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Detection and Partial Molecular Characterization of Rickettsia and Bartonella from Southern African Bat Species

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Detection and Partial Molecular Characterization of Rickettsia and Bartonella from Southern African Bat Species Detection and partial molecular characterization of Rickettsia and Bartonella from southern African bat species by Tjale Mabotse Augustine (29685690) Submitted in partial fulfillment of the requirements for the degree MAGISTER SCIENTIAE (MICROBIOLOGY) in the Department of Microbiology and Plant Pathology Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria, South Africa Supervisor: Dr Wanda Markotter Co-supervisors: Prof Louis H. Nel Dr Jacqueline Weyer May, 2012 I declare that the thesis, which I hereby submit for the degree MSc (Microbiology) at the University of Pretoria, South Africa, is my own work and has not been submitted by me for a degree at another university ________________________________ Tjale Mabotse Augustine i Acknowledgements I would like send my sincere gratitude to the following people: Dr Wanda Markotter (University of Pretoria), Dr Jacqueline Weyer (National Institute for Communicable Diseases-National Health Laboratory Service) and Prof Louis H Nel (University of Pretoria) for their supervision and guidance during the project. Dr Jacqueline Weyer (Centre for Zoonotic and Emerging diseases (Previously Special Pathogens Unit), National Institute for Communicable Diseases (National Heath Laboratory Service), for providing the positive control DNA for Rickettsia and Dr Jenny Rossouw (Special Bacterial Pathogens Reference Unit, National Institute for Communicable Diseases-National Health Laboratory Service), for providing the positive control DNA for Bartonella. Dr Teresa Kearney (Ditsong Museum of Natural Science), Gauteng and Northern Region Bat Interest Group, Kwa-Zulu Natal Bat Interest Group, Prof Ara Monadjem (University of Swaziland), Werner Marias (University of Johannesburg), Dr Francois du Rand (University of Johannesburg) and Prof David Jacobs (University of Cape Town) for collection of blood samples. The National Research Foundation for financial support. To my colleagues at the University of Pretoria, for their support during my studies. To my family especially my parents, for their love, encouragement and for giving me the opportunity to pursue my studies. ii Summary Detection and partial molecular characterization of Rickettsia and Bartonella from southern African bat species By Tjale Mabotse Augustine Supervisor: Dr Wanda Markotter Co-supervisors: Prof Louis H. Nel Dr Jacqueline Weyer Department of Microbiology and Plant Pathology Faculty of Natural and Agricultural Sciences University of Pretoria For the degree MSc (Microbiology) In Africa, bats have been implicated in a number of emerging and re-emerging diseases, mostly associated with viral infections – such as caused by rabies-related lyssaviruses, paramyxoviruses and coronaviruses. Whereas most infectious agents reported in bats have been viruses, bacterial species have rarely been reported. The main objective of this study was to identify bacterial species that may circulate in southern African bats by using nucleic acid detection methods. Two bacterial species were targeted: Rickettsia and Bartonella. These were chosen because they are capable of infecting and causing diseases in a wide range of hosts including humans. We evaluated and optimized several polymerase chain reaction (PCR) assays to detect Rickettsia and Bartonella . These included PCRs targeting the citrate synthase gene ( gltA) and 16S rRNA gene for Rickettsia and the citrate synthase ( gltA ) gene for Bartonella. A panel of 354 bat blood samples, collected from different sites in South Africa and Swaziland, were tested using these assays. Rickettsia and Bartonella DNA was detected in 6/354 and 13/354 bats, respectively, and characterized using DNA sequencing. All the Rickettsias were closely related to iii other Rickettsia species circulating in these areas and all the Bartonellas clustered together, but were distantly related to Bartonella species from the same geographical area. This study reports for the first time the detection of Rickettsia and Bartonella DNA in southern African bats. This finding contributes to the knowledge regarding Rickettsia and Bartonella diversity and host distribution. The epidemiology and transmission pathways of these bacteria in bat populations remains to be elucidated as is the public health importance of the circulation of these potential pathogens in bats. A likely source of infection is unknown, but since bats carry ectoparasites (flies, fleas, ticks and mites); surveillance for these pathogens in ectoparasites should be a first step in elucidating epidemiology and transmission pathways to other hosts including humans. Given the potential for human disease, the surveillance and characterization of these pathogens will be in the interest of good public health practices. iv List of figures Figure 1.1 : Phylogenetic relationships of the organisms of the order Rickettsiales and their reservoir hosts based on 16S rRNA and 18S rRNA 3 gene sequences. Only the bootstrap values higher than 75% are shown in the branch nodes. From Merhej and Raoult (2011), license number: 2878240534804. Figure 1.2: Guidelines for the classification of Rickettsia at genus, group, and species level using genotypic, phenotypic and phylogenetic characteristics. 5 From Fournier et al., (2009), licence number: 2878250837050. Figure 1.3: Time course of markers of rickettsial infection with association with detection and diagnosis. From Richards (2011), license number: 29044720303354. 11 Figure 1.4: Guidelines used for diagnosis of rickettsial infections as described in Raoult et al., (1997) and La Scola et al., (1997). 19 Figure 1.5: Map of Africa (From Mongab.com/date accesed: 23-09-2011) showing the known distribution of rickettsial species in Africa. Rickettsia species are indicated by the coloured dots. The location of the dots does not indicate the exact area in which the rickettsial species was identified, it indicates only the country. 21 Figure 1.6 : Phylogenetic analysis of the genus Bartonella on multilocus sequence analysis. From Saenz et al., (2007), licence number: 2878761140434. 28 Figure 1.7: Guidelines for diagnosis of Bartonella infections as described by Anderson and Newmann, (1997) and Breitschwerdt et al ., (2010). Figure 2.1 : Multiple alignment of representative African Rickettsia species indicating the primer binding site on the gltA gene. The dots indicate identical base pairs. 35 v Figure 2.2 : Multiple alignment of representative African Rickettsia species representing the primer binding site on the 16S rRNA gene. The dots indicate 69 identical base pairs. Figure 2.3 : The real time PCR amplification curve generated during the amplification of the partial gltA gene of Rickettsia conorii . Sample 1 : R. 71 conorii DNA, Sample 2: No template negative control. Figure 2.4 : Agarose gel electrophoresis analysis illustrating the partial 16S rRNA (400 bp) and gltA (645 and 202 bp) genes of Rickettsia conorii amplified during conventional and nested PCR, respectively. Lane 1: 100bp DNA ladder (Fermentas), Lane 2: No template negative control, Lane 3: 72 Conventional PCR targeting the partial gltA gene of R. conorii , Lane 4: Conventional PCR targeting the partial 16S rRNA gene of R. conorii , Lane 5: Nested PCR targetting the partial gltA gene of R. conorii . Figure 2.5 : Pairwise alignment analysis of the partial gltA gene of Rickettsia conorii with the recombinant plasmid DNA containing partial gltA gene of interest. C-product: Conventional PCR product of the partial gltA gene (645 bp), Rec-product: Recombinant plasmid DNA containg the partial gltA gene of 73 interest, N-product: nested PCR product of the partial gltA gene (202 bp), RT- product: Real time PCR product of the partial gltA gene (70bp). The colored dots indicate identical base pairs. Figure 2.6 : Pairwise alignment analysis of the partial 16S rRNA gene of Rickettsia conorii with the recombinant plasmid containing partial 16S rRNA gene. C-product: Conventional PCR product of the partial 16S rRNA gene , 74 Rec-plasmid: Recombinant plasmid DNA containing the partial 16S rRNA gene. The coloured dots indicate identical base pairs. Figure 2.7 : Agarose gel electrophoresis analysis illustrating the copies of the partial gltA and 16S rRNA genes of Rickettsia conorii that were generated in different PCRs when the recombinant plasmid DNA is serially diluted (10 -1 to 75 10 -10 ). A: Copies of the partial gltA gene (200bp) of R. conorii generated in nested PCR, B: Copies of the partial gltA (645 bp) of R. conorii generated in a conventional PCR. C: Copies of the partial the 16S rRNA of R. conorrii generated in conventional PCR. M-100bp DNA ladder (Fermentas, USA), N: vi No template negative control. The values at each lane indicate the copy number of the respective genes. Figure 2.8 : Standard curve constructed by plotting the Cp values against the 77 log concentration of the serially diluted recombinant plasmid containing the partial gltA gene amplified in real time PCR. The reaction was performed in triplicate. Figure 2.9 : Multiple alignment of representative sequences of Bartonella species indicating the primer binding site on the gltA gene. The dots indicate 79 identical base pairs. Figure 2.10 : Agarose gel electrophoresis indicating the partial fragment of the gltA gene of Bartonella spp. amplified using conventional PCR. Lane 1: 100bp DNA ladder (Fermentas), Lane 2: No template negative control, Lane 80 3: Partial gltA gene of B. henselae , Lane 4: Partial gltA gene of B. quintana , Lane 5 : Partial gltA gene of B. vinsonii , Lane 6: Partial gltA gene of B.
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