Rickettsiales and rickettsial diseases in Australia - Murdoch ...

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1.4.3. Isolation ...................................................................................... 14 1.4.4. Polymerase Chain Reaction (PCR) ............................................ 14 1.4.4.1. Conventional PCR ............................................................... 15 1.4.4.2. Real-time PCR (qPCR) ........................................................ 15 1.4.4.3. Molecular speciation ............................................................ 16 1.5. Geographic distribution of the Rickettsiales ..................................... 18 1.6. Rickettsiales species in Australia ..................................................... 18 1.6.1. Anaplasma ................................................................................. 18 1.6.1.1. Australian (cattle) tick fever (Anaplasma marginale) .............. 18 1.6.1.2. ... Canine infectious cyclic thrombocytopenia (Anaplasma platys) ............................................................................................................ 19 1.6.2. Ehrlichia ..................................................................................... 20 1.6.3. Rickettsia .................................................................................... 20 1.6.3.1. Murine typhus (Rickettsia typhi) ........................................... 22 1.6.3.2. Queensland tick typhus (Rickettsia australis) ...................... 25 1.6.3.3. Flinders island spotted fever (R. honei) ............................... 27 1.6.3.4. Australian spotted fever (R. honei strain marmionii) ............ 28 1.6.4. Orientia ....................................................................................... 30 1.6.4.1. Scrub typhus (Orientia tsutsugamushi) ................................ 30 1.6.5. This Study .................................................................................. 32 Chapter 2. Materials and Methods ......................................................... 34 2.1. Rickettsial Isolation .......................................................................... 34 2.1.1. Blood sample processing ........................................................... 34 2.1.2. Tick sample processing .............................................................. 34 2.1.3. Flea sample processing ............................................................. 35 v
2.2. Cell Culture ...................................................................................... 35 2.3. Freezing Samples ............................................................................ 36 2.4. Molecular Methods .......................................................................... 37 2.4.1. DNA sample preparation ............................................................ 37 2.4.2. Primer/probe design and validation ............................................ 37 2.4.2.1. Primer/probe set design ....................................................... 37 2.4.2.2. Testing Sensitivity ................................................................ 38 2.4.2.3. Testing Specificity ................................................................ 39 2.4.3. qPCR detection .......................................................................... 39 2.4.4. Conventional PCR ...................................................................... 41 2.4.5. Sequencing ................................................................................ 45 2.4.6. Bioinformatics ............................................................................. 45 2.5. Serology .......................................................................................... 46 2.5.1. Microimmunofluorescence ......................................................... 46 Chapter 3. A serological prevalence study for rickettsial exposure of cats and dogs in Launceston, Tasmania, Australia .................................. 48 3.1. Abstract ........................................................................................... 48 3.2. Introduction ...................................................................................... 49 3.3. Materials and methods .................................................................... 50 3.3.1. Sample and data collection ........................................................ 50 3.3.2. Detection of antibodies to Spotted Fever Group Rickettsia ........ 51 3.3.3. Statistical analysis of serological results .................................... 51 3.4. Results ............................................................................................. 51 3.4.1. Serology Results ........................................................................ 51 3.4.2. Statistical analysis ...................................................................... 52 vi
Page 1 and 2: Rickettsiales and rickettsial disea
Page 3 and 4: Abstract Currently, there are 12 kn
Page 5: Table of Contents Rickettsiales and
Page 9 and 10: 6.5. Results ......................
Page 11 and 12: List of Figures Figure 1. Phylogene
Page 13 and 14: software. Bootstrap values are indi
Page 15 and 16: Acknowledgements I would first like
Page 17 and 18: Preface The work in this thesis is
Page 19 and 20: Izzard L, Fuller A, Blacksell S, Pa
Page 21 and 22: Abbreviations AG - Ancestral group
Page 23 and 24: sca4 - The rickettsial gene D / PS-
Page 25 and 26: In 1989, Coxiella burnetii was remo
Page 27 and 28: 1.2. Taxonomy and Pathogenicity of
Page 29 and 30: Amblyomma 13, 31, 232 , Rhipicephal
Page 31 and 32: inflammation around the brain and f
Page 33 and 34: eplaced by transport systems in Ric
Page 35 and 36: Table 2. Current list of validated
Page 37 and 38: Anaplasmataceae 136, 179, 181 . The
Page 39 and 40: 1.5.4.3. Molecular speciation Initi
Page 41 and 42: 1.6. Geographic distribution of the
Page 43 and 44: The brown dog tick (Rhipicephalus s
Page 45 and 46: marmionii) was detected in Victoria
Page 47 and 48: of material and of rodent populatio
Page 49 and 50: third article was published that co
Page 51 and 52: positive for SFG rickettsia, wherea
Page 53 and 54: 1.7.4. Orientia 1.7.4.1. Scrub typh
Page 55 and 56: eported the presence of L. deliense
Page 57 and 58:
Chapter 2. Materials and Methods 2.
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All cell lines except XTC-2 were gr
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system that took into consideration
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depending on the specific condition
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Australia). A 100 bp DNA ladder (In
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Table 3 continued. Reference Anneal
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method within the PHYLIP software p
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Chapter 3. A serological prevalence
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The study was focused around Launce
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Overall, 53% of the dogs and 63% of
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off. There was however no statistic
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from these animals could also be us
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4.2. Introduction Currently in Aust
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Figure 3. Map of Tasmania, Australi
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Figure 4. Phylogenetic tree showing
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East (more than 10,000 kilometres n
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Worldwide, the presence of SFG rick
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Page 93 and 94:
Page 95 and 96:
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ather than a new species. Both the
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named the “ELB agent” after the
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She was appropriately immunised, wi
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igors and had been admitted to anot
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Animal inoculation was also attempt
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Figure 10. A condensed phylogenetic
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cats to R. felis positive fleas res
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This new isolate appears to be the
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7.3. Clinical case history A 52-yea
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CRP was elevated. Liver function te
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egions. The specific primers that t
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7.5.2. Culture The Vero cell cultur
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Figure 14. Phylogenetic trees showi
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When tested with O. tsutsugamushi s
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specify that a 16S rRNA sequence di
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Chapter 8. Anaplasma platys in Aust
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8.3. Methods 8.3.1. Probe Design Al
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above. The assay was able to detect
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RNA gene of A. platys was published
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Chapter 9. Concluding Remarks Littl
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which has been tentatively named Ri
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A new species of Orientia was isola
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Appendices Appendix 1: Mathematical
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8. Andrew R, 1994. Queensland tick
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immunogenicity and transmissibility
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36. Brumpt E, 1932. Longevite de vi
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52. Currie B, O'Connor L, Rhodes F,
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subjective synonyms of Ehrlichia ph
Page 153 and 154:
82. Fournier PE, Roux V, Raoult D,
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99. Grove DI, 1988. African tick ty
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118. Ishikura M, Ando S, Shinagawa
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131. Kelly PJ, Beati L, Mason PR, M
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147. Marquez FJ, Muniain MA, Perez
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162. Neilson GH, 1955. A case of Qu
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177. Paddock CD, Sumner JW, Comer J
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191. Pinn TG, Sowden D, 1998. Queen
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207. Renesto P, Ogata H, Audic S, C
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225. Seong SY, Choi MS, Kim IS, 200
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242. Stewart RS, 1991. Flinders Isl
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258. Unsworth NB, Stenos J, Faa AG,
Page 177 and 178:
273. Watts MR, Benn RA, Hudson BJ,
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