) gltA [n] ompA [n] ompB [n]
Rickettsia sp. RF2125 [AF516333, Rickettsia sp. TCM1 R0005L Rodent (liver) Positive (poor sequence quality) 278/278 bp (99 %)] [AB359459, 454/454 bp (100 %)]
Rickettsia sp. RF2125 [AF516333, Rickettsia sp. TCM1 R0011S Rodent (spleen) negative 284/286 bp (99 %)] [AB359459,Malaya 397/397 bp (100 %)] Rickettsia sp. TCM1 Rickettsia sp. TCM1 Rickettsia sp. RF2125 R0013K Rodent (kidney) [AB359458, 286/286 bp (100 %)] of[AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)] R. raoultii (CP010969)/ R. conorii Rickettsia sp. TCM1 Rickettsia sp. RF2125 R0013S Rodent (spleen) (AE006914)/ R. honei NTT-118 [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)] (U59726) [280/286 (98 %)]
R. raoultii (DQ365803)/R. heilongjiangensis (CP002912)/ B0014L Bat (liver) Positive (unable to be sequenced) negative Rickettsia sp. TCM1 (AB359458) [286/286 bp (100 %)] Rickettsia sp. RF2125 B0014S Bat (spleen) negative negative [AF516333, 286/286 bp (100 %)] R. raoultii (CP010969)/ R. conorii Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0014K Bat (kidney) (AE006914)/ R. honei NTT-118 [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)] (U59726) [283/286 (99 %)]
126 University
126 Table 4.19, continued
Animal BLAST analyses (closest relative) Sample ID species (organ gltA [n] ompA [n] ompB [n] examined) Rickettsia asiatica Rickettsia sp. TCM1 B0015L Bat (liver) negative [AF394901, 281/286 bp (98 %)] [AB359459, 491/491 bp (100 %)]
Rickettsia sp. TCM1 Rickettsia raoultii B0015S Bat (spleen) negative [AB359459, Malaya491/491 bp (100 %)] [CP010969, 762/775 bp (98 %)] Rickettsia sp. TCM1 Rickettsia sp. TCM1 [AB359459, R0028K Rodent (kidney) negative [AB359458, 280/286 bp (98 %)] of491/491 bp (100 %)] Rickettsia sp. TCM1 R0028S Rodent (spleen) negative negative [AB359459, 457/457 bp (100 %)] R. raoultii (DQ365803)/R. heilongjiangensis (CP002912)/ B0031L Bat (liver) negative negative Rickettsia sp. TCM1 [286/286 bp (100 %)]
Rickettsia asiatica Rickettsia sp. TCM1 B0031K Bat (kidney) negative [AF394901, 267/269 bp (99 %)] [AB359459, 423/423 bp (100 %)]
R. raoultii (CP010969)/ R. conorii Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0032L Bat (liver) (AE006914)/ R. honei NTT-118 [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)] (U59726) [285/286 (99 %)]
127 Rickettsia sp. RF2125 [AF516333, Rickettsia sp. TCM1 B0032K Bat (kidney) University negative 285/286 bp (99 %)] [AB359459, 491/491 bp (100 %)]
127 Table 4.19, continued
Animal BLAST analyses (closest relative) Sample ID species (organ gltA [n] ompA [n] ompB [n] examined)
R. raoultii (CP010969)/ R. conorii Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0032S Bat (spleen) (AE006914)/ R. honei NTT-118 [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)] (U59726) [282/286 (99 %)] Malaya Rickettsia sp. TCM1 Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0034L Bat (liver) [AB359458, 284/286 bp (99 %)] [AB359459,of 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)] Rickettsia sp. RF2125 Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0034K Bat (kidney) [AF516333, 281/286 bp (98 %)] [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)]
Rickettsia sp. RF2125 Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0034S Bat (spleen) [AF516333, 280/286 bp (97 %)] [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)]
Rickettsia sp. TCM1 [AB359458, Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0037L Bat (liver) 283/286 bp (99 %)] [(AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)]
Rickettsia sp. RF2125 [AF516333, Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0037K Bat (kidney) 247/248 bp (99 %)] [AB359459, 491/491 bp (100 %)] [JX183538, 754/754 bp (100 %)]
Rickettsia sp. TCM1 [AB359458, Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0037S Bat (spleen) 268/269 bp (99 %)] [AB359459, 327/327 bp (100 %)] [JX183538, 754/754 bp (100 %)]
Rickettsia sp. TCM1 [AB359458, Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0038L Bat (liver) 285/286 bp (99 %)] [AB359459, 491/491 bp (100 %)] [JX183538, 750/754 bp (99 %)] 128 128 University
128 Table 4.19, continued
Animal BLAST analyses (closest relative) Sample ID species (organ gltA [n] ompA [n] ompB [n] examined) Rickettsia sp. RF2125 Rickettsia sp. TCM1 Rickettsia sp. RF2125 B0038K Bat (kidney) [AF516333, 282/286 bp (98 %)] [AB359459, 491/491 bp (100 %))] [JX183538, 750/754 bp (99 %)]
Rickettsia sp. RF2125 Rickettsia sp. TCM1 Rickettsia raoultii B0038S Bat (spleen) [AF516333, 284/286 bp (99%)] [AB359459, 491/491Malaya bp (100 %)] [CP010969, 762/775 bp (98 %)] Rickettsia sp. TCM1 Rickettsia sp. TCM1 S0040L Squirrel (liver) Positive (bad sequences) [AB359458, 286/286 bp (100%)] [AB359459,of 491/491 bp (100 %)] Squirrel Rickettsia sp. TCM1 [AB359458, Rickettsia sp. TCM1 Rickettsia sp. RF2125 [JX183538, S0040K (kidney) 286/286 bp (99%)] [AB359459, 491/491 bp (100 %)] 750/754 bp (99 %)] Rickettsia sp. TCM1 Rickettsia sp. TCM1 [AB359459, Rickettsia sp. RF2125 [JX183538, S0040S Squirrel (spleen) [AB359458, 286/286 bp (100%)] 491/491 bp (100 %)] 750/754 bp (99 %)]
129 129 University
129 None of the 71 wildlife blood samples on FTA cards collected from Pulau Tioman,
Pahang (including 40 bat samples, nine rats, seven squirrels, ten monitor lizards, four
primates and a toddy cat, Appendix P) provided by PERHILITAN, were positive upon
screening for rickettsial DNA using primers targeting on rickettsial gltA, ompA and ompB
genes.
4.5 Phylogenetic analysis of tick and flea-borne rickettsiae
4.5.1 Phylogenetic analysis based on partial fragments of gltA gene
Figure 4.21 shows the phylogenetic placement of gltA sequences (368 bp) amplified
from human, ticks, fleas, and animal hosts collected from different geographical regions
in Peninsular Malaysia. Only one representative of sequence type from different locations
and sample types was included in the phylogenetic analysis. Sequences with low quality were not included in the analysis. Hence, 37 sequencesMalaya of the rickettsial species detected in the present study were aligned with those retrieved from the GenBank database. R.
belli was included as an outgroup. of
Phylogenetic analysis revealed that the gltA sequences were clustered into two major
groups, i.e.: spotted fever group rickettsiae and Rickettsia felis-like organisms. SFG
rickettsiae are frequently detected in tick samples collected from different geographical
regions in this study (including animal shelters, cattle farms, rural villages, snake ticks in
Sepang, and Kuala Lompat forest reserve). They were grouped in the same cluster with
those of R. raoultii, R. heilongjiangensis, Rickettsia sp. TCM1, Rickettsia sp.
UniversityKagoshima6, and R. tamurae. It was difficult to differentiate species within SFG
rickettsiae as reflected by the low bootstrap values between branches in the dendrogram
(Figure 4.21) owing to the close sequence similarity between the species.
130 Malaya of
University
Figure 4.21: Phylogenetic placement of rickettsiae based on partial sequences of rickettsial gltA (368bp) gene.
The gltA sequences of the rickettsiae detected in the present study were aligned with those retrieved from the GenBank database. Phylogenetic analysis was performed by using the neighbour-joining method (Kimura 2-parameter model) and tree were tested by bootstrapping (1000 replications). Scale bar indicates the nucleotide substitutions per site. R. belli str. HJ7 was used as an outgroup. * denotes the rickettsia detected in this study.
131 There were nine sequence variants (one R. felis variant and eight RFLO variants)
exhibiting one to 12 nucleotide differences (96.7 % - 99.7 %) in the gltA partial sequence,
as compared to that of R. felis URRWXCal2 (GenBank accession no.: CP000053). Five
gltA sequences obtained from three C. felis fleas (only one representative was shown in
Figure 4.21) from Kelantan, one Haemaphysalis tick from Perak, and one Rh. microplus
cattle tick from Kedah are in the same cluster with R. felis type strain (URRWXCal2).
RFLO gltA genes were amplified from nine ticks and 23 representative fleas collected in
rural areas, a cattle tick in Terengganu farm, a dog tick from Kuala Lumpur, a patient in
UMMC, 12 monkey bloods (sample 0095 as representative), nine organs in small
mammals from Kuala Lompat, and five ticks from Kuala Lompat. RFLO amplified from
patient A are clustered into the same branch with RFLOs amplified from the monkey blood sample, a dog flea collected in Perak and someMalaya ticks from cat, cattle, rodent and questing ticks.
4.5.2 Phylogenetic analysis based on ompBof genes
Dendrograms were constructed for the ompB partial sequences obtained in this study,
using R. raoultii and R. felis/RFLO as the reference strains, respectively (Figure 4.22 and
Figure 4.23). Figure 4.22 shows the phylogenetic analysis based on the ompB gene of R.
raoultii. The ompB sequences of R. raoultii (of those Rickettsia closely related to
Rickettsia sp. Kagoshima in gltA analysis, section 4.3.4.2) detected from ticks collected
from animal Farm 1, Farm 4 and Farm 6 which showed 93 % identities to R. raoultii Universitystrain Khabarovsk (GenBank accession no.: DQ365798) are grouped in a single cluster in the dendrogram. This further support that the rickettsia could be a new species. R.
raoultii detected from an Amblyomma sp. tick collected from a skink in Pahang is
clustered with R. raoultii detected in Amblyomma snake ticks collected in Johor with 100
bootstrap value. Based on the sequence similarity of ompB, R. raoultii detected in
patients, ticks and animal tissue organs from urban and rural areas are grouped in a branch
132 closely related to R. raoultii reference strains (R. raoultii strain Khabarovsk, R. raoultii
strain Elanda-23/95, R. raoultii strain Marne). Notably, R. raoultii amplified from patient
C clustered with R. raoultii amplified from H. wellingtoni ticks from chicken and goat.
Additionally, R. raoultii amplified from patient D is in the same cluster with R. raoultii
amplified from H. bispinosa tick collected from a chicken (DK021) (Appendix H).
Malaya of
UniversityFigure 4.22: Phylogenetic placement of rickettsial based on the partial sequences of R. raoultii (749 bp) ompB gene.
The ompB gene sequences of the rickettsiae species detected in the present study were aligned with those retrieved from the GenBank database. Phylogenetic analysis was performed by using the neighbour-joining method (Kimura 2-parameter model) and tree were tested by bootstrapping (1000 replications). Scale bar indicates the nucleotide substitutions per site. R. typhi str, Wilmington was included as an outgroup. * denote the rickettsia detected in this study.
133 Figure 4.23 shows the phylogenetic analysis based on ompB gene of rickettsia closely
related to R. felis/RFLO identified in this study. The sequences were differentiated into
two distinct types, of which one cluster was closely related to R. felis URRWXCal2, and
another cluster was more closely related to Rickettsia sp. RF2125 and Rickettsia
asemboensis (RFLO). The phylogenetic tree clearly demonstrated that RFLOs were
detected in a wider range of samples (patient, ticks, C. felis orientis fleas and wildlife) as
compared to R. felis (only detected in ticks and C. felis felis fleas). These findings show
the need of isolation and characterization of the rickettsiae.
Malaya of
UniversityFigure 4.23: Phylogenetic placement of rickettsiae based on the partial sequences of R. felis and RFLO ompB (749 bp) gene.
Sequences of the Rickettsia species detected in the present study were aligned with those retrieved from the GenBank database. Phylogenetic analysis was performed by using the neighbour-joining method (Kimura 2-parameter model) and tree were tested by bootstrapping (1000 replications). Scale bar indicates the nucleotide substitutions per site. R. typhi str, Wilmington was included as an outgroup. * denotes the rickettsia detected in this study.
134 4.6 Isolation of R. felis-like organisms from infected fleas
The homogenates of rickettsia-positive ticks and fleas from the previous section
(section 4.3) were used for the isolation of Rickettsia. The homogenates of eight
rickettsia-positive individual ticks (five Rh. sanguineus, two Rh. microplus, and an A.
varanense) and three pools of rickettsia-positive ticks (two pools of Haemaphysalis spp.
and a pool of A. helvolum; five to six individuals) which were kept in a -80 oC freezer
(for about a year) were used for the isolation of rickettsia. A total of two individual fleas
(a C. felis felis and a C. felis orientis) and 21 pools of flea (pool of four to 11 individuals
of C. felis orientis) sourced from the same host of the positive fleas (screened in section
4.3.5.2) were also attempted for the isolation of R. felis/ RFLO. However, none of the
isolation attempt was successful. The cultures were either contaminated by fast growing bacteria or no bacterial growth at all. Malaya A second sampling trip was conducted to collect fleas from rural villages with high
rickettsial seropositivity and high detection ofrates of rickettsia in fleas (Perak and Pahang).
A total of nine individuals of C. felis orientis, a pool of C. felis felis (six fleas) and 11
pools (two to seven individuals) of C. felis orientis were used for the rickettsial isolation
by using C3/36 mosquito cell line in shell vials. One day following inoculation, the
cultures were assessed for contaminations under an inverted microscope. PCR targeting
rickettsial gltA and ompB genes were used to confirm the presence of rickettsia in the
triturated fleas (used for initial inoculation) and the infected cells on day 1, 6 and 13 days Universityfollowing the initial inoculation. Rickettsial DNA was consistently detected in three passages of C6/36 cells infected by a pool of five C. felis orientis flea collected from a
dog (TH2014) in a rural village of Perak (4o 19’ 51.5208’’N, 100o 54’ 10.0296’’). The
rickettsia was tentatively named as Rickettsia sp. TH2014.
135 The partial sequences of the gltA (307 bp), ompB (793 bp) and 16S rRNA genes (1368
bp) amplified from the isolate were determined and subjected to BLAST analysis against
the NCBI database in order to confirm the identity of the rickettsial isolate. Table 4.20
shows the results of the BLAST analyses. The sequences for gltA, ompB and 16S rRNA
gene fragments were 100 % identical to the corresponding sequences of Candidatus
Rickettsia senegalensis strain PU01-02, reported in C. felis in West Africa (Mediannikov
et al., 2015). The sequences for gltA, ompB and 16S rRNA gene fragments showed 98.3
%, 94.8 % and 99.6 % similarity, respectively, as compared to R. felis URRWXCal2
(GenBank accession no.: CP000053). The sequences for gltA and ompB partial fragments
were also identical with the gltA (265 bp; GenBank accession no.: KF963603) and ompB
(797 bp; GenBank accession no.: KF963608) sequences of uncultured Rickettsia sp. clone HL15c detected from a cat flea previously (Tay et al.,Malaya 2014). Table 4.20: BLAST analyses of the amplified gene fragments of Rickettsia sp. TH2014 isolated from ofC. felis orientis . Gene BLAST analyses % identity (bp) gltA Candidatus Rickettsia senegalensis strain PU01-02 100 % (307/307 bp) (KF666472) Rickettsia sp. Rf31 (AF516331) 100 % (307/307 bp) Rickettsia felis URRWXCal2 (CP000053) 98 % (302/307 bp) Uncultured Rickettsia sp. clone HL15c 100 % (265/265 bp) * (KF963608)
ompB Candidatus Rickettsia senegalensis strain PU01-02 100% (793/793 bp) (KF666470) Uncultured Rickettsia sp. clone HL15c 100% (789/789 bp) ** (KF963608) Rickettsia felis URRWXCal2 (CP000053) 95 % (752/793 bp)
University16S Candidatus Rickettsia senegalensis strain PU01-02 100 % (1368/1368 bp) rRNA (KF666476) Rickettsia secondary endosymbiont Curculio 99 % (1364/1368 bp) hilgendorfi Rickettsia felis URRWXCal2 (CP000053) 99 % (1363/1368 bp)
* The available sequences for the strain were shorter than 307 bp. **The available sequences for the strain were shorter than 793 bp.
136 4.7 Study on the growth characteristics of Rickettsia sp. TH2014 in C6/36 cells
Three independent time course infections (experiment 1-3) were employed for the
quantification of rickettsiae present in the infected C6/36 cells (Figure 4.24 to Figure
4.29). A standard curve was constructed for every run of real-time PCR (Figure 4.24,
Figure 4.26, Figure 4.28). The number of Rickettsia sp. TH2014 per ml of cells was
calculated using a real-time PCR assay based on the standard curve generated.
In this study, a total of 1.73 × 105 and 2.3 × 109 of rickettsial organisms were used to
initiate the infection in T75 flasks for the first (C6/36-1, Figure 4.25) and second (C6/36-
2 and C6/36-3, Figure 4.27) time-course experiments, respectively. After day 1,
exponential growth of the organisms with the maximum number of replicates on the 4
dpi was observed, and this was followed by a slow growing phase or stationary phase. By 7 dpi, there were 2.1 × 105, 1.0 × 1010 and 3.4 × 1010 RickettsiaMalaya sp. TH2014 copies/mL of rickettsial DNA quantified in time-course experiment 1 (C6/36-1), 2 (C6/36-2) and 2
(C6/36-3), respectively. of
In the time-course experiment 3, the infections were carried out in 24 well plates, A
total of 2.03 × 108 and 4.96 × 108 of rickettsial organisms were used to initiate the
infection for C6/36-4 and C6/36-5, respectively. A continual increase in the number of
Rickettsia sp. TH2014 DNA copies were observed in one of the replicate (C6/36-5).
Another replicate (C6/36-4) showed a decreasing copies number of rickettsial DNA at 4
and 8 dpi, respectively, followed by a continual increase until 14 dpi (Figure 4.29).
UniversityHowever, the mean value of the replicates showed that the number of Rickettsia sp.
TH2014 DNA copies increased gradually. At 14 dpi, the number of DNA copies sampled
for Rickettsia sp. TH2014 were 6.0 × 109 copies/mL and 3.7 × 109 copies/mL in both
replicates (C6/36-4 and C6/36-5, respectively).
137 Taken together, the Rickettsia sp. TH2014 copies/mL of cell sampled at the end of
experiments increased as compared to the inoculating dose for all of the experiments
(Figure 4.25, Figure 4.27, and Figure 4.29). This data suggests that Rickettsia sp. TH2014
was able to grow and replicate in Ae. albopictus C6/36 cells.
Malaya of
Figure 4.24: Standard curve for quantitation of Rickettsia sp. TH2014. The plasmid pCR4-TOPO cloning vector showed linearity in the dilution series, for 1.65 × 106 to 1.65 × 1011 copies (R2=0.9923, Efficiency: 99 %, y=-3.3416x+45.812) of gltA gene in the real-time PCR experiment for time-course experiment 1 (C6/36-1).
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Figure 4.25: Growth of Rickettsia sp. TH2014 in Ae. albopictus C6/36 cells for seven days (time-course experiment 1, C6/36-1) in T75 flask. A total of 1.73 × 105 of rickettsial DNA copies were used to initiate the infection. Rickettsial DNA was not detected in uninfected/negative control cells. Malaya of
University Figure 4.26: Standard curve for quantitation of Rickettsia sp. TH2014. The plasmid pCR4-TOPO cloning vector showed linearity in the dilution series, for 1.65 × 106 to 1.65 × 1011 copies of gltA gene (R2=0.99, Efficiency= 99 %, y=-3.3472x+46.553) in the real-time PCR experiment for time-course experiment 2 (C6/36-2 and C6/36-3).
139
Figure 4.27: Growth of Rickettsia sp. TH2014 in Ae. albopictus C6/36 cells for seven days (time course experiment 2, C6/36-2 and C6/36-3) in T75 flasks. A total of 2.3 × 109 of rickettsial DNA copies were used to initiate bothMalaya of the infection. Rickettsial DNA was not detected in uninfected/negative control cells. of
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Figure 4.28: Standard curve for quantitation of Rickettsia sp. TH2014. The plasmid pCR4-TOPO cloning vector showed linearity in the dilution series, for 1.65 × 106 to 1.65 × 1011 copies of gltA gene (R2=0.99, efficiency= 93 % y=-3.5376x+48.111) in the real-time PCR experiment for time-course experiment 3 (C6/36-4 and C6/36-5).
140
Figure 4.29: Growth of Rickettsia sp. TH2014 in Ae. albopictus C6/36 cells for 14 days (time-course experiment 3, C6/36-4 and C6/36-5) in 24 well plates. A total of 2.03 × 108 and 4.96 × 108 of rickettsial DNA copies were used to initiate the infection for C6/36-4 and C6/36-5, respectively. Rickettsial DNA was not detected in uninfected/negative controlMalaya cells.
In general, the rounding of infected C6/36of cells (2 dpi) was first observed under an
inverted microscope (Figure 4.30). The infected cells began to detach from the
flask/culture plate surface when an abundant of free rickettsiae were observed towards
the end of the time-course experiments.
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Figure 4.30: Uninfected C6/36 cells (left), infected C6/36 cells, 2 dpi (Right), 400X magnification.
141 4.8 Microscopic study of the morphological characteristics of Rickettsia sp.
TH2014 in C6/36 cells
4.8.1 Giemsa stain
Morphologically, the rickettsiae are highly pleomorphic (coccobacillary to short rods) and stained pink under Giemsa stain (Figure 4.31). Diplococci and short chains were occasionally seen.
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Figure 4.31: Giemsa stain of Rickettsia sp. TH2014 (11 dpi), magnification of 1000X, Universityscale: 10 µm.
142 4.8.2 Transmission electron microscopy (TEM)
TEM was used to examine the growth characteristics of Rickettsia sp. TH2014 in the
infected C6/36 cells for 1, 4 and 7 dpi. Electron micrographs revealed intracytosolic
rickettsia with some undergoing binary fission (Figure 4.32 and Figure 4.33). Intracellular
rickettsiae grew either in small clusters or as solitary organisms in spherical or elongated
shape in the cytoplasm of the infected cells (Figure 4.33). The rickettsia demonstrated an
electron-lucent “halos”, presumably a slime layer, within the cytoplasm of the infected
cells (Figure 4.34). TEM analysis of this study did not provide evidence that Rickettsia
sp. TH2014 grows within the nucleus of the host cells. However, additional analysis of
samples prepared at more different time points will be necessary to make a definitive
conclusion. Higher numbers of rickettsiae were observed at 4 dpi (Figure 4.33 and Figure 4.35) compared to those at 1 dpi (Figure 4.32). At 7 dpi,Malaya C6/36 cells were heavily infected with Rickettsia sp. TH2014 and ruptured cells were observed (Figure 4.36). No rickettsiae were observed in uninfected C6/36 cells (Figureof 4.37).
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143 Malaya of
Figure 4.32: Transmission electron micrograph of Rickettsia sp. TH2014 in C6/36 Universitymosquito cells (1 dpi). A small number o rickettsiae were present in the host cells. The intracytosolic rickettsia (arrow) was in the process of binary fission. Bar: 2 µm (19,000X magnification). N, nucleus.
144 Malaya of
Figure 4.33: Transmission electron micrograph of Rickettsia sp. TH2014 infected C6/36 cells (4 dpi).
The C6/36 cells were heavily infected with a higher number of rickettsiae. The small arrows indicate examples of round and rod-shape rickettsiae (the round rickettsiae could be attributed to the orientation of the rickettsiae during the processing of the sample); the large arrow indicates the release of rickettsia from a cell; the large arrow head indicates the binary fission of the bacteria. Bar: 2 µm (19,000X magnification). N, nucleus; V, vacuole.
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Figure 4.34: Close-up of the arrow head area in Figure 4.33.
The small arrows indicate the electron-lucent “halo” or a slime layer surrounding the rickettsiae. Bar: 500 nm (48,000X magnification). N, nucleus.
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146 Malaya of
Figure 4.35: Transmission electron micrograph of Rickettsia sp. TH2014 infected C6/36 cells (4 dpi).
The small arrows indicate examples of round and elongated rickettsiae, Bar: 2 µm (18,000X magnification). N, nucleus; G, Golgi apparatus/endoplasmic reticulum. Rickettsia was not observed in the nuclei of the infected host cells.
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147 Malaya of
Figure 4.36: Transmission electron micrograph of Rickettsia sp. TH2014 infected C6/36 cells (7 dpi).
The host cell was ruptured and many indifferent stages of cytoplasmic organic decomposition were observed. Bar: 1 µm (24,000X magnification).
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148 Malaya of
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Figure 4.37: Transmission electron micrograph of uninfected C6/36 cells at day 7.
Bar: top, 1 µm (24,000X magnification) and bottom, 2 µm (15,000X magnification). N, nucleus; V, vacuole.
149 4.8.3 Toluidine blue staining of semi-thin section of infected cells
The rickettsiae were also observed in the toluidine blue-stained infected cells using a
light microscope. More rickettsiae were observed in cells infected at 4 dpi (Figure 4.38B)
as compared to 1 dpi (Figure 4.38A). The cells were heavily infected by Rickettsia sp.
TH2014 at 7 dpi (Figure 4.39A). There were no bacteria observed in an uninfected C6/36
throughout the 7 days (Figure 4.39B).
Malaya of
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Figure 4.38: Light photomicrograph showing toluidine blue stain of the infected cells at 1 dpi (A) and 4 dpi (B), magnification: 1000X. Arrow heads indicate the bacteria observed at 4 dpi.
150 Malaya of
Figure 4.39: Light photomicrograph showing toluidine blue stain of the infected cell at 7 dpi (A) and uninfected cell at day 7 (B), magnification: 1000X. Arrow heads indicate the bacteria observed.
University 4.9 Genome properties of Rickettsia sp. TH2014
The genome assembly of Rickettsia sp. TH2014 genome was performed using three
different assembly software packages: SPAdes (Bankevich et al., 2012), ABySS
(Simpson et al., 2009) and Velvet (Zerbino & Birney, 2008) after removing the low
quality raw reads. The assemblies generated using different software packages were
151 compared, and the best assembly in terms of N50 size, and number of contigs was chosen.
The genome assembly generated by ABySS 2.0.2 using Kmer 31 was chosen for final
analysis based on the fact that it has a larger N50 with less contig numbers. This whole
genome sequencing project has been submitted at GenBank database under the accession
number MXAX00000000 (BioProject ID: PRJNA378585).
The estimated genome size and GC content of the Rickettsia sp. TH2014 draft genome
were 1, 368, 580 bp and 32.9 %, respectively. The draft genome of rickettsia contained a
total of 308 contigs ranging from 502 bp to 32, 167 bp and assembled with ABySS
software to a depth of 945X coverage and N50 of 7, 884 bp. Functional annotation by
RAST analysis showed 1, 469 predicted protein-coding sequences, 32 tRNA genes and 3
rRNA genes. BLAST analyses of 5S rRNA, 16S rRNA and 23S rRNA of Rickettsia sp. TH2014 showed 99.12 % (113/114 bp), 99.66 % (1482/1487Malaya bp) and 99.1 % (2735/2760 bp) similarity to R. felis URRWXCal2 (GenBankof accession no.: CP000053), respectively. A schematic circular diagram of the Rickettsia sp. TH2014 is depicted in Figure 4.40.
There is no significant match of plasmid found in the genome as predicted by the
PlasmidFinder software. The absence of plasmid was further confirmed by mapping all
the contigs to the reference plasmids sequence of R. felis URRWXCal2, pRF and pRF
delta (GenBank accession no.: NC_007110 and NC_007111).
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Figure 4.40: Schematic circular diagram of Rickettsia sp. TH2014 genome generated using DNA plotter software.
Key for the diagram (outer to inner): 1st ring: forward coding sequences (CDS, blue), 2nd ring: reverse coding sequences (CDs, pink), 3rd ring: tRNA genes (black), 4th ring: rRNA genes (red), 5th ring: GC plot, where green and dark purple correspond to above and below average GC content, respectively.
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Figure 4.41: Functional categories of the RAST predicted genes of Rickettsia sp. TH2014. Figure 4.41 shows the distribution of the rickettsialMalaya genes in different functional categories as assigned by the RAST pipeline. The RAST-predicted protein-coding genes
in the genome of Rickettsia sp. TH2014 areof enriched in protein metabolism (177 genes);
followed by respiration (77 genes); cofactors, vitamins, prosthetic group pigments (69
genes); DNA metabolism (63 genes); RNA metabolism (65 genes); cell wall and capsules
(59 genes); carbohydrates (52 genes), and fatty acids, lipids and isoprenoids (44 genes).
All of these functions are mainly responsible for the basic biological function of the
rickettsia.
Table 4.21 shows the genes involved in the disease virulence defense and intracellular Universityresistance of Rickettsia sp. TH2014. There are 20 genes involved in the resistance to antibiotics and toxic compounds including lysozyme inhibitors (one gene), cobalt-zinc-
cadmium resistance (one gene), resistance to fluoroquinolones (four genes) and beta
lactamase (five genes). Another nine genes are involved in virulence, disease and defense
functions under the subsystem of invasion and intracellular resistance.
154 Table 4.21: Virulence, disease, defense category of Rickettsia sp. TH2014.
Subcategory Subsystem Predicted roles Copper homeostasis: Copper homeostasis protein CutE copper tolerance Cobalt-zinc- Cobalt-zinc-cadmium resistance cadmium resistance protein
DNA gyrase subunit A (EC 5.99.1.3), Resistance to DNA gyrase subunit B (EC 5.99.1.3), antibiotics and toxic Resistance to Topoisomerase IV subunit B (EC compounds fluoroquinolones 5.99.1.-), Topoisomerase IV subunit A (EC 5.99.1.-)
Beta-lactamase (EC 3.5.2.6), Metal- dependent hydrolases of the beta- Beta-lactamase lactamase superfamily I, Beta-lactamase class D SSU ribosomal protein S7p (S5e), TranslationMalaya elongation factor G, Translation elongation factor Tu, Mycobacterium SSU ribosomal protein S12p (S23e), virulence operon DNA-directed RNA polymerase beta' Invasion and involved in proteinof subunit (EC 2.7.7.6), intracellular resistance synthesis (SSU DNA-directed RNA polymerase beta ribosomal proteins) subunit (EC 2.7.7.6), LSU ribosomal protein L35p, Translation initiation factor 3, LSU ribosomal protein L20p.
Additionally, Rickettsia sp. TH2014 genome exhibited two spoT genes (SpoTd and
SpoTb in RAST annotation) encoding for guanosine-3’, 5’-bis(diphosphate) 3’- Universitypyrophosphohydrolase which allow bacterial adaptation to nutritional stress (Cashel et al., 1996). BLAST analyses on NCBI further confirmed the identity of the two spoT genes
of Rickettsia sp. TH2014 by showing the highest identity of 99 % (598/606 bp) and 95 %
(677/716 bp) to R. felis URRWXCal2 (GenBank accession no.: CP000053) spoT11 and
R. amblyommatis isolate An13 (GenBank accession no.: CP015012) spoT, respectively.
155 4.10 Phylogenetic position of Rickettsia sp. TH2014
The genome properties of Rickettsia sp. TH2014 were compared with those of R. felis
and RFLOs, i.e., R. hoogstraalii and Rickettsia asemboensis (Table 4.22).
Table 4.22: Comparison of genome information for Rickettsia sp. TH2014 with R. felis and RFLOs.
Category Rickettsia sp. R. felis R. R. asemboensis TH2014 URRWXCal2 hoogstraalii strain NMRCii (PRJNA378585) (PRJNA13884) (PRJEB7296) (PRJNA271102) Genome 1.368580 MB 1.49 MB 1.48481 MB 1.37674 MB Size Gene/ Cds 1469 1479 1540 1408 G+C 32.9 32.5 32.4 32.3 content (%) rRNAs 3 3 3 3 tRNA 32 33 33 33 Other RNA - 4 1 5 Isolation C. felis orientis C. felis Hard and soft Ctenocephalides source ticks cat and dog Malayafleas Country Malaysia California, Croatia Kenya USA Reference - (Ogata et al., (Sentausa et (Jima et al., 2005b) of al., 2014) 2015)
The full length 16S rRNA gene of Rickettsia sp. TH2014 (1389 bp) was aligned with
R. felis URRWXCal2 type strain and RFLOs including Candidatus Rickettsia
senegalensis (GenBank accession no.: KF666476), R. hoogstraalii strain Croatica
(GenBank accession no.: NR104877) and Rickettsia asemboensis isolate F30 (GenBank
accession no.: JN315967). Multiple alignment and nucleotide differences of 16S rRNA
genes for Rickettsia sp. TH2014, R. felis and RFLOs is depicted in Figure 4.42. Rickettsia
Universitysp. TH2014 16S rRNA showed 100 % (1389/1389 bp), 99.6 % (1384/1389 bp), 99.4 %
(1380/1389 bp) and 99.4 % (1381/1389 bp) sequence identities to that of Candidatus
Rickettsia senegalensis (GenBank accession no.: KF666476), R. felis URRWXCal2
(GenBank accession no.: CP000053), R. hoogstraalii strain Croatica (GenBank accession
156 no.: NR104877) and Rickettsia asemboensis isolate F30 (GenBank accession no.:
JN315967), respectively.
Malaya of
University
Figure 4.42: Multiple alignment of the 16S rRNA genes (1389 bp) for Rickettsia sp. TH2014, R. felis and RFLOs. Nucleotide variations are marked in red box.
157 Malaya of
University Figure 4.42, continued
158 Malaya of
UniversityFigure 4.42, continued
159 A phylogenetic tree showing the placement of Rickettsia sp. TH2014 was constructed
based on the concatenated sequences of the almost full length sequence of gltA (1250 bp)
and 16S rRNA (1421 – 1424 bp) genes derived from the genome sequencing (Figure
4.43). Sequences of Rickettsia sp. TH2014 were aligned with the genome reference
strains retrieved from the GenBank database. The phylogenetic analysis highlights the
position of Rickettsia sp. TH2014 isolate next to Candidatus Rickettsia senegalensis in
the R. felis/RFLO cluster with a bootstrap value of 100 %.
Malaya of
Figure 4.43: Phylogenetic placement of Rickettsia sp. TH2014 based on the concatenated sequences (2668 bp – 2674 bp) for rickettsial gltA (1250 bp) and rickettsial 16S rRNA gene (1421 bp – 1424 bp).
The phylogenetic analysis was performed by using the neighbour-joining method (Kimura 2-parameter model). The tree was tested using bootstrapping (1000 replications). Scale bar (0.005) indicates the nucleotide substitutions per site. * denotes the Rickettsia Universitysp. TH2014 isolated in this study.
160 4.11 Comparative whole-genome single nucleotide polymorphism (SNP) analysis
Table 4.23 listed the rickettsial reference genomes which are available for comparative
whole-genome SNP analysis and pairwise comparison using JSpeciesWS (section 4.11).
Figure 4.44 shows the SNP-based phylogenetic tree of Rickettsia spp. using TG
rickettsiae as an outgroup. The SNP-based phylogenetic tree shows that Rickettsia sp.
TH2014 was grouped in R. felis/RFLO subgroup with high bootstrap values (931).
Table 4.23: Accession number for rickettsial species included in whole-genome kSNP analysis and pairwise genome comparison.
Genome Chromosome Plasmid Size (bp) Contigs GC (%) R. bellii RML369-C NC_007940 - 1522076 1 31.6
R. canadensis McKiel NC_009879 - 1159772 1 31.1
R. typhi Wilmington NC_006142 - 1111496 1 28.9 R. australis Cutlack NC_017058 NC_017041Malaya 1323280 2 32.3 R. japonica YH NC_016050 1283087 1 32.4 NZ_CCXM00 R. hoogstraalii Croatica - 1484812 2 32.4 000000 R. felis URRWXCal2; NC_007110/of NC_007109 1587240 3 32.5 California 2 NC_007111 NZ_JWSW01 NZ_CP0115 R. asemboensis NMRCii 1375546 88 32.2 000001 17 R. heilongjiangensis NC_015866 - 1278471 1 32.3 054 R. conorii Malish 7 NC_003103 - 1268755 1 32.4 R. rickettsii Sheila NC_009882 - 1257710 1 32.5 Smith R. akari Hartford NC_009881 - 1231060 1 32.3
R. slovaca D-CWPP NC_017065 - 1275720 1 32.5
R. massiliae MTU5 NC_009900 NC_009897 1376184 2 32.5 UniversityR. africae ESF-5 NC_012633 NC_012634 1290917 2 32.4 R. prowazekii NMRC NC_000963 1111520 1 29 Madrid E NZ_CM00146 NZ_CM0014 R. helvetica C9P9 1417115 2 32.2 7 68 R. philipii 364D NC_016930 - 1287740 1 32.5 Candidatus R. NZ_LAOH00 - 1438374 68 32.8 amblyommii Darkwater 000000 R. peacockii Rustic NC_012730 NC_012732 1314898 2 32.6
161 Table 4.23, continued
Genome Chromosome Plasmid Size (bp) Contigs GC (%) R. montanensis OSU NC_017043 NC_017055 1279798 1 32.6 85-930 R. parkeri Portsmouth NC_017044 - 1300386 1 32.4 Rickettsia endosymbiont of Ixodes CM000770 - 2100092 20 30.6 scapularis R. rhipicephali 3-7- NC_017042 - 1305467 2 32.4 female6-CWPP Rickettsia sp. TH2014 - - 1368580 308 32.8
Malaya of
Figure 4.44: SNP-based phylogenetic tree with TG (R. prowazekii and R. typhi) as Universityoutgroup. The phylogenetic tree was tested using 1000 bootstrap. * denotes the Rickettsia sp. TH2014 isolated in this study.
162 4.12 Pairwise genome comparison of Rickettsia sp. TH2014
The average nucleotide identity based on BLAST (ANIb) (Table 4.24) showed that
Rickettsia sp. TH2014 had the highest sequence similarity with R. felis URRWXCal2
(95.02 %) followed by R. asemboensis (94.18 %) and R. hoogstraalii (93.61 %). In the
pairwise genome alignment based on MUMmer (Table 4.25), Rickettsia sp. TH2014
genome also showed the highest average nucleotide identity with R. felis URRWXCal2
(96.05 %) followed by R. asemboensis (95.35 %) and R. hoogstraalii (94.91 %). The
tetranucleotide frequency correlation coefficients (TETRA) of Rickettsia sp. TH2014 and
R. felis URRWXCal2 are high (0.99255) (Table 4.26). Rickettsia sp. TH2014 genome
also has high tetranucleotide frequency correlation coefficient with R. hoogstraalii
(0.99147). However, the tetranucleotide frequency correlation coefficient between Rickettsia sp. TH2014 and R. asemboensis is lower (0.97241).Malaya According to Richter and Rossello-Mora (2009), TETRA values > 0.99 may support
the species circumscription based on the ANIof range > 95-96 %. Hence, Rickettsia sp.
TH2014 is suggested to be closely related to R. felis URRWXCal2 with ANI value of
95.02-96.05 % and TETRA value of 0.99255. RFLO (Rickettsia asemboensis and R.
hoogstraalii) are the next closest species for Rickettsia sp. TH2014.
University
163 Table 4.24: Average Nucleotide Identity of Rickettsia sp. TH2014 genome with other rickettsial genome based on BLAST(ANIb).
Genome ANIb [%] Aligned [%] Aligned [bp] R. felis URRWXCal2; California 2 95.02 74.68 1022092 R. asemboensis NMRCii 94.18 62.61 856824 R. hoogstraalii Croatica 93.61 66.27 906976 R. australis Cutlack 91.9 64.11 877363 Rickettsia endosymbiont of Ixodes 91.83 53.66 734399 scapularis R. montanensis OSU 85-930 91.8 59.56 815150 R. slovaca D-CWPP 91.77 59.56 815145 R. japonica YH 91.64 59.77 818003 R. rhipicephali 3-7-female6-CWPP 91.64 59.17 809725 R. heilongjiangensis 054 91.59 59.53 814695 R. peacockii Rustic 91.59 58.62 802286 R. conorii Malish 7 91.56 59.24 810787 R. akari Hartford 91.48 Malaya 60.79 831924 R. parkeri Portsmouth 91.48 59.7 816999 R. rickettsii Sheila Smith 91.41 59.24 810697 R. africae ESF-5 of91.4 59.82 818693 R. massiliae MTU5 91.33 60.32 825539 R. helvetica C9P9 91.33 60.19 823768 R. philipii 364D 91.31 59.52 814561 Candidatus R. amblyommii Darkwater 90.86 54.77 749585 R. canadensis McKiel 87.82 52.2 714389 R. prowazekii NMRC Madrid E 86.53 52.35 716475 R. typhi Wilmington 86.3 51.97 711319 R. bellii RML369-C 80.64 51.47 704400 UniversityANIb: Average nucleotide identity based on BLAST.
164 Table 4.25: Average Nucleotide Identity of Rickettsia sp. TH2014 genome with other rickettsial genome based on MUMmer (ANIm).
Genome ANIm [%] Aligned [%] Aligned [bp] R. felis URRWXCal2; California 2 96.05 82.21 1125075 R. asemboensis NMRCii 95.35 74.05 1013477 R. hoogstraalii Croatica 94.91 77.19 1056354 R. australis Cutlack 93.33 73.67 1008300 Rickettsia endosymbiont of Ixodes 93.29 67.02 917160 scapularis R. montanensis OSU 85-930 92.92 70.37 963062 R. helvetica C9P9 92.85 71.09 972878 R. massiliae MTU5 92.84 71.25 975175 R. rhipicephali 3-7-female6-CWPP 92.81 70.98 971377 R. slovaca D-CWPP 92.79 70.98 971416 R. japonica YH 92.78 70.7 967631 R. parkeri Portsmouth 92.72 70.53 965258 R. peacockii Rustic 92.71 Malaya 70.05 958728 R. heilongjiangensis 054 92.7 70.74 968173 R. conorii Malish 7 92.7 70.66 967039 Candidatus R. amblyommii Darkwater of 92.67 64.18 878355 R. africae ESF-5 92.59 71.11 973230 R. philipii 364D 92.57 70.1 959361 R. rickettsii Sheila Smith 92.55 69.99 957879 R. akari Hartford 92.48 71.15 973750 R. canadensis McKiel 89.71 60.16 823341 R. prowazekii NMRC Madrid E 88.4 56.25 769791 R. typhi Wilmington 88.14 56.12 768037 R. bellii RML369-C 84.21 29.98 410233 UniversityANIm: Average nucleotide identity based on MUMmer.
165 Table 4.26: Pairwise comparison of rickettsial species based on tetra nucleotide composition.
Genome Pearson correlation coefficient R. felis URRWXCal2; California 2 0.99255 R. hoogstraalii Croatica 0.99147 R. massiliae MTU5 0.98742 Candidatus R. amblyommii Darkwater 0.98739 R. montanensis OSU 85-930 0.98724 R. rhipicephali 3-7-female6-CWPP 0.98716 R. parkeri Portsmouth 0.98656 R. africae ESF-5 0.9861 R. conorii Malish 7 0.98599 R. slovaca D-CWPP 0.98598 R. japonica YH 0.98513 R. heilongjiangensis 054 0.98499 R. rickettsii Sheila Smith Malaya0.98452 R. philipii 364D 0.9843 R. helvetica C9P9 0.98343 R. australis Cutlack of 0.98327 R. peacockii Rustic 0.98275 R. akari Hartford 0.98181 R. asemboensis NMRCii 0.97241 R. canadensis McKiel 0.95886 Rickettsia endosymbiont of Ixodes 0.94285 scapularis R. bellii RML369-C 0.92939 R. prowazekii NMRC Madrid E 0.91153 R. typhi Wilmington 0.90827 University
166 4.13 Comparative analysis of gene functions with R. felis and RFLO
The comparison of the genomic functional annotation of Rickettsia sp. TH2014 with
three closely related species, i.e., R. felis URRWXCal2 type strain, R. hoogstraalii and R.
asemboensis is shown in Table 4.27. The whole genome sequence data for Candidatus
Rickettsia senegalensis is not available at the time of genome analysis. Genes associated
with photosynthesis, iron acquisition and metabolism, motility and chemotaxis and
secondary metabolisms are not found in all the rickettsial genome studied (Table 4.27).
Of the 12 major rickettsial virulence factors reported in VFDB, all except enzyme
phospholipase A2 were found in Rickettsia sp. TH2014. The list of virulence factors and
percent similarity of the virulence genes against R. felis URRWXCal2 are presented in
Table 4.28. All of the 18 genes encoding for rickettsial type IV secretion system (T4SS) are present in Rickettsia sp. TH2014, demonstrating highMalaya sequence similarity (96.3-99.7 %) to that of R. felis URRWXCal2. Phospholipase A2 was not annotated in Rickettsia sp.
TH2014. of
Genomic analyses of Rickettsia sp. TH2014 also revealed genes responsible for
programmed cell death and toxin-antitoxin (TA) systems under the subcategory of
regulation and cell signaling. The TA systems of Rickettsia sp. TH2014 were compared
to R. felis URRWXCal2 by using function-based comparison method in RAST pipeline
and search on annotation files (Table 4.29). The system includes vapB-vapC (virulence-
associated protein) loci, phd-doc toxin-antitoxin (programmed cell death) system, YdcE- UniversityYdcD toxin-antitoxin (programmed cell death) system, MazEF toxin-antitoxin (programmed cell death) system, and toxin-antitoxin replicon stabilization systems
(RelE/StbE-RelE/StbD). Overall. Rickettsia sp. TH2014 has fewer number of TA systems
as compared to R. felis except for YdcE, YcdD, and YefM.
167 Table 4.27: Comparison of the gene functions between Rickettsia sp. TH2014 with R. felis and two RFLOs. Functional assignments of all genes were performed using RAST pipeline.
Number of genes Rickettsia R. felis strain R. R. asemboensis Categories sp. URRWXCal2 hoogstraalii (taxonomy ID: TH2014 (taxonomy ID: (taxonomy 1068590) 315456) ID: 467174) Cofactors, Vitamins, 69 68 85 92 Prosthetic Groups, Pigments Cell Wall and Capsule 59 59 61 56 Virulence, Disease and 20 20 20 20 Defense Potassium metabolism 1 1 1 1 Photosynthesis 0 0 0 0 Miscellaneous 2 2 2 2 Phages, Prophages, 0 2 7 0* Transposable elements, Plasmids Membrane Transport 31 20 27 24 Iron acquisition and 0 0 0 0 metabolism Malaya RNA Metabolism 65 66 81 94 Nucleosides and 20 19 21 17 Nucleotides Protein Metabolism 177 of178 177 172 Cell Division and Cell 31 27 25 22 Cycle Motility and Chemotaxis 0 0 0 0 Regulation and Cell 19 39 35 21 signalling Secondary Metabolism 0 0 0 0 DNA Metabolism 63 57 73 58 Fatty Acids, Lipids, and 44 30 80 34 Isoprenoids Nitrogen Metabolism 2 3 3 2 Dormancy and 1 1 1 1 Sporulation Respiration 77 76 77 76 Stress Response 32 38 36 34 UniversityMetabolism of Aromatic 0 2 2 2 Compounds Amino Acids and 35 51 62 48 Derivatives Sulfur Metabolism 3 3 3 3 Phosphorus Metabolism 6 6 6 6 Carbohydrates 52 60 74 53 *Plasmid for the genome were reported in GenBank database (Accession: CO011517) but not in RAST pipeline.
168 Table 4.28: Virulence factors in Rickettsia sp. TH2014 and similarity against R. felis URRWXCal2.
Virulence % similarity to R. felis Genes Functions factors URRWXCal2 Actin-based motility RickA (Actin Activates host Arp2/3 polymerase rickA complexes, resulting in 1438/1161 bp (89.6 %)* protein) nucleation Promotes both actin filament nucleation and profilin-dependent elongation, mimicking eukaryotic formins to Sca2 (Cell sca2 assemble actin comet tails 4620/4911 bp (94.1 %)* surface antigen) for Rickettsia motility. Potential role in mediating the association of Rickettsia pathogens with mammalian host cells Adherence & invasion Recruitment of human vitronectin, a mammalianMalaya glycoprotein that inhibits the membrane-damaging Adr1 adr1 633/873 bp (72.5 %)* effect of the terminal cytolytic complementof pathway, thus mediating resistance to serum killing
Cell surface protein Adr2 adr2 involved in bacterial 662/675 bp (98.1 %) adherence to host cells
Previous studies suggested that rOmpA plays a role in adhesion, but recent studies indicate that rOmpA/Sca0 ompA rOmpA is not critical for 2954/3181 bp (92.9 %) virulence but may play a role in survival or Universitytransmission from the tick vector Mediates both adhesion and invasion of host cells, with the Ku70 subunit of rOmpB/Sca5 ompB 4752/4992 bp (95.2 %) nuclear DNA-dependent protein kinase identified as the primary host receptor
169 Table 4.28, continued
Related % similarity to R. felis Virulence factors Functions genes URRWXCal2 Adherence & invasion Mediates adhesion to various types of Sca1 sca1 4052/5450 bp (74.3 %)* epithelial and endothelial cells Harbors two α-helices that bind and activate host vinculin, a membrane-cytoskeletal Sca4 sca4 2940/3196 bp (91.9 %)* protein associated with focal adhesions that anchors F-actin to the membrane Enzyme phagosomal escape by Hemolysin C tlyc 886/900 bp (98.4 %) rickettsiae Mediates processes of pat1 and host entry, phagosomal Phospholipase A2 not found pat2 escape, and host lysis for cell-to-cell spreadMalaya Phospholipase D pld phagosomal escape 583/600 bp (97.2 %) Secretion system rvhB1 of 809/840 bp (96.3 %) rvhB2 352/360 bp (97.8 %) rvhB3 Putative translocated 287/288 bp (99.7 %) rvhB4a effector RalF, known in 2389/2418 bp (98.8 %) rvhB4b prokaryotes only from 2394/2433 bp (98.4 %) rvhB6a some Rickettsia and 3310/3417 bp (96.9 %) rvhB6b Legionella spp. contains 1900/1941 bp (97.9 %) rvhB6c a Sec7 domain. This 2845/2934 bp (97.0 %) rvhB6d 2614/2676 bp (97.7 %) Rvh T4SS domain is conserved rvhB6e across eukaryotic 3418/3468 bp (98.6 %) rvhB7 guanine nucleotide 143/144 bp (99.3 %) rvhB8a exchange factors 734/745 bp (98.5 %) rvhB8b (GEF), which activate 720/732 bp (98.4 %) rvhB9a ADP-ribosylation 636/651 bp (97.7 %) rvhB9b factors (Arfs), proteins 473/477 bp (99.2 %) UniversityrvhB10 involved in vesicle 1424/1446 bp (98.5 %) rvhB11 trafficking and actin 986/1005 bp (98.1 %) rvhD4 remodeling 1747/1776 bp (98.4 %) Rvh T4SS ralF 1383/1416 bp (97.7 %) effector *Gaps (>10 bps) were detected in Rickettsia sp. TH2014
170 Table 4.29: Comparison of metabolic reconstruction of programmed cell death and TA systems for Rickettsia sp. TH2014 and R. felis URRWXCal2.
Number of genes Subsystem Role Rickettsia sp. R. felis TH2014 URRWXCal2
Death on curing 1 2 protein, Doc toxin Phd-Doc, YdcE-YdcD toxin-antitoxin Programmed cell death 3 1 (programmed cell death toxin YdcE systems) Prevent host death 1 3 protein, Phd antitoxin
HigA protein (antitoxin 1 1 to HigB)
RelB/StbD replicon stabilization protein 3 9 (antitoxin to RelE/StbE)Malaya RelE/StbE replicon 2 9 stabilization toxin VapB protein (antitoxin of 1 2 to VapC)
VapC toxin protein 2 2
Toxin-antitoxin replicon stabilization systems VapC-like protein 1 1
YefM protein 1 0 (antitioxin to YoeB)
ParD protein (antitioxin 0 1 to ParE)
UniversityParE toxin protein 0 1
YoeB toxin 0 1
MazEF toxin-antitoxin Programmed cell death (programmed cell death) 1 0 antitoxin YdcD system
171 CHAPTER 5: DISCUSSION
5.1 Molecular investigation of suspected cases of rickettsioses in febrile patients
attending to UMMC
The impact of rickettsioses as the leading causes of treatable fever for unknown origin
has been documented in Southeast Asian countries (Phongmany et al., 2006). However;
as most studies were based on serological diagnosis (Lai et al., 2014; Lewthwaite &
Savoor, 1936; Parola et al., 2003a), information is lacking on the genetic and biology of
pathogenic rickettsiae in this region. Although rickettsioses have been known to occur in
Malaysia for many years, data is scarce on Rickettsia spp. associated with human
infections. Evidence is not yet available on the direct transmission of rickettsiae from
arthropods to humans in Malaysia, nevertheless; it is believed that exposure to Rickettsia- infected animals and ectoparasites may pose some riskMalaya to human health (Imaoka et al., 2011; Phongmany et al., 2006; Souza et al., 2016).
5.1.1 Serological and molecular findingsof
The IgM serological findings (17.1 % and 9.8 % against R. typhi and R. rickettsii,
respectively, based on a cut off titer of 1:64) in this study suggest recent exposure of our
febrile patients to TG and SFG rickettsiae. However, interpretation for serological
diagnosis of rickettsioses is challenging (McQuiston et al., 2014; Premaratna et al., 2012).
In some SFG cases encountered in the United States, high IgM titers observed during the
first visit did not rise within the first few weeks of infection. Additionally, the presence Universityof IgM was frequently without a corresponding development of IgG antibodies (McQuiston et al., 2014). In a Sri Lanka study, it has been reported that most patients
who had a 1:64 or 1:128 titer obtained after the seventh day of illness had no clinical
rickettsioses (Premaratna et al., 2012). Furthermore, specific identification of rickettsiae
is not possible merely by IFA alone due to the serological cross-reactivity of rickettsiae
within the same group. For instance, some R. typhi seropositive cases may be due to R.
172 felis (Schriefer et al., 1994), while R. rickettsii seropositive cases may be due to a wide
variety of SFG rickettsiae, especially when patients are having low antibody titers
(Anacker et al., 1987). The unavailability of specific rickettsial antigens, especially for
those which are yet to be isolated, also hampers the accurate diagnosis of rickettsioses.
In this study, using a IgM cut-off titer of 1:64 (also recommended by the CDC in the
presumptive diagnosis of acute rickettsioses and commercially available IFA kits), four
and seven patients in this study were presumed to have acute rickettsioses due to SFG and
TG rickettsiae, respectively. However, none of the patients were suggestive of acute SFG
rickettsioses when the cut-off titer was raised to 1:128. Of the three patients seropositive
for TG antigen, R. felis DNA was amplified from one of the patient (patient A) with high
IgM and IgG titers against TG antigen. This study highlights the difficulty in interpreting the results of serological assays which may potentiallyMalaya impact the diagnosis and reporting of rickettsioses in this study. of Following the first report of R. felis infection amongst rural residents of the central
Thai-Myanmar border (Parola et al., 2003a), R. felis has been identified from febrile
patients in several Asian countries, including Bangladesh (Faria et al., 2015), Korea (Choi
et al., 2005a), Thailand (Sophie et al., 2014) and Laos (Dittrich et al., 2014a). This study
reports for the first time the detection of Rickettsia sp. RF2125, a R. felis-like organism
(RFLO) in the blood sample of a febrile patient in Malaysia.
UniversityPhylogenetic analysis of the Rickettsia spp. using ompB sequences in this study showed the closest genetic relatedness of the rickettsia detected from patient C and D
with R. raoultii (Figure 4.1), a new species of SFG rickettsia associated with scalp eschars
and neck lymphadenopathy following tick bites in patients from France (Parola et al.,
2009), Slovakia (Sekeyova et al., 2012), and Poland (Switaj et al., 2012). The clinical
entity of the rickettsial infection, first named as TIBOLA (tick-borne lymphadenopathy)
173 in a female patient in France in 1997 (Raoult et al., 1997), can be due to R. slovaca and
R. raoultii (Parola et al., 2009). So far, infection caused by R. raoultii has been reported
to be less pathogenic in humans than with R. slovaca (Parola et al., 2009). R. raoultii
infection has been reported in two individuals from China who had painful rashes around
the site of tick bites, but no lymphadenopathy (Jia et al., 2014). The presence of R. raoultii
in Dermacentor, Haemaphysalis and Amblyomma ticks has been reported in China, Japan
and Thailand (Parola et al., 2013). In this study, the infection caused by R. raoultii was
mild and patients recovered without any specific medication for rickettsioses. The typical
features such as eschar and neck lymphadenopathy were not noted from our patients. Low
WBC and platelet counts were the only laboratory findings observed in the patients of
this study (Table 4.2).
5.1.2 Clinical presentations of rickettsial PCR-positiveMalaya patients Taken together, fever, headache, arthralgia, and respiratory symptoms are the common
clinical findings of the febrile patients in thisof study. Elevated amino transferase levels
(alanine aminotransferase and aspartate aminotransferase) is the most common
abnormalities in patients infected R. felis (Hun & Troyo, 2012). In Mexico and Australia
(Williams et al., 2011; Zavala-Castro et al., 2009), patients infected with R. felis were
reported to have thrombocytopenia, elevated alanine aminotransferase and aspartate
aminotransferase level which are also observed in patient A of this study. Although the
rash is a typical feature of rickettsioses, only one patient (A) presented with petechial Universityrash. Rash can be difficult to be seen especially in patients with darker complexion (Silpapojakul et al., 1993). Cutaneous rash has been reported in R. felis infection (Parola
et al., 2005b; Renvoisé et al., 2009a). However, a lack of cutaneous rash amongst
Senegalese patients has been reported (Socolovschi et al., 2010). The rash associated with
murine typhus is variable (non-pruritic, macular, or maculopapular) and has been reported
in 20-80 % of infected patients, according to a review by Civen and Ngo (2008). For R.
174 raoultii infection, localized rashes around sites of tick bites had been described in two
(100 %) patients in China (Jia et al., 2014), but only one (20 %) of the five patients in
France diagnosed with R. raoultii infection developed rash (Parola et al., 2009).
The most severe presentation noted in this study was pneumonia and septic shock in
patient B who was diagnosed with murine typhus. However, as the patient also had
underlying interstitial pulmonary fibrosis and precipitated by the existing lung pathology,
it is difficult to conclude that her respiratory problems are solely related to murine typhus.
The most recent systemic review showed that complications due to R. typhi infection were
recorded in 26.1 % patients and the most frequent were pulmonary (pneumonia or
pulmonary infiltrates), followed by pulmonary effusion, respiratory failure, central
nervous system involvement, meningism, seizures, ataxia, and acute kidney injury (Tsioutis et al., 2017). Other complications which areMalaya less frequently reported include ocular manifestation, septic shock, multi-organ failure and hemophagocytic syndrome
(Tsioutis et al., 2017). Cases of murine typhusof have been reported in the neighboring
countries of Malaysia including Laos (Paris et al., 2012), Vietnam (Hamaguchi et al.,
2015), Singapore (Chen et al., 2001) and travelers returning from Thailand (Sakamoto et
al., 2013) and Indonesia (Yoshimura et al., 2015). In a review by Tsioutis et al. (2014),
elderly patients have been reported to have more severe clinical pictures, as evidenced by
a higher complication rate and longer duration of fever.
Thrombocytopenia, often known as a predictive marker for early diagnosis of dengue, Universityis a common laboratory finding in these patients. The detection of rickettsiae from the blood samples of three patients clinically suspected for dengue fever suggests that
differential diagnosis of rickettsioses and dengue could be difficult. Additionally, it is
important to have confirmatory laboratory tests for diagnosis of rickettsioses when both
diseases are endemic in this region. This finding also highlights the importance for
175 clinicians to have awareness/clinical suspicion about rickettsioses including febrile
patients from the urban areas.
5.2 Determination rickettsial seropositivity in urban blood donors, animal farm
workers, and indigenous community
This study provides an update on the exposure of Malaysian indigenous community
and animal farm workers to rickettsioses through serological assessment against R.
conorii and R. felis. Molecular detection of rickettsiae was also conducted in ticks and
fleas infesting domestic animals in the respective surveyed areas to identify possible
rickettsial agents that were circulating in our environment. Antigenic cross-reactivity has
been reported among SFG rickettsiae (Hun & Troyo, 2012; Parola et al., 2013). The cross-
reactivity between members of SFG rickettsiae, including R. conorii, R. rickettsii, R. helvetica, R. slovaca, R. massiliae, R. africae and others,Malaya has been highlighted by the manufacturer of the IFA kit used for this study. Hence, it is possible that IgG for other
SFG rickettsiae members is present in theof participants of this study. Similarly, cross-
reactivity between R. felis and typhus group Rickettsia, R. akari and R. australis was also
stated by the manufacturer of the IFA assays. However, Znazen et al. (2006) hypothesized
that many reactions due to R. conorii could be caused by R. felis. In this study, individuals
seropositive for both rickettsial species were noted in 5.7 % of farm workers, 17.6 % of
the indigenous community and none of the urban blood donors. However, it is difficult to
differentiate R. felis and other SFG rickettsiae without the use of further serological Universityassays, such as cross-absorption techniques and Western blot (Hun & Troyo, 2012). The findings of this study indicate that SFG rickettsioses is prevalent in the indigenous
community. Up to 50 % of the individuals exhibit seropositivity against R. conorii,
whereas approximately one-fourth of the population was previously exposed to R. felis.
The seroprevalence of rickettsioses is affected by the geographical differences, lifestyle
176 and occupation of subjects investigated (Tay et al., 2013). According to a recent survey
by Chandren et al. (2015), a majority of the indigenous people in Malaysia live in wooden
houses or simple cement homes in close proximity to jungle and plantation areas. The
close-to nature living environment exposes them to infected animal ectoparasites, such as
ticks and fleas. Additionally, their close contact with animals and working environment
also enhance the risk of contracting tick and flea-borne diseases. Additionally, there is a
lack of awareness about rickettsioses, which hampers the prevention practices in the
community. In contrast, the seropositivity to R. conorii in urban blood donors was
relatively low (3.3 %) compared with that obtained in a previous serosurvey (1.7 %) (Tay
et al., 2003).
Farm workers may be subjected to increasing risk of tick and flea-borne diseases (Centers for Disease Control and Prevention, 2011). MalayaFor instance, exposures to several tick and flea-borne pathogens have been reported among farm workers in Tianjin, China
(Zhang et al., 2008). The presence of variousof SFG rickettsiae and R. felis in cattle ticks
(H. bispinosa and Rh. microplus) was demonstrated in the vector surveillance in this study
(Table 4.18). Although the vectorial capacity of the infected ticks is yet to be established,
the results in this study highlight the potential exposure of farm workers to rickettsioses.
Increased seropositivity rates against R. conorii and R. felis were observed in older age
group (>50 years old) compared with younger age groups (Table 4.6) in this study. This
result could be due to long-term persistence of antibody, as also noted for scrub typhus in UniversityMalaysia (Tay et al., 2013). Low seroprevalences (3.3 % and 0.0 % for R. conorii and R. felis, respectively) were noted in urban blood donors, and these findings could be due to
low exposure of the urban population to ticks in the urban areas as compared with those
in the rural areas. The occurrences of Rickettsia spp. in fleas collected from urban areas
were significantly lower compared with fleas collected from rural areas (Table 4.16), and
177 these results may be an explanation for the relatively lower seropositivity observed
amongst urban blood donors.
High seroprevalences of SFG rickettsiae have been documented in several
investigations in Southeast Asia. A high seropositivity rate of SFG rickettsial infection
has been reported in Thai patients from Chiangrai (33.0 %) and Mae Sot (27.3 %), who
presented with undifferentiated febrile illness (Blacksell et al., 2015). A seroprevalence
of 20.4 % towards R. conorii was reported in healthy rural residents from Gag Island,
Indonesia (Richards et al., 2003). Since the first report of R. felis infection amongst rural
residents of the central Thai Myanmar border (Parola et al., 2003a), R. felis has been
identified in febrile patients in several Asian countries, including Korea, Thailand, and Laos (Hun & Troyo, 2012). Malaya The findings of this study indicate that 22.5 % of the indigenous population and 16.1 % of farm workers were previously exposedof to R. felis. In contrast, none of the urban blood donors tested were seropositive for R. felis (Table 4.4). In agreement with our study,
two teams investigating the etiology of unexplained fevers in Senegal and Kenya also
reported high incidence of R. felis infection among indigenous population (Richards et
al., 2010; Socolovschi et al., 2010; Socolovschi et al., 2012c). Similar study conducted
in Spain documented an increased seroprevalence of R. felis in rural areas (7.1 %)
compared with urban (3.5 %) and semirural areas (1.7 %) (Nogueras et al., 2006). In
Tianjin. China, high seroprevalence rates towards rickettsial agents (Anaplasma Universityphagocytophilum, Coxiella burnetii, Bartonella henselae, and R. typhi) were reported for healthy farm workers (Zhang et al., 2008).
In this study, the high R. felis seropositivity in the indigenous community correlates
with high detection rates of R. felis/RFLO in fleas collected from rural areas compared
with urban areas (Table 4.16). Additionally, detection of SFG rickettsiae in the ticks
178 collected from rural villages may explain for the higher seroprevalence of R. felis and R.
conorii in the indigenous community.
5.3 Determination of the type and distribution of rickettsiae in arthropods and
animals
5.3.1 Rickettsiae in ticks from urban areas
Rh. sanguineus, a three-host tick mainly infesting dogs, is the main reservoir of R.
conorii (Levin et al., 2012). It is well adapted to human rural and urban environments
because of its association with dogs (Levin et al., 2012). The vector surveillance in this
study indicates that Rh. sanguineus was the only tick species collected and identified from
urban dogs in this study. Phylogenetic analysis of the tick mitochondrial 12S rDNA gene
sequences placed Rh. sanguineus ticks collected in this study into the same clade as those reported in Thailand, Taiwan, Iran and Brazil (Figure Malaya4.6). Rickettsia sp. 2125, R. heilongjiangensisof, Rickettsia sp. HL-93, R. raoultii and R. conorii were detected in 16.3 % of Rh. sanguineus (including three unfed ticks) in this
study. The detection of R. conorii in unfed ticks (Table 4.8) suggests that possible
transovarian transmission of the rickettsia in the tick population. In a recent report by Yu
et al. (2016), Rickettsia spp. (species not determined) were detected from 8.1 % and 42.9
% of Rh. sanguineus collected from grass and cattle, respectively, in Guangdong, China.
The detection of a rickettsia closely related to RFLO (resembling Rickettsia sp. RF2125)
in Rh. sanguineus dog ticks has also been reported in China (Zhang et al., 2014). In UniversityPalestine, R. massiliae, Candidatus Rickettsia barbariae, and Candidatus Rickettsia goldwasserii were reported in 11.6 % of the Rh. sanguineus collected from different
animal hosts (dog, sheep, goat, horse, and wolf) (Ereqat et al., 2016). In Peru, RFLO and
Rickettsia asemboensis were identified in 5 % of the Rh. sanguineus collected from cat
and dogs (Kocher et al., 2016). However, rickettsiae were not detected from the Rh.
179 sanguineus in a study in Bangkok (Foongladda et al., 2011) and a previous study
conducted by University Veterinary Hospital in Malaysia (Watanabe et al., 2015). The
discrepancy observed among the reported prevalences of rickettsiae could be due to the
differences in methodologies adopted, the biotope of the vectors, and the abundance of
Rickettsia reservoirs (Khrouf et al., 2014).
5.3.2 Rickettsiae in ticks from animal farms
Rh. microplus is a common tick infesting cattle while H. bispinosa tick has been
reported to parasitize both cattle and goats (Brahma et al., 2014). As early as 1969, H.
bispinosa had been reported to be widely distributed throughout Peninsular Malaysia
(Kedah, Perak, Pahang, Selangor, Negeri Sembilan, Malacca and Johore) infesting wild Carnivora, Artiodactyla and domestic animals (HoogstraalMalaya et al., 1969). There are several studies reported the presence of rickettsiae in cattle ticks in the Asia- Pacific region. Recently, a rickettsia closelyof related to R. tamurae has been detected in 6 % of the Rh. microplus ticks collected from sambar deer in Thailand (Sumrandee et al.,
2016). Rickettsiae exhibiting high sequence similarities with R. heilongjiangensis, and
Rickettsia sp. LON-13 were identified in Rh. microplus in Laos (Kernif et al., 2012).
Other than that, an uncharacterized rickettsia has been reported in H. longicornis (12.4
%) from grazing cattle in Korea (Kang et al., 2013). In North-eastern China, rickettsiae
exhibiting a close phylogenetic relationship with R. raoultii (0.6 %) and R. japonica (3.3
%) were reported in H. longicornis ticks collected from domestic animals (sheep and Universitycattle) (Dong et al., 2014). In this study, rickettsiae closely related to R. raoultii, R. heilongjiangensis, and R. felis/RFLO were identified from 9.3 % cattle ticks (21 H.
bispinosa and four Rh. microplus) (Table 4.18). Similarly, a RFLO, Rickettsia
asemboensis was identified from 6.6 % (3/45) Rickettsia positive-Rh. microplus in Costa
Rica recently (Troyo et al., 2016).
180 In this study, a rickettsia closely related to R. raoultii was identified in 12 H. bispinosa
ticks. The gltA and ompB genes amplified from these ticks revealed 98 % and 93 %
sequence similarity to R. raoultii (also known as Rickettsia sp. Kagoshima6; JQ697956)
and R. raoultii strain Khabarovsk (DQ365798), respectively. According to the naming
criteria for rickettsiae (Raoult et al., 2005), the low sequence similarity of both gltA and
ompB genes (less than 99.9 % and 99.2 %, respectively) suggests that it could be a novel
species. Rickettsia sp. Kagoshima6 gltA gene has been reported in both A. testudinarium
and Haemaphysalis G1 recently in a tickborne bacterial survey in Laos (Taylor et al.,
2016). Rickettsia sp. Kagoshima6 has also been identified in H. bispinosa in Thailand, a
neighboring country of Malaysia (Malaisri et al., 2015). Collectively, these findings
suggest that the rickettsial strain could have a wide distribution in the Haemaphysalis ticks in Southeast Asia. Isolation would be importantMalaya for further identification and characterization of the rickettsial species. Although the findings in this study suggest that cattle ticks could be a potential vector for SFGof rickettsiae; however, further investigation is required to determine the vectorial capability of the ticks.
R. raoultii, the causative agent for tick-borne lymphadenopathy (TIBOLA), was
reported in Haemaphysalis ticks from Thailand and is widely distributed in Dermacentor
ticks in northern China (Ahantarig et al., 2011; Parola et al., 2009; Tian et al., 2012). In
addition to cattle ticks, this study also reports the identification of a closely related strain
of R. raoultii in both Rh. sanguineus and Haemaphysalis ticks infesting peri-domestic Universityanimals in the rural villages, as well as in the Amblyomma ticks (snake and skink) and H. exigua flies (Table 4.18). The observation of four R. raoultii gltA sequence types (98 %
to 100 %) in cattle ticks suggest the presence of new genetic variants in this region.
Further study to investigate the biology and virulence of these variants are needed.
181 5.3.3 Rickettsiae in ticks from rural villages
Haemaphysalis ticks were the predominant ticks found in the peri-domestic animals
(cats, chickens, dogs, and goat) in rural villages in this study (Table 4.7). Other species
of ticks collected include Rh. sanguineus from dogs and Rh. microplus from cattle.
Rickettsial DNA was detected from 40.6 % of ticks (23 pools of Haemaphysalis spp., one
Rh. sanguineus, and two Rh. microplus) collected from rural villages. Haemaphysalis spp.
ticks were the major tick species found in rural villages infesting cats, chickens, dogs and
goats.
Sequence analyses of amplified fragments of rickettsial gltA, ompA and ompB genes
from ticks collected in rural villages showed the identification of a number of rickettsiae
that had been previously reported, including R. felis, R. raoultii, R. tamurae, Rickettsia sp. TCM1 and Rickettsia sp. RF2125 (Table 4.18). In additionMalaya to these rickettsial species, an R. heilongjiangensis-like organism was detected for the first time from Haemaphysalis
ticks in chicken, cats, and dogs in rural villages;of Rh. sanguineus from dogs in urban areas;
and cattle tick in animal farms in this study. R. heilongjiangensis is distributed in the
Russian Far East and Northern China. A phylogenetically related strain (PMK94) had
been isolated from a patient with septic shock in Thailand (Gaywee et al., 2007; Takada
et al., 2009).
Rickettsia sp. TCM1, an aetiological agent of Japanese spotted fever-like illness, was
isolated and described in Thailand (Takada et al., 2009). In this study, Rickettsia sp. UniversityTCM1 was also identified from Haemaphysalis ticks and rodents in the forest reserve (Table 4.19). The rickettsia has also been previously identified from two wild rats
captured at the wet markers in Kuala Lumpur (Tay et al., 2014) and Ixodes lividus tick
from migratory birds in United Kingdom (Graham et al., 2010).
182 To the best of our knowledge, R. felis was first discovered from Rh. microplus cattle
tick and Haemaphysalis dog tick collected in rural villages in Malaysia. R. felis is
increasingly found in diverse arthropods particularly in C. felis cat flea, ticks and
mosquitoes (Brown & Macaluso, 2016). Lately, the organism has been reported in H.
leporispalustris ticks in Northern California (Roth et al., 2016). Other tick species that
have been reported to be infected by R. felis include H. flava, Haemaphysalis kitaokai, I.
ovatus, R. sanguineus, A. cajennense, and Amblyomma humerale (Cardoso et al., 2006;
Ishikura et al., 2003; Soares et al., 2015). The detection of R. felis/RFLO from fleas (C.
felis felis and C. felis orientis), and various tick species (Haemaphysalis sp., Rh.
microplus, and Rh. sanguineus) collected from cattle, sheep, chickens, cats and dogs from
different study sites in Peninsular Malaysia (Table 4.18) suggests the widespread distribution of the rickettsial organisms. Malaya Rickettsia sp. LON-13 (closely related to R. japonica) (Hanaoka et al., 2009) was
reported for the first time from a Haemaphysalisof cat tick in this study. A mixed rickettsial
infection was suspected in the Haemaphysalis cat tick as R. hulinensis (first isolated from
H. concinna ticks collected in Hulin Country, China) (Yu et al., 1993) was also detected
from the same tick through sequence analysis of the ompB gene (776/882 bp). However,
due to the lack of genetic information (sequence data on Rickettsia sp. LON-13 ompB
gene was not available in NCBI database), further validation study is required to confirm
whether this was indeed a case of mixed rickettsial infection in the tick sample.
University5.3.4 Rickettsiae in ticks from a forest reserve Dermacentor and Haemaphysalis ticks were the predominant feeding ticks and
questing ticks collected from the vegetation in the Kuala Lompat forest reserve, Pahang.
There were more Dermacentor ticks (61 %) recovered from the vegetation, as compared
to Haemaphysalis spp. and Amblyomma spp. ticks. The tick of the genus Haemaphysalis
183 had been reported to be widely distributed across Southeast Asia (Petney et al., 2007).
Sambar deer and wild pig have been identified as the common animal hosts for
Dermacentor ticks in tropical Asia including Malaysia (Mariana et al., 2008). In a survey
of ticks and other ectoparasites in Kedah, both Dermacentor and Haemaphysalis ticks
have been identified as the most common tick genera recovered from flagging vegetation
and non-volant small mammals (Mariana et al., 2008) which is in concordance with the
findings in this study.
SFG rickettsiae (closely related to R. asiatica, R. heilongjiangensis, R. raoultii,
Rickettsia sp. TCM1, Rickettsia sp. RF2125 and R. tamurae) were detected from 31.8 %
and 32.9 % of feeding and questing ticks from the forest reserve, respectively (Table
4.13). Recently, 43.1 % of the unfed Haemaphysalis ticks (H. concinna and H. japonica) collected in the forest area from Chinese-Russian borderMalaya have been reported to harbor R. heilongjiangensis, R. raoultii and Candidatus R. tarasevichiae (Cheng et al., 2016). In
another study conducted in China, Haemaphysalisof spp. collected from vegetation, for
instance, H. longicornis and H. concinna, have been reported as a vector for R.
heilongjiangensis (Liu et al., 2016). R. raoultii was predominant in the Dermacentor spp.
(i.e. D. silvarum, D. nuttalli) collected in the same study (Liu et al., 2016). Likewise, R.
raoultii was detected from three Dermacentor (possibly D. andersoni) and a H. hystricis
ticks collected from vegetation in this study. Additionally, R. heilongjiangensis was
detected from a H. bispinosa feeding on a rodent.
UniversityIn Laos, Rickettsia closely related to R. raoultii and R. japonica were also reported in 5.7 % positive pools of ticks collected in protected forest areas including H. hystricis,
Haemaphysalis G1 and A. testudinarium (Taylor et al., 2016). In this study, R. raoultii
was detected in an A. helvolum feeding on a skink collected from the forest reserve (Table
4.13). Additionally, two other A. testudinarium ticks collected from vegetation were
184 positive for R. tamurae. A rickettsial study in Thailand revealed the detection of rickettsia
closely related to R. tamurae in Amblyomma integrum and A. testudinarium collected
from forest floor (Malaisri et al., 2015). Taken together, the findings of most studies in
other Asian countries as well as the current study suggest that Amblyomma and
Haemaphysalis ticks are the potential vectors for R. raoultii and R. heilongjiangensis in
this region.
5.3.5 Rickettsiae in snake ticks
A. helvolum ticks have been identified from different snakes including Python sp.,
Ptyas (Zamensis) korros and Naja naja (Kohls) in Malaysia (Kohls, 1957). A. varanense
is also one of the ticks species most widespread in large snakes in Southeast Asia
(Burridge, 2001). As P. molurus and N. sumatrana snakes are native to Southeast Asia (Barker & Barker, 2010; Wuster, 1996), ticks parasitizingMalaya the snakes could be endemic where the animal hosts are available. Human infestation by A. testudinarium ticks has
been reported in Selangor, Malaysia (Yamauchiof et al., 2012). Hence, the presence of
rickettsial agents in the snake ticks may pose a risk to both wildlife and human.
Rickettsia closely related to R. tamurae (Candidatus Rickettsia sepangensis) and
Rickettsia closely related to R. raoultii (Candidatus Rickettsia johorensis) were identified
from A. varanense and A. helvolum snake ticks in this study. Closely related species of R.
raoultii have also been detected from A. helvolum from a lizard (Varanus salvator) in
Thailand (Doornbos et al., 2013). Exposure to infected snake ticks may pose risks to Universityhuman health as R. tamurae and R. raoultii have been implicated in human infections (Imaoka et al., 2011; Phongmany et al., 2006). Rickettsia closely related R. raoultii have
been frequently detected in this study including patients (C and patient D) whereby their
ompB sequences showed 97.5-97.6 % identities with Candidatus Rickettsia johorensis.
The same strain of Candidatus Rickettsia johorensis was detected in Amblyomma tick
185 collected from a skink in forest reserve in Pahang, in which both their gltA and ompB
sequences are identical. Other than that, a rickettsia closely related to R. tamurae (gltA
showing 97 % sequence identity to Candidatus Rickettsia sepangensis) was also detected
in two Amblyomma ticks collected in the vegetation of the forest reserve.
To date, there is no report on the human infections caused by tickborne pathogens with
reptile as a host in Southeast Asia. The presence of SFG rickettsiae (Rickettsia species
closely related to R. raoultii, R. tamurae and R. bellii) has been recently reported in A.
varanense and A. helvolum in snakes in a Thailand study (Sumrandee et al., 2014).
Detection of other SFG rickettsiae in reptilian ticks include: R. honei in Bothriocroton
hydrosauri ticks (formerly Aponoma hydrosauri) collected from lizards and snakes in
Australia (Stenos et al., 2003); Rickettsia spp. closely related to R. tamurae in A. fimbriatum ticks collected from reptiles (yellow-spottedMalaya monitor, water python and green- tree snake) in the Northern Territory of Australia (Vilcins et al., 2009) and A. exornatum
tick from a lizard (Varanus olivaceus) in theof United States of America (Reeves et al.,
2006c). In the South America, Rickettsia sp. strain Colombianensi has been identified
from A. dissimile ticks parasitizing iguanas in Colombia (Miranda et al., 2012). The
findings obtained in this study suggest the existing of a reservoir for SFG rickettsial
infections that are potentially transmitted by Amblyomma ticks and reptiles in different
geographical regions of Peninsular Malaysia.
5.3.6 Rickettsiae in fleas UniversityResults obtained from vector surveillance in this study (Table 4.18) showed the detection of R. felis and RFLO not only in tick species (Haemaphysalis sp., Rh. microplus,
and Rh. sanguineus collected from cattle, sheep, chickens, cats and dogs), but also in the
fleas (C. felis felis and C. felis orientis) from different study sites in Peninsular Malaysia
with high occurrences (Table 4.16). The identification of rickettsiae in this research is
186 strongly supported by the sequence analyses of two rickettsial genes (gltA and ompB).
The results in this study suggest that R. felis and RFLO are prevalent (66.2 %) in the fleas
collected from cats and dogs in several rural villages. Due to the high occurrence of fleas
infected with R. felis/RFLO in this study, the rural villagers may have high degree of
exposure to the rickettsial pathogens as C. felis fleas are not host-specific (Perez-Osorio
et al., 2008).
R. felis has been reported in a wide range of arthropods across the world including
fleas. The occurrence of R. felis/RFLO in fleas of this study is aligned to a report by
Foongladda et al. (2011) where Rickettsia sp. related to R. felis was detected in 67.4 %
(66/98) of flea specimens from dogs in Thailand. In a recent study in China, R. felis has
been detected from a variety of arthropod hosts including louse (Linognathus setosus) and mosquitoes (Culex pipiens pallens, Anopheles sinensisMalaya) (Zhang et al., 2014). In Laos, a high occurrence (76.6 %, 69/90) of R. felis was reported in C. felis fleas from dogs
(Kernif et al., 2012). R. felis-infected fleasof are also reported in other Asian countries
including Taiwan (Hsu et al., 2011; Tsai et al., 2011).
Majority of the C. felis orientis fleas collected from rural villages in this study was
infected by Rickettsia sp. RF2125, rickettsia closely related to R. felis. Rickettsia sp.
RF2125 has been reported in C. canis and C. felis fleas at the Thailand-Myanmar border
(Parola et al., 2003a); C. canis and C. felis in Uruguay (Venzal et al., 2006); C. felis in
Malaysia (Mokhtar & Tay, 2011); and C. felis in Costa Rica (Troyo et al., 2012). It was Universitylater described in a different flea species including A. erinacei sourced from hedgehogs in Algeria (Bitam et al., 2006); A. erinacei from foxes in France (Rolain et al., 2009); E.
gallinacea from rats in Egypt (Loftis et al., 2006) and P. irritans from a dog in Hungary
(Hornok et al., 2010), reflecting the widespread of Rickettsia sp. RF2125 in different
arthropods.
187 Interestingly, the study also observed that R. felis was detected only from C. felis felis
while RFLO, Rickettsia sp. RF2125 was detected only in C. felis orientis (Table 4.17)
collected from rural villages. Similarly, in India, only Rickettsia sp. RF2125 was detected
in C. felis orientis but not R. felis (Hii et al., 2015). Meanwhile, the absence of Rickettsia
sp. RF2125 from C. felis was observed in Australia. Hence, Hii et al. (2015) suggested a
specific vector-endosymbiont adaptation and co-evolution of the RFLO within the
subspecies of C. felis. On the other hand, Kernif et al. (2012) discovered significantly
higher frequency of R. felis in C. felis orientis (89.4 %; 59/68) than in C. felis felis (52.6
%; 10/19) collected from Laos. Rickettsia sp. RF2125 has also been report in C. felis flea
in Malaysia previously Mokhtar and Tay (2011). Although it is not known the actual
reason which cause the differences in the distribution of R. felis in fleas, further investigation is required as the interaction of endosymbiontsMalaya within arthropods can have an impact on the dissemination of vertically-transmitted pathogenic bacteria (Pornwiroon et al., 2007). of
5.3.7 Rickettsiae in other arthropod samples
In addition to infection in C. felis fleas, R. felis has also been detected in both An.
gambiae and Ae. albopictus mosquitoes in Africa (Socolovschi et al., 2012c; 2012a). An.
gambiae was demonstrated as a competent vector of R. felis through an experimental
model (Dieme et al., 2015). Ae. albopictus mosquitoes are known to be native to
Southeast Asia, colonizing rural and peri-urban areas (Socolovschi et al., 2012a). None Universityof the 165 Ae. albopictus and 70 Cx. quinquefasciatus collected from different localities in this study was positive for rickettsia. Likewise, none of the male (10) and female (14)
pools of Ae. albopictus collected from Yangzhou, China, were positive for any rickettsial
species (Zhang et al., 2016). The little DNA material extracted from each individual
mosquito may explain for the lower sensitivity in the detection of R. felis which possibly
contributed to the absence of R. felis in the mosquitoes in this study.
188 The DNA of a rickettsia closely related to R. raoultii was detected from 4.0 % of 53
H. exigua flies collected from animal farms in this study. Interestingly, one of the
rickettsia detected from the flies captured in a Johore animal farm (Farm 8) was identical
to Candidatus Rickettsia johorensis (closely related to R. raoultii) following the detection
of the organism from Amblyomma snake tick collected from Johor, Amblyomma skink
tick collected in Pahang and a H. bispinosa cattle tick in Terengganu. The finding
suggests that R. raoultii may be present in different arthropods across Peninsular
Malaysia. This would be the first report on the detection of rickettsiae from H. exigua
flies. H. exigua is an obligate blood feeder that feed on animal host for most of the time
and may affect the health and productivity of cattle and buffaloes (Macqueen & Doube,
1988; Mullen & Durden, 2009). Haematobia flies are reported as potential vectors for several pathogens (Bartonella, Morganella morganiiMalaya, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus hyicus, Serratia marcescens) in addition to causing reduction in weight and milk productionof (Chung et al., 2004; Palavesam et al., 2012; Torres et al., 2011). The finding in this study suggests the possibility of H. exigua
flies as a potential vector for rickettsia apart from the pathogens listed above. However,
it is also likely that the rickettsia was present in the fly just after a blood meal from the
infected cattle. Thus, further validation of the vectorial capacity of ticks, fleas and flies is
necessary to illustrate the involvement of animal ectoparasites in the transmission of
rickettsioses.
University5.3.8 Rickettsiae in animal hosts A wide range of animal blood samples (including cats, cattle, goats, buffaloes, deer,
horses, pangolins, rodents, squirrels, monitor lizard and monkey) were examined for the
presence of rickettsial DNA in this study. There are two blood samples from buffaloes, a
horse (provided by VRI) and 12 monkey blood samples (provided by PERHILITAN)
were positive for rickettsial DNA. The low detection rate of rickettsial DNA (1.1 %) in
189 the animal blood samples is similar with a recent study conducted by Ndeereh et al.
(2017) who reported 3.2 % detection rate of SFG rickettsiae (rickettsial species not
determined) in a buffalo from Kenya.
Cats have been indicated as a potential reservoir for R. felis in a recent study in
Bangladesh whereby R. felis DNA was detected in 28 % of stray cat blood (Ahmed et al.,
2016). In this study, the 150 cat blood samples were collected from pet cats visiting a
veterinary clinic in Serdang. The negative results in rickettsial screening could be due to
the less exposure of pet cats to ectoparasites as compared to stray cats. Our finding is in
agreement with those reported previously whereby rickettsial DNA was not amplified
from any blood samples of client-owned cat presented to veterinary hospitals in Bangkok
(Assarasakorn et al., 2012) as well as both healthy cats in shelters and pet cat presented to veterinary clinics in China (Zhang et al., 2014). TheMalaya same outcome was observed in two studies conducted in Australia (Barrs et al., 2010), and the United States of America
(Bayliss et al., 2009; Hawley et al., 2007). of
All the animal (cattle, sheep and goat) blood samples collected from farm 1 to farm 8
were free from rickettsial infection despite of the detection of rickettsia from the feeding
ticks. Other reports showed that infestation of the animals with rickettsia-infected
arthropods did not always correlate to rickettsial infection in the animal blood (Reif &
Macaluso, 2009). The absence of rickettsia in the domesticated ruminants of this study is
in consistent with a study by Maina (2012) where rickettsia was not detected in any of the Universitycattle, sheep and goats examined in Asembo, western Kenya. These findings show that ruminants are less likely to serve as primary reservoir for rickettsiae.
However, the low detection rates of rickettsia in blood sample could be attributed to
the transient and intermittent nature of bacteremia of some vector-borne pathogens as
reported for Rickettsia spp. in dogs which makes them difficult to be detected in the blood
190 streams (Persichetti et al., 2016). Other hypothesis about the lack of rickettsia in animal
blood could be related to a sequestration of rickettsia in other tissues such as endothelial
cells, spleen and dermal tissues. It is also possible that the concentration of rickettsia in
blood was low and not within the detection limit of the PCR assays (Barrs et al., 2010;
Hawley et al., 2007).
In this study, surveillance for rickettsial infections in various geographical regions was
also extended to wildlife animals. As tick-borne diseases are becoming important
worldwide, monitoring the causative agents in wildlife may serve as a useful indicator for
potential human exposure (Castellaw et al., 2011). A total of 24.0 % blood samples
collected from cynomolgus monkey (Macaca fascicularis) were positive for Rickettsia
sp. RF2125 in this study (Tay et al., 2015). The cynomolgus monkey, also known as long- tailed macaque or crab-eating monkey, is widely distributedMalaya in the Southeast Asia region (Gumert, 2011). The macaque has been associated with bacterial infections such as
hemotropic Mycoplasma and Bartonella quintanaof (Huang et al., 2011; Maggi et al.,
2013). Cynomolgus monkeys are found living in close proximity to human populated
areas due to rapid deforestation and changes in land use patterns (Gumert, 2011). R. felis
has been reported in the C. felis fleas sourced from Cercopithecus cephus monkeys in
Gabon (Rolain et al., 2005) and the fecal specimens (22 %, 25/113) of wild-living African
apes (Keita et al., 2013) suggesting the potential risk of primates as a reservoir host for
R. felis. Both R. felis and RFLO have also been reported in Ctenocephalides fleas and Universityticks from various geographical regions in this study. With the finding of Rickettsia sp. RF2125 in human infection in this study, it will be interesting to investigate whether an
enzoonotic cycle of Rickettsia sp. RF2125 involving fleas, monkey and human exists
here. More extensive molecular investigation and isolation of the RFLO from the relevant
animal hosts and vectors are necessary to provide further evidence.
191 Rickettsia sp. RF2125, Rickettsia sp. TCM1, R. honei/R. conorii/R. raoultii, and R.
asiatica were detected from the organs tissues harvested from all 12 small mammals (bats,
rodents and squirrel) caught in the Kuala Lompat forest reserve. Previously, rickettsiae
demonstrating close similarity to those of R. honei/R. conorii/R. raoultii and Rickettsia
sp. TCM1 have been reported in Rattus spp. (wild rats) in Malaysia (Tay et al., 2014).
The detection of Rickettsia sp. TCM1 and R. honei/R. conorii/R. raoultii in four of the
rodents in this study strengthens our previous finding that these SFG rickettsiae are
circulating in the rodents in this region. SFG rickettsiae have also been extensively
reported in wild rats in other countries. In Germany, R. helvetica DNA, was detected in
1.1 % (3/268) ear pinna samples from Norway rats (Heuser et al., 2017). R. felis and R.
helvetica have also been detected from wild rodents in South-eastern Germany (Schex et al., 2011). Wild rodents were reported as the source ofMalaya R. sibirica obtained across a large territory known for the endemicity of North Asian tick typhus (Eremeeva & Dasch, 2015).
Bats are often infested by numerous typeof of ectoparasites such as ticks, fleas, flies and
mites (Reeves et al., 2016). A wide range of ectoparasites from bats have been directly
linked to pathogens such as Anaplasma, Borrelia, Bartonella and Rickettsia in ticks and
mites (Brook et al., 2015; Loftis et al., 2005; Reeves et al., 2006b; Socolovschi, 2012).
In this study, the DNA of Rickettsia sp. RF2125, Rickettsia sp. TCM1, R. honei/R.
conorii/R. raoultii, and R. asiatica were detected from various organ tissues (kidney,
liver, spleen) of the bats (Table 4.19). Amongst these rickettsial species, R. asiatica was Universityfirst isolated from I. ovatus ticks in Japan in 1993 (Fujita et al., 2006) and later reported in sika deer in Japan (Jilintai et al., 2008). In a study conducted in United States, R. conorii
and R. rickettsii antibodies have been detected in a bat (Reeves et al., 2006a). Antibodies
against R. rickettsii, R. parkeri, R. amblyommii, and R. rhipicephali have also been
detected in Brazilian bats (D'Auria et al., 2010). R. rickettsii has been isolated from spleen
and liver tissues of wild mice, squirrel and chipmunks in Virginia and Western Montana
192 (Bozeman et al., 1967; Burgdorfer et al., 1962). Serologic evidence of the exposure of
small mammals to SFG rickettsiae has also been documented in Brazil (Coelho et al.,
2016). All these findings suggested the potential role of wild small mammals as a
reservoir for SFG rickettsiae. The sequestration of the rickettsia in tissues other than blood
has been hypothesized by Barrs et al. (2010) and Hawley et al. (2007). However, as
observed in this study, not all the animal organ tissues (liver, spleen, kidney) harvested
from an animal were positive for rickettsial DNA (Table 4.19). Similar finding has been
reported in a study of opossum whereby R. typhi DNA was amplified from the spleen but
not the liver using PCR assay (Williams et al., 1992).
In this study, rickettsial DNA was detected in 31.8 % and 32.9 % of the feeding (on
rodents and skink) and questing ticks collected from the same location. The DNA of both Rickettsia sp. TCM1 and Rickettsia sp. RF2125 were detectedMalaya in ticks feeding on rodents, questing ticks and rodent’s organs, while the DNA of Rickettsia sp. TCM1, R. raoultii
and Rickettsia sp. RF2125 was detected in ofboth bats and questing ticks. The distribution
of rickettsial species in the wild animals and ticks suggest possible transmission of the
rickettsial species in the ticks and animal hosts in the forest reserve.
5.3.9 Limitation and future study
The high detection rate of rickettsial DNA in the small mammals in this study could
be attributed to the organ sampling method in which the animals were dissected in fields.
Hence, there is a possibility that cross-contamination between sample might occur during Universitythe dissection.
Based on BLAST analyses, rickettsial gltA, ompA and ompB gene sequences were
obtained from different tick species in various locations as well as from animal organ
tissues. The presence of more than one rickettsial organism in a single sample was
observed in this study. As rickettsiae are detected mainly by a PCR approach, it is possible
193 that some might have remained undetected due to the bias of PCR assays in amplifying
certain rickettsiae (Kanagawa, 2003). Hence, other microbial detection methods such as
16S metagenomic sequencing would be helpful to complement the findings, especially
when more than one type of rickettsial species is present in the samples. It is challenging
to determine the species status of rickettsiae merely based on sequences of the partial
fragments of rickettsial gltA, ompA and ompB genes. Therefore, the species status of
rickettsiae should be further confirmed by isolation of the rickettsiae.
5.4 Phylogenetic analysis of tick/flea-borne rickettsiae
Rickettsial surveillance in this study provides an overview and updates of rickettsial
species circulating in this region which could be possibly linked to the identification of
potential vectors and maintenance hosts. In this study, rickettsiae are found in association with a wide range of arthropods which feed on differentMalaya species of animals. Based on the phylogenetic analysis of rickettsial gltA gene (Figure 4.21), this study observed that SFG
rickettsiae particularly R. raoultii and RFLOof (Rickettsia sp. RF2125) are the rickettsial
species that were implicated in the febrile patients and infect various animal species and
arthropods samples. Only one patient sample was positive for R. typhi during the
molecular screening. However, R. typhi was not detected in the arthropod and animal
samples in this study although the organism can also be detected through PCR screening
targeting on gltA gene (Roux et al., 1997). The prevalence of R. typhi was probably very
low in the sample investigated. Further investigation on the source of the organism Universityleading to murine typhus in this region is necessary. A wide distribution of SFG rickettsiae in arthropod samples across several states in
Peninsular Malaysia was demonstrated in this study (Figure 4.20). However, there was
no correlation between the distribution of rickettsial species and the geographical regions.
In the present study, the identification of several potentially novel rickettsiae, including
194 Candidatus Rickettsia sepangensis and Candidatus Rickettsia johorensis in Amblyomma
snake tick; R. raoultii (Rickettsia sp. Kagoshima6) in H. bispinosa cattle tick; R. felis in
Rh. microplus cattle tick and Haemaphysalis dog tick in rural villages; and Rickettsia sp.
LON-13 in Haemaphysalis cat tick has expanded the current knowledge concerning the
type and contribution of rickettsiae infecting tick species in this region.
Phylogenetic analysis based on ompB genes of R. felis and RFLO in this study showed
that the clustering of RFLO detected in patient A with RFLO detected in monkey, dog
flea and ticks collected from animal and vegetation (Figure 4.21). This finding indicated
the possible circulation and enzoonotic cycle of RFLO in a wide range of environment in
this region. In addition, the dendrogram constructed based on ompB sequences showed
that R. raoultii detected from patient C and patient D are clustered in the same branch with R. raoultii detected in H. wellingtoni and H. bispinosaMalaya ticks collected from peri- domestic animals, respectively (Figure 4.22). These findings suggest that ticks from peri-
domestic animals could be a potential vectorof for R. raoultii, in particular, Haemaphysalis
ticks. Haemaphysalis species are one of the tick species commonly reported from human
exposed to vegetation (Estrada-Pena & Jongejan, 1999). Human exposure to tick bite (H.
wellingtoni) has been reported in Thailand (Parola et al., 2003c) while H. bispinosa is
known has been reported to have affinity to human (Arunachalam & Harikrishnan, 2009).
The potential role of Haemaphysalis ticks as a vector in transmitting rickettsioses in this
region warrants further investigation.
University5.5 Growth characteristic and comparative phylogenetic analysis of Rickettsia sp. TH2014
This zoonotic surveillance in this study indicated that Rickettsia closely related to R.
raoultii and R. felis/ RFLO are the predominant rickettsial species prevalent in this region.
Both RFLO and R. raoultii were detected in the febrile patients, animal, ticks and fleas
195 examined in this study. The identification of several potential novel species including
Candidatus Rickettsia johorensis, Candidatus Rickettsia sepangensis, and Rickettsia
closely related R. raoultii (Rickettsia sp. Kagoshima6), are noteworthy for further study.
To date, isolation and culture of rickettsiae remain as the major hinder in the research
due to the intracellular nature of the bacteria. Yet, isolation of rickettsiae is a prerequisite
for characterization of new strain found in this region. In this study. none of the attempts
to culture rickettsiae from PCR-positive tick and flea samples were successful. It is
generally believed that the freeze-thaw process of the sample may decrease the bacterial
viability (Sleight et al., 2006).
The isolation of Rickettsia sp. TH2014, was successfully cultured from the homogenates of a pool of C. felis orientis fleas, freshlyMalaya collected from a dog in a rural village with high detection rate of rickettsia in fleas (68.4 %). The isolation and growth of the bacteria in Ae. albopictus C6/36 cellsof was confirmed by using molecular detection methods. The partial gltA, ompB and 16S rRNA genes amplified from Rickettsia sp.
TH2014 showed 100 % sequence identities to Candidatus Rickettsia senegalensis strain
PU01-02. Previously, Mediannikov et al. (2015) reported that the ompB sequence of the
Candidatus Rickettsia senegalensis strain PU01-02 was identical to the small portion (15
% of the total sequence) of ompB sequence for Rickettsia sp. clone HL15c identified in
C. felis flea in Malaysia (Tay et al., 2014). The partial sequences of gltA and ompB for
Rickettsia sp. TH2014 were also identical to the uncultured Rickettsia sp. clone HL15c University(Table 4.20). Unfortunately, 16S rRNA gene sequence for uncultured Rickettsia sp. clone HL15c is not available for further comparison.
Candidatus Rickettsia senegalensis was first described in C. felis collected from a cat
in Senegal (Mediannikov et al., 2015). Since then, it has been reported in C. felis fleas in
Australia (Lawrence et al., 2015a), California (Billeter et al., 2016), Texas (Blanton et
196 al., 2016) and Northeast India (Khan et al., 2016), suggesting its emergence and
widespread in different parts of the world. In Australia, Candidatus Rickettsia
senegalensis DNA (100 % identity) was detected in cat fleas that were not surface-
sterilized (Lawrence et al., 2015a). Additionally, Candidatus Rickettsia senegalensis
DNA was also reported in 25 % of the C. felis fleas in India based on analysis of gltA and
ompB genes (Khan et al., 2016). In United states, Candidatus Rickettsia senegalensis
(demonstrating 99 % similarity) were detected in 10 C. felis flea pools in California. A
total of 46.2 % of opossums infested with fleas in Texas demonstrated molecular evidence
of Candidatus Rickettsia senegalensis (Billeter et al., 2016; Blanton et al., 2016).
However, the roles of Candidatus Rickettsia senegalensis in human and animal diseases
and ecology are still unknown (Mediannikov et al., 2015). Similarly, Rickettsia sp. TH2014 was only detected from fleas, suggestingMalaya that it is possibly a rickettsial endosymbiont in the flea. The pathogenic role of the organism is not known to either human or animal. of
The presence of Rickettsia sp. TH2014 was consider rare in previous Malaysian
investigation as only one flea sample (HL15c) out of 177 fleas examined was found to
harbor the rickettsia (demonstrating identical gltA and ompB sequences to that of
Rickettsia sp. TH2014). The rickettsia was not detected all the flea samples collected from
both urban and rural villages in the first phase of this study (Table 4.17). The rickettsia
was only detected from two C. felis orientis fleas collected in the second field study from Universityone of the rural village in Perak. This could be due to only a small number of positive flea samples were sequenced. The reason for the lower prevalence of Rickettsia sp. TH2014
as compared to Rickettsia sp. RF2125 is still under investigation.
Phylogenetic analysis of concatenated sequences of gltA and rrs gene (Figure 4.43)
shows that Rickettsia sp. TH2014 forms an isolated clade with R. felis, R. asemboensis,
197 Candidatus Rickettsia senegalensis and R. hoogstraalii, in adjacent with R. australis and
R. akari. R. asemboensis, is a rickettsial species closely related to R. felis which was
recently isolated from C. canis and C. felis fleas collected from cats and dogs in Kenya,
Africa (Luce-Fedrow et al., 2015b), whereas R. hoogstraalii was isolated from H. sulcata
ticks in Croatia (Duh et al., 2010). The high sequence similarity between Rickettsia sp.
TH2014 and Candidatus Rickettsia senegalensis was confirmed with a high bootstrap
value (100 %) (Figure 4.43).
Determination of genome pairwise comparisons using ANI is one of the robust
methods for the investigation and confirmation of genomic relatedness between bacterial
strains as a substitute for the more labor intensive DNA-DNA hybridization (DDH)
techniques (Kim et al., 2014; Richter & Rossello-Mora, 2009). The findings from ANI and TETRA analyses (ANIb= 95.02 %, ANIm=96.05 %,Malaya TETRA=0.99255 with reference to R. felis URRWXCal2) confirm the close genetic relatedness of Rickettsia sp. TH2014
to R. felis URRWXCal2. Unfortunately,of the genome of Candidatus Rickettsia
senegalensis is not available for comparison purpose. Overall, the taxonomic finding of
Rickettsia sp. TH2014 based on kSNP confirms the clustering of Rickettsia sp. TH2014
with R. felis, R. hoogstraalii and R. asemboensis (Figure 4.44), consistent with the
phylogenetic analysis based on concatenated sequences (gltA and ompB) using
neighbour-joining method (Figure 4.43).
In order to improve our knowledge regarding the growth characteristics of Rickettsia Universitysp. TH2014, the number of rickettsial DNA copies was determined using a quantitative real-time PCR assay based on the amplification of gltA gene. Bacterial growth is usually
characterized by four phases, i.e., lag, log (exponential), stationary and death. In this
study, a decreasing number of DNA copies was observed after the initial inoculation of
Rickettsia sp. TH2014 in C6/36 cells. It represents a lag phase which is required by the
198 rickettsial organisms to adapt its growth in the host cell. On day 2 onwards, an exponential
growth of rickettsia with rapid replication of rickettsia was observed until day 4. This was
then followed by a slow growing phase or stationary phase (Figure 4.25 and Figure 4.27).
A lag phase of 2 to 3 days has been described for R. helvetica cultivated in Vero cells,
and 0-7.5 hours for R. prowazekii in chicken embryo cells (Elfving et al., 2012; Wisseman
et al., 1976). There were about 6 days of lag phase for R. felis strain LSU in C6/36 and
Drosophila melanogaster S2 cells as the number of DNA copies decreased from the
initiation of infection (0 dpi) until approximately 6 dpi, followed by a subsequent increase
until 20 dpi (Luce-Fedrow et al., 2014). Similar growth pattern of approximately 7 days
of lag phase followed by exponential phase was observed in R. asemboensis cultured in
C6/36 cells (Luce-Fedrow et al., 2015b). The duration of lag phase is associated with bacterial growth phase and/or the morphological formMalaya from which the infecting seeds were isolated (Luce-Fedrow et al., 2014; Wisseman et al., 1976). Quantitation of rickettsial growth in the 24 well tissue cultureof plates showed that the mean lag phase was approximately 4 dpi (Figure 4.29) and the exponential phase was 4- 6 dpi before the
growth slowed down at 6-10 dpi and increased gradually at 10-14 dpi. The longer lag
phase observed in the rickettsia cultured in the 24 well tissue culture plates could be due
to the effect of growth environment whereby there was more inoculum but less cell
surface area and nutrient as compared to the culture condition in T75 tissue culture flasks.
Bright field microscopy (after Giemsa staining) and TEM were used to observe the Universitymorphological characteristics of Rickettsia sp. TH2014 within the host cells. Cytopathic effects due to rickettsial growth were observed at 7 dpi where cell lysis occurred (Figure
4.36). Noticeable vacuolation of cells was seen at 4 dpi (Figure 4.33). TEM analysis at 4
dpi reveals host cells with free rickettsiae in the cytoplasm and vacuole as well as
rickettsiae undergoing binary fission (Figure 4.32 and Figure 4.33). Rickettsiae are known
to replicate by binary fission once they are free in the cytosol (Schaechter et al., 1957).
199 The intracellular rickettsiae appeared to be in both rod and round shape. Both forms are
within the range of normal size for rickettsiae (0.3-0.5 µm by 0.8-2.0 µm) and the round
shape of rickettsiae most likely represent cross-sectional view of the elongated forms
(Figure 4.33-Figure 4.35). Similar observation of other rickettsial species has been
reported in Rickettsia asemboensis (Luce-Fedrow et al., 2015b), R. felis (Luce-Fedrow et
al., 2014), R. bellii (Labruna et al., 2004) and R. prowazeki (Wisseman et al., 1976). One
of the features of SFG rickettsiae is their ability to grow intranuclearly as reported for R.
helvetica (Burgdorfer et al., 1979). However, intranuclear growth of Rickettsia sp.
TH2014 was not seen in the TEM analysis. According to Elfving et al. (2012), the
incidence of intranuclear growth phenomenon was low and may not be an optimal
criterion for differentiation between rickettsial species. More experiments will be necessary to establish whether Rickettsia sp. TH2014Malaya is able to grow intranuclearly in C6/36 cells or other cells.
5.6 Genome properties and comparativeof pathogenomics of Rickettsia sp.
TH2014
A draft genome of Rickettsia sp. TH2014 was described in this study. The estimated
genome size of Rickettsia sp. TH2014 (1.37 MB) is within the range of typical genome
size for rickettsiae. Rickettsia spp. are strict intracellular bacteria which are well known
for their small genomes size (1 to 2.1 Mb) due to genome degradation (Duan et al., 2014).
Intracellular bacteria typically possess small genomes as a consequence of the reduction Universityof originally larger genomes invariably accompanying the adaptation to parasitic lifestyle (Blanc et al., 2007a). Previous studies revealed extreme genome reduction and massive
gene loss in highly-vertebrate-pathogenic rickettsial organisms compared with less
virulent species implying that gene loss is the primary feature of evolution and
specialization in obligate intracellular pathogenic bacteria (Georgiades et al., 2011b).
200 There is no significant plasmid recognized in the draft genome of Rickettsia sp.
TH2014. Plasmids are diversely distributed among bacterial strains of a species with some
having no plasmid and some having one or several plasmids. Rickettsial plasmids were
first detected in R. felis and subsequently reported in another 10 SFG rickettsiae species,
i.e., R. africae, R. amblyommii, R. australis, R. endosymbiont of Ixodes, R. helvetica, R.
massiliae, R. monacensis, R. peacockii, R. raoultii, and R. rhipicephalii. (El Karkouri et
al., 2016). On the other hand, plasmid has not been identified in 12 other SFG rickettsiae
and TG rickettsiae, including R. akari, R. bellii, R. canadensis, R. conorii, R.
heilongjiangensis, R. japonica, R. montanensis, R. parkeri, R. prowazeki, R. philipii, R.
rickettsii, R. slovaca, R. sibirica, and R. typhi.
A few factors are known to contribute to the presence or absence of plasmid in rickettsial genome. The presence of two plasmid formsMalaya (pRF and pRF delta) in R. felis URRWXCal2 has been unambiguously confirmed by five independent laboratories
worldwide. It has been observed that the plasmidof content of R. felis, from none to two
plasmid forms, may vary according to their culture passage history as the small plasmid
form are unstable (Fournier et al., 2008). Plasmid loss has been demonstrated in serially
passaged R. peacockii (Baldridge et al., 2008). In this study, the genome of Rickettsia sp.
TH2014 was derived from an established culture which has undergone 14 passages.
Hence, it is not possible to determine whether there is any plasmid loss without genome
sequencing of the earlier passages.
UniversityIn addition to culture conditions, the plasmid content of R. felis may vary from one strain to another. This is evidenced by the presence of pRF but not pRF delta in R. felis
strain LSU and Rickettsia sp. RF2125 in A. erinacei fleas from Algeria (Bitam et al.,
2006; Pornwiroon et al., 2006). In contrast, Rolain et al. (2009) failed to detect any
plasmid form, neither pRF nor pRF delta, from Rickettsia sp. RF2125 in A. erinacei fleas
201 from France and Rickettsia sp. RF2125 in C. canis fleas in Gabon. Their data indicated
that the two genotypes may differ by the presence or the absence of such conjugative
plasmids depending on the source of fleas and on the area of collection (Rolain et al.,
2009).
5.6.1 RAST prediction o virulence genes in Rickettsia sp. TH2014
RAST analysis provides prediction and annotation for genes with a role in virulence,
defence; regulation and cell signaling of Rickettsia sp. TH2014. The genes associated
with virulence, defence, and intracellular resistance include copper homeostasis, cobalt-
zinc-cadmium resistance, resistance to fluoroquinolones, Beta-lactamase, and
Mycobacterium virulence operon involved in protein synthesis (SSU ribosomal proteins)
(Table 4.21). The genes involved in this system are also found in R. felis URRWXCal2 (taxonomy ID: 315456.3) through function-based comparisonMalaya in RAST pipeline. The resistance profile obtained from genome analysis explains the inefficacy of some
antibiotics against rickettsioses. Beta-lactamsof that are frequently used to treat febrile
patient is known to be ineffective against tickborne rickettsioses (Biggs, 2016). Although
some fluoroquinolones show in vitro activity against rickettsiae, however, their use in
certain rickettsial infections are associated with deleterious effect, i.e., delayed
subsidence of fever, increased disease severity, and longer clinical onset of the disease
(Botelho-Nevers et al., 2011; Rolain et al., 1998).
Genes encoding programmed cell death and toxin-antitoxin (TA) systems are Universitycategorized under regulation and cell signaling category in RAST analysis. The number of TA systems are higher in pathogenic bacteria such as Yersinia pestis (Buts et al., 2005).
Overall, Rickettsia sp. TH2014 has less number of TA genes as compared to R. felis
URRWXCal2 except for YdcE, YdcD and YefM (Table 4.29), suggesting that Rickettsia
sp. TH2014 is possibly less virulent as compared to R. felis URRWXCal2. YdcE gene
202 encodes for EndoA activity (also known as cleavage activity) which is toxic to the growth
of both Escherichia coli and Bacillus subtilis. This toxicity is reversed by co-expression
of YdcD (Pellegrini et al., 2005). YefM is the antitoxin to the YoeB toxin which is an
endoribonuclease and an autorepressor that recognizes a long palindrome containing the
core hexamer, followed by binding to the short repeat (Bailey & Hayes, 2009). The gene
encoded for YoeB has to be reexamined after the complete genome of Rickettsia sp.
TH2014 is available. The ParE-ParD toxin-antitoxin annotated in R. felis URRWXCal2
genome was not annotated in Rickettsia sp. TH2014. ParE, acts as DNA gyrase inhibitor
induces DNA-gyrase covalent complexes formation, and inhibits replication and damage
chromosome integrity (Jiang et al., 2002). The absence of ParE-ParD in Rickettsia sp.
TH2014 could be due to the incomplete genome sequence. On the other hand, RelE and
Doc toxins which are inhibitors of translation are annotated in both Rickettsia sp. TH2014
and R. felis URRWXCal2 (Christensen et al., 2001; Hazan et al., 2001). Additionally,
VapC toxin which is present in R. felis URRWXCal2 and toxic to eukaryotes upon
injection into the cytoplasm as a free toxin is also identified in the Rickettsia sp. TH2014
genome (Audoly et al., 2011). Rickettsial vapC gene is over transcribed and over-
expressed and is released in the host cytoplasm to cause early cell death upon exposure
to chloramphenicol (Audoly et al., 2011).
The bacterial TA systems participating in the cascade of the stringent response
pathway are induced by alarmones [guanosine tetra- (ppGpp) and pentaphosphate University(pppGpp)] whose intracellular concentration of is regulated Malaya by SpoT/RelA enzymes (Ogata et al., 2006). Two genes encoding for SpoT (spoT and spoT11), a hydrolase that involved
in the metabolism of guanosine tetra- (ppGpp) and pentaphosphate (pppGpp), are
annotated in Rickettsia sp. TH2014 genome. Intracellular parasitic rickettsiae undergo
dormancy and multiplication following activation during host starvation and feeding
(Burgdorfer & Brinton, 1975; Fournier et al., 2009). Accumulation of guanosine
203 nucleotides pppGpp (guanosine 3’-diphosphate 5’ triphosphate) and ppGpp (guanosine
3’-diphosphate 5’-diphosphate) are mediated during bacterial adaptation to nutritional
stress which is known as stringent response (Cashel et al., 1996). Rickettsia sp. TH2014
had more spoT genes (2) than R. prowazekii (1 gene), but fewer genes than R. felis (14
genes), R. bellii (10), R. akari (7), R. rickettsii (5), R. conorii (4), R. sibirica (4), and R.
typhi (4) as reported by Fournier et al. (2009). The presence of a high number of TA loci
and spoT genes in SFG rickettsiae, suggests that the stringent response pathway is
important for obligate intracellular bacteria (Ogata et al., 2006).
5.6.2 Virulence genes for Rickettsia sp. TH2014 searched against VFDB
The detection of typical profile of virulence genes as R. felis (identified in the VFDB
database) suggests that Rickettsia sp. TH2014 may possess the similar pathogenicity pattern and adaptation ability in the eukaryotic host. MalayaThe virulence factors of Rickettsia sp. TH2014 was determined by BLAST search in the VFDB database. All the major
virulence factors in Rickettsia listed in VFDBof database were annotated in Rickettsia sp.
TH2014 except for the enzyme phospholipase A2. The virulence factors include proteins
involved in actin based motility, adherence and invasion, phagosomal escape enzyme and
vir homolog (rvh) type IV secretion systems (T4SS). SFG rickettsiae gain entry to the
host cells by adhesion whereby there are several rickettsial adhesins (Adr1, Adr2, rOmpA,
rOmpB, Sca1 and Sca4) that bind to the host cell receptors. Subsequently, activation of
intracellular signaling pathways induces actin polymerization and membrane Universityrearrangement causing the attached rickettsiae to be engulfed. Rickettsia escaped from phagosomes that engulf them by secreting phospholipases TlyC and Pld. Surface protein
RickA and Sca2 form an actin tail which aids the movement of the bacteria. T4SS help
rickettsiae to survive intracellularly as it allows them to secrete effector molecules
(Uchiyama, 2012).
204 Bacterial cell surface antigen (sca) proteins play important roles in the interaction with
host cells. OmpA (Sca0), Sca1, Sca2, Sca4 and ompB (Sca5) are five sca genes that appear
to have evolved under positive selection and present in nearly all the SFG rickettsiae
(Blanc et al., 2005). Adr1, Adr2, ompA, Sca1, Sca4 and ompB are genes encoding
adhesins that are categorized under adherence and invasion in the VFDB database. Based
on the genome analysis of Rickettsia sp. TH2014, the sequence similarities of these
virulence genes ranged from 72.5 % to 98.1 % when comparing with R. felis
URRWXCal2 (Table 4.28). The lower sequence similarity observed in some of the
virulence genes (rickA, sca2, adr1, sca1 and sca4) could be due to the gaps present in
Rickettsia sp. TH2014 genome.
Phospholipase A2 (PLA2) has been implicated in both rickettsial entry into the host cell and escape from phagosome (Walker et al., 2001).Malaya The expression and activity of PLA2 homologs in SFG rickettsiae have yet to be determined (Welch et al., 2012). It has
been difficult to identify genes that encode ofPLA2 enzymes (Welch et al., 2012). Putative
PLA2 motifs of various rickettsial proteins and Pseudomonas aeruginosa ExoU as
published by Rahman et al. (2010) were searched against Rickettsia sp. TH2014 genome
but none of the PLA2 genes (pat1 and pat2) was found. Other genes with potential
membranolytic activities in rickettsial genomes include hemolysin A (tlyA), hemolysin C
(tlyC) and phospholipase D (pld) (Whitworth et al., 2005). It is possible that Rickettsia
sp. TH2014 utilizes these virulence factors for membrane entry and phagosomal escape Universityin replacement with PLA2. The absence of PLA2 genes in Rickettsia sp. TH2014 could be due to the gaps or incomplete genes in the draft genome. Further confirmation is
needed to confirm the presence or any mutations in genes encoding for enzyme
phospholipase A2 in Rickettsia sp. TH2014.
205 Intracellular bacterial pathogens usually undergo actin-based motility to facilitate cell-
bacteria spread during infection (Haglund & Welch, 2011). SFG rickettsiae possess two
actin polymerizing proteins, i.e. RickA and Sca2 which are also found in Rickettsia sp.
TH2014. RickA acts as an activator of the host Arp2/3 complex involved in early actin-
based motility after infection. The mode of motility is faster with bacterial Sca2 later in
the infection and independent of RickA and Arp2/3 (Reed et al., 2014).
All of the 18 genes involved in rickettsial vir homolog (rvh) T4SS were annotated in
Rickettsia sp. TH014 draft genome with 96.3 % to 99.7 % sequence identity to those of
rvh in R. felis URRWXCal2 (Table 4.28). As in concordance with other rickettsial
species, Rickettsia sp. TH2014 is lacking of the VirB5 homolog, a minor pili playing a
role in T-pilus formation. The lack of VirB5 in rickettsiae is correlated with rickettsial intracellular lifestyle whereby a VirB5 protein wouldMalaya be redundant as substrates would be secreted and imported directly from the host environment (Gillespie et al., 2009;
Schmidt-Eisenlohr et al., 1999). of
The components of F-T4SS annotated in Rickettsia sp. TH2014 include IncF plasmid
conjugative transfer proteins (TraD, TraG, and TraN) and IncF plasmid conjugative
transfer pilus assembly proteins (TraB TraC, TraH, TraF, TraU and TraW). F-T4SS are
mating pair formation proteins essential for conjugation which are common throughout
the genus Rickettsia (Lawley et al., 2003; Weinert et al., 2009). Nearly full set of genes
related to the tra-trb operon of F-plasmid of E. coli was observed in R. belli strain RML University369-C, R. felis strain LSU-Lb and R. massiliae despite of the fact that R. belli does not harbour plasmid (Blanc et al., 2007b; Gillespie et al., 2015b; Ogata et al., 2006). Genes
involved in the conjugative DNA transfer are encoded on R. bellii chromosome and these
includes traDTi, traATi, traD, traG, traH, traF, traN, traC, traU, traW, traV, traB, and
traE (Ogata et al., 2006). However, other rickettsiae harboring plasmids (R. africae, R.
206 monacensis and R. peacockii) do not carry full tra-trb-like operon. These partial tra
clusters are non-functional in some cases (Baldridge et al., 2007; Ogata et al., 2005b).
Similarly, Rickettsia sp. TH2014 does not carry all the conjugative Tra components.
5.6.3 Limitations and future study
For future sequencing study, the number of scaffolds should be reduced to 20. The
functional analysis of rickettsia is not complete due to the presence of the gaps in the
genome. Hence, the sequences for phospholipase A2 enzyme (PLA2) and some TA
systems may not be annotated. A near future study on the gaps-filling in the Rickettsia sp.
TH2014 genome should be carried out. The captured gaps can be closed with primer
walking on the clones while uncaptured gaps can be closed by using multiplex PCR,
inverse PCR, restriction site PCR, capture PCR and adaptor-PCR (Rogers et al., 2005). Additionally, primers can be obtained to complete the Malayagenome by mapping to the existing genomes of R. felis. Gaps in the genome can be filled by using bioinformatic tools such
as Sealer (Paulino et al., 2015), GapCloser ofin SOAPdenovo2 package (Luo et al., 2012)
and GapFiller (Boetzer & Pirovano, 2012). A complete comparative analysis and detail
insight of Rickettsia sp. TH2014 with other RFLO, particularly Candidatus Rickettsia
senegalensis which has been reported in other geographical regions (India and Australia),
can be carried out upon the availability of the complete genome. The integration of these
data in an international database could help to understand the circulation of RFLO and
their emergence or reemergence.
UniversityWith the availability of the isolate and genome data, some of the virulence genes can be further explored for identification of specific genetic markers for rapid detection of
RFLO or development of serological tools. Apart from that, a continuous surveillance of
rickettsiae using larger sample size in this country is important to understand the
epidemiology and transmission dynamics of these organisms. This information is needed
207 for monitoring of disease outbreak, identification of novel strains as well as developing
comprehensive approaches for modern surveillance, diagnosis, prevention and control
measures for rickettsioses.
Malaya of
University
208 CHAPTER 6: CONCLUSION
This study gives an overview of various rickettsioses implicated in Malaysian febrile
patients, and determines the extent of exposure to SFG rickettsiae in two populations
(indigenous community and animal farm workers) who are at high risk of tick bites in
Peninsular Malaysia. A R. felis-like organism, R. typhi and a rickettsia closely related to
R. raoultii were detected in four febrile patients admitted to UMMC between 2012-2014
using molecular approaches. The clinical presentations of the patients were nonspecific
with petechiae observed only in a patient infected with Rickettsia sp. RF2125. The finding
of this study suggests that clinicians should consider SFG rickettsioses in the differential
diagnoses of febrile patients. Laboratory diagnosis using molecular approach is essential to establish the diagnosis for rickettsioses. Malaya This study shows that the seropositivity of SFG rickettsiae (R. conorii and R. felis) were significantly higher in the indigenousof community and animal farm workers, as compared to urban blood donors. This finding indicates that SFG rickettsioses is a health
concern to the indigenous community and farm workers. In the vector and zoonotic
surveillance study, various tick species (Amblyomma spp., Dermacentor spp.,
Haemaphysalis spp., Rh. sanguineus, and Rh. microplus) were identified from different
ecosystems. C. felis felis and C. felis orientis are the predominant flea species infesting
cats and dogs examined in this study. PCR screening targeting on gltA, ompA and ompB
genes revealed the presence of rickettsial DNA in 16.3 %, 9.3 % and 40.6 %, of ticks Universitycollected from urban areas (stray dogs), animal farms (cattle and sheep) and rural villages (peri domestic animals), respectively. In the forest area, 31.8 % and 32.9 % of the feeding
ticks and questing ticks (Amblyomma spp., Dermacentor spp., and Haemaphysalis spp.)
respectively, were positive for Rickettsia sp. asiatica, R. heilongjiangensis, R. raoultii,
Rickettsia sp. RF2125, R. tamurae, and Rickettsia sp. TCM1 in this surveillance study.
209 Rickettsial DNA detection rates were higher in the fleas collected from rural villages
(66.2 %) as compared to urban areas (8.1 %). The higher occurrences of rickettsiae in the
ticks/fleas collected in rural villages may explain the relatively high seropositivity
observed in the indigenous community who reside at the rural villages.
Several potentially novel species, including Candidatus Rickettsia johorensis in
Amblyomma snake ticks and H. exigua flies, Candidatus Rickettsia sepangensis in
Amblyomma snake ticks, and Rickettsia sp. Kagoshima 6 (R. raoultii) in cattle ticks have
been identified. Conversely, none of the mosquitoes, Ae. albopictus and Cx.
quinquefasciatus, were positive for rickettsial DNA in this study. A low detection rate of
rickettsia (1.1 %) was observed in the blood samples of domestic animals investigated in
this study. The DNA of Rickettsia sp. TCM1 was detected from two buffaloes and a horse. Examination of wildlife samples show the detection Malayaof rickettsial DNA (Rickettsia sp. RF2125) in 24.0 % of 50 cynomolgus monkey (Macaca fascicularis) blood samples and
Rickettsia sp. RF2125, Rickettsia sp. TCM1,of R. honei/R. conorii/R. raoultii, and R.
asiatica from the organ tissue samples of four rats, seven bats and a squirrel in a forest
reserve. Further investigation and characterization of these potentially novel rickettsiae
are required.
In summary, Rickettsia closely related to R. raoultii and RFLO (Rickettsia sp. RF2125)
are the predominant Rickettsia species circulating in Peninsular Malaysia as they are
detected in human, ticks, fleas, and wildlife. A R. felis-like organism (designated as UniversityRickettsia sp. TH2014) was isolated from C. felis orientis flea collected from a dog flea in a rural village in this study. The gltA, ompB and rrs sequences of Rickettsia sp. TH2014
were identical to Candidatus Rickettsia senegalensis strain PU01-02 in Senegal and
uncultured Rickettsia sp. clone HL15c from a flea in previous Malaysian study. The gltA,
210 ompB and rrs gene fragments of Rickettsia sp. TH2014 showed 98.3 %, 94.8 % and 99.6
% similarity, respectively, as compared to R. felis URRWXCal2 type strain.
The draft genome of Rickettsia sp. TH2014 (GenBank accession no.
MXAX00000000) was determined by Hi-seq sequencing and assembled using ABySS
software to a depth of 954X coverage. The estimated size and GC content of the rickettsia
are 1.37 Mb and 32.9 %, respectively. RAST analysis showed 1, 465 predicted coding
sequences, 32 tRNA genes and 2 rRNA genes. Plasmid was not detected using
PlasmidFinder and mapping of the assembled genome to the reference plasmids. Pairwise
genome comparisons confirmed the closely genetic relatedness of Rickettsia sp. TH2014
with Rickettsia felis URRWXCal2 (ANIb=95.02 %, ANIm=96.05 %, TETRA=0.99255).
The grouping of Rickettsia sp. TH2014 in the same cluster with R. felis URRWXCal2 and other RFLOs (R. asemboensis and R. hoogstraalii)Malaya was demonstrated by comparative whole-genome single nucleotide polymorphism (SNP) analysis. Genes responsible for
the basic function of Rickettsia sp. TH2014,of resistance to antibiotics and toxic compounds
and virulence genes were annotated through RAST analysis. An array of virulence genes
including those associated with actin-based motility, adherence and invasion, type IV
secretion systems and toxin-antitoxin systems were also annotated in the genome. Further
works on gap-filling of the Rickettsia sp. TH2014 genome are required for precise
understanding and identification of gene marker or development of new serological and
molecular detection tools.
UniversityThe research described in this study sought to determine the overall distribution of rickettsial species in different human populations, arthropods, and animals from different
localities in Peninsular Malaysia. The findings are important in illustrating the
involvement of ticks/fleas and animals as vectors and maintenance hosts in the
transmission of rickettsioses and would be beneficial to public health authority in
211 formulating prevention and control strategies for rickettsioses. The establishment of
Rickettsia sp. TH2014 in cell culture facilitates future investigations of its transmission,
distribution and pathogenic potential to human and animal.
Malaya of
University
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LIST OF PUBLICATIONS AND PAPERS PRESENTED
Journals (ISI-cited, Tier-1) related to thesis
1. K.L. Kho, F.X. Koh, L.I. Mohd Hasan, L.P. Wong, M.G. Kisomi, A. Bulgiba, Q. N. Hassan Nizam and S. T. Tay. (2017). Rickettsial seropositivity in the indigenous community and animal farm workers, and vector surveillance in Peninsular Malaysia. Emerging Microbes and Infections, 6, e18, doi:10.1038/emi.2017.4. 2. K.L. Kho, F.X. Koh, H.K. Lakhbeer Singh, H.A. Mohamed Zan, S. Ponnampalavanar, A. Kukreja, and S. T. Tay. (2016). Case report: spotted fever group rickettsioses and murine typhus in a Malaysian teaching hospital. American Journal of Tropical Medicine and Hygiene, 95(4):765-768. Doi:10.4269/ajtmh.16-0199. 3. K.L. Kho, F.X. Koh, and S.T. Tay. (2015). Molecular evidence of potential novel spotted fever group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks parasitizing wild snakes. Parasites and Vectors, 8: 112. DOI: 10.1186/s13071-015- 0719-3. 4. S.T. Tay, F.X. Koh, K.L. Kho, and F.T. Sitam. (2015). Rickettsial infections in Monkeys, Malaysia. Emerging Infectious DiseasesMalaya, 21(3), 545-547. Other Journals of 1. M. Ghane Kisomi, L. P. Wong, S.T. Tay, A. Bulgiba, K. Zandi, K.L. Kho, et al. (2016) Factors Associated with Tick Bite Preventive Practices among Farm workers in Malaysia. PLoS ONE, 11(6): e0157987. doi:10.1371/journal.pone.0157987. 2. F.X. Koh, K.L. Kho, C. Panchadcharam, F.T. Sitam, and S.T. Tay. (2016). Molecular detection of Anaplasma spp. in pangolins (Manis javanica) and wild boars (Sus scrofa) in Peninsular Malaysia. Veterinary Parasitology, 227, 73-76. ISSN 0304- 4017, http://dx.doi.org/10.1016/j.vetpar.2016.05.025. 3. J.J Khoo, F. Chen, K.L. Kho, AI. Ahmad Shanizza, F.S. Lim, K.K. Tan, L.Y. Chang and S. Abu Bakar. (2016). Bacterial community in Haemaphysalis ticks of domesticated animals from the orang asli communities in Malaysia. Ticks and Tick- Borne Diseases, 7, 929-937. doi:10.1016/j.ttbdis.2016.04.013. 4. S.T. Tay, K.L. Kho, W.Y. Wee, and S.W. Choo. (2016). Whole-genome sequence Universityanalysis and exploration of the zoonotic potential of a rat-borne Bartonella elizabethae. Acta Tropica, 155, 25-33. 5. M. Mohd Shukri, K.L. Kho, M. G. Kisomi, R. Lani, S. Marlina, S. F. Muhd Radzi, S. T. Tay, L. P. Wong, A. B. Awang Mahmud, Q.N.H. Nizam, S. Abu Bakar, and K. Zandi. (2015). Seroprevalence report on tick-borne encephalitis virus and Crimean- Congo hemorrhagic fever virus among Malaysian’s farm workers. (2015). BMC Public Health, 15, doi: 10.1186/s12889-015-1901-4. 6. K.L. Kho, F.X. Koh, T. Jaafar, Q.N.H. Nizam, and S.T. Tay. (2015). Prevalence and molecular heterogeneity of Bartonella bovis in cattle and Haemaphysalis bispinosa ticks in Peninsular Malaysia. BMC Veterinary Research, 11:153.
261 7. V.L Low, S.T, Tay, K.L. Kho, F.X. Koh, T.K. Tan, Y.A.L. Lim, C. Panchadcharam, B.L. Ong, Y. Norma-Rashid and M. Sofian-Azirun. (2015). Molecular characterization of the tick Rhipicephalus microplus in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world. Parasites & Vectors, 8: 341. 8. T.K. Tan, V.L. Low, S.C. Lee, C. Panchadcharam, S.T. Tay, R. Ngui, P. Bathmanaban, K.L. Kho, F.X. Koh, R.S.K. Sharma, T. Jaafar, Q.N.H. Nizam and Y.A.L. Lim. (2015). Detection of Schistosoma spindale ova and associated risk factors among Malaysian cattle through coprological survey. The Japanese Journal of Veterinary Research, 63: 63-71. 9. S.T. Tay, A.S. Moktar, K.C. Low, S.N. Mohd Zain, J. Jeffery, N. Abdul Aziz and K.L. Kho. (2014). Short communication: Identification of rickettsiae from wild rats and cat fleas in Malaysia. Medical and Veterinary Entomology, 28, 104-108. 10. S.T. Tay, F.X. Koh, K.L. Kho, B.L. Ong. (2014). Molecular survey and sequence analysis of Anaplasma spp. in cattle and ticks in a Malaysian Farm. Tropical Biomedicine, 31(4): pp 769-776.
Conference proceedings Malaya 1. K.L. Kho, F.X. Koh, L.I. Mohd Hasan, H. Kaur, T. Jaafar, Q.N. Hassan Nizam, and T.S. Tay. (2015). Antibody prevalence of rickettsial infections in different human populations and molecular detection of rickettsiaeof in animal ectoparasites in Malaysia. 25th International Conference of the World Association for the Advancement of Veterinary Parasitology, United Kingdom, pg. 345. 2. K.L. Kho and S.T. Tay (2013). Molecular detection of rickettsioses in animal and arthropod vectors in Malaysia. 24th International Conference of the World Association for the Advancement of Veterinary Parasitology, Australia, pg. 636. 3. K.L. Kho and S.T. Tay (2013). Molecular investigation of Rickettsia and Bartonella species in cat fleas (Ctenocephalides felis) from domestic cats and dogs in west Malaysia. Proceedings of 49th Annual Scientific Conference of the Malaysian Society of Parasitology and Tropical Medicine, pg. 72.
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