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1.5. Methods of identification and characterisation of Rickettsiales 1.5.1. Staining Organisms in the order Rickettsiales are considered Gram-negative-like, however they stain very poorly with the Gram staining method 136 and are typically stained using Giemsa or Gimenez staining methods 92 . This technique works particularly well with members of the Anaplasmataceae family, as they form clusters within vacuoles in the host cell and are therefore quite clearly visible when stained 31, 76 . The exception to this is Anaplasma platys, which is often difficult to detect due to low or absent numbers during certain stages of its cyclic parasitaemia 28, 101, 148 . Immunohistochemical staining of biopsy samples can also be an effective method of rickettsial detection; however this method has limited specificity in detecting different species 176 . 1.5.2. Serology The first serological method for detection of rickettsial disease (the Weil-Felix test) was designed in 1916 276 . This method relied on the cross-reaction of rickettsial antibodies with Proteus vulgaris strains OX2 and OX19 for SFG and TG and P. mirabilis strain OXK for STG rickettsia 201 . This test however has both low sensitivity and specificity 97 . Rickettsial antibodies in human and animal specimens have since been detected using various methods including complement fixation (CF) 229 , enzyme-linked immunosorbent assay (ELISA) 100 and immunofluorescence assay (IFA) 97 . Of these, IFA is considered the reference method for the detection of antibodies to both Rickettsiaceae and 13
Anaplasmataceae 136, 179, 181 . The main limitations with this method are the degree of cross-reactivity between species of the same genus 181 , and that it is a retrospective analysis, as it can take several weeks to detect antibodies, usually well after the patient has recovered. 1.5.3. Isolation Isolation of Rickettsiales from a clinical sample is the most definitive diagnostic method for detection of a rickettsial infection 181 . It can be performed using various samples including buffy coat from heparinised or EDTA-treated whole blood, triturated blood clot, skin biopsy and necropsy tissue. Alternatively, if a suspect arthropod is available, cultures can be performed directly on it 136, 181 . In the past animal inoculation and yolk sac culture were the commonly used isolation techniques 48, 98 . Cell culture methods are now the main techniques used for isolation of Rickettsiaceae and Anaplasmataceae 106, 136 , although animal inoculation is still useful for the removal of contaminants, as cell cultures are typically antibiotic-free 71 . Cell lines used include Vero, L929, HEL, XTC-2, MRC5 and DH-82 54, 136, 181 . Again, this method is retrospective, as it can take weeks to obtain an isolate, usually well after the patient has recovered. 1.5.4. Polymerase Chain Reaction (PCR) Clinical manifestations of rickettsial disease are typically non-specific; therefore laboratory testing of samples is crucial for accurate diagnosis. Typically this is performed by serology, although this method has limitations, with antibodies not present at the early stages of infection and a degree of cross-reactivity among species 77, 78 . Advancements in molecular methods have allowed the 14
Page 1 and 2: Rickettsiales and rickettsial disea
Page 3 and 4: Abstract Currently, there are 12 kn
Page 5 and 6: Table of Contents Rickettsiales and
Page 7 and 8: 2.2. Cell Culture .................
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: Table 2. Current list of validated
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.
Page 59 and 60: All cell lines except XTC-2 were gr
Page 61 and 62: system that took into consideration
Page 63 and 64: depending on the specific condition
Page 65 and 66: Australia). A 100 bp DNA ladder (In
Page 67 and 68: Table 3 continued. Reference Anneal
Page 69 and 70: method within the PHYLIP software p
Page 71 and 72: Chapter 3. A serological prevalence
Page 73 and 74: The study was focused around Launce
Page 75 and 76: Overall, 53% of the dogs and 63% of
Page 77 and 78: off. There was however no statistic
Page 79 and 80: from these animals could also be us
Page 81 and 82: 4.2. Introduction Currently in Aust
Page 83 and 84: Figure 3. Map of Tasmania, Australi
Page 85 and 86: Figure 4. Phylogenetic tree showing
Page 87 and 88:
East (more than 10,000 kilometres n
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Worldwide, the presence of SFG rick
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Figure 5. Phylogenetic tree showing
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Page 95 and 96:
Page 97 and 98:
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
Page 127 and 128:
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
Page 133 and 134:
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
Page 173 and 174:
242. Stewart RS, 1991. Flinders Isl
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258. Unsworth NB, Stenos J, Faa AG,
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273. Watts MR, Benn RA, Hudson BJ,
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