Wongserepipatana, Manun (2016) Prevalence and associations of Trypanosoma spp. and Sodalis glossinidius with intrinsic factors of tsetse flies. PhD thesis. http://theses.gla.ac.uk/7537/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Prevalence and associations of Trypanosoma spp. and Sodalis glossinidius with intrinsic factors of tsetse flies Manun Wongserepipatana This thesis is submitted in part fulfilment of the requirements for the Degree of Doctor of Philosophy. Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical, Veterinary and Life Sciences University of Glasgow August 2016 Abstract Trypanosomiasis has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. Whilst assessments of the biology of trypanosomes, vectors, vertebrate hosts and the environment have provided useful information about life cycles, transmission, and pathogenesis of the parasites that could be used for treatment and control, less information is available about the effects of interactions among multiple intrinsic factors on trypanosome presence in tsetse flies from different sites. It is known that multiple species of tsetse flies can transmit trypanosomes but differences in their vector competence has normally been studied in relation to individual factors in isolation, such as: intrinsic factors of the flies (e.g. age, sex); habitat characteristics; presence of endosymbionts (e.g. Wigglesworthia glossinidia, Sodalis glossinidius); feeding pattern; host communities that the flies feed on; and which species of trypanosomes are transmitted. The purpose of this study was to take a more integrated approach to investigate trypanosome prevalence in tsetse flies. In chapter 2, techniques were optimised for using the Polymerase Chain Reaction (PCR) to identify species of trypanosomes (Trypanosoma vivax, T. congolense, T. brucei, T. simiae, and T. godfreyi) present in four species of tsetse flies (Glossina austeni, G. brevipalpis, G. longipennis and G. pallidipes) from two regions of eastern Kenya (the Shimba Hills and Nguruman). Based on universal primers targeting the internal transcribed spacer 1 region (ITS-1), T. vivax was the predominant pathogenic species detected in flies, both singly and in combination with other species of trypanosomes. Using Generalised Linear Models (GLMs) and likelihood ratio tests to choose the best-fitting models, presence of T. vivax was significantly associated with an interaction between subpopulation (a combination between collection sites and species of Glossina) and sex of the flies (2 = 7.52, df = 21, P-value = 0.0061); prevalence in females overall was higher than in males but this was not consistent across subpopulations. Similarly, T. congolense was significantly associated only with subpopulation (2 = 18.77, df = 1, P-value = 0.0046); prevalence was higher overall in the Shimba Hills than in Nguruman but this pattern varied by species of tsetse fly. When associations were analysed in individual species of tsetse flies, there were no consistent associations between trypanosome prevalence and any single factor (site, sex, age) and different ii combinations of interactions were found to be significant for each. The results thus demonstrated complex interactions between vectors and trypanosome prevalence related to both the distribution and intrinsic factors of tsetse flies. The potential influence of the presence of S. glossinidius on trypanosome presence in tsetse flies was studied in chapter 3. A high number of Sodalis positive flies was found in the Shimba Hills, while there were only two positive flies from Nguruman. Presence or absence of Sodalis was significantly associated with subpopulation while trypanosome presence showed a significant association with age (2 = 4.65, df = 14, P-value = 0.0310) and an interaction between subpopulation and sex (2 = 18.94, df = 10, P-value = 0.0043). However, the specific associations that were significant varied across species of trypanosomes, with T. congolense and T. brucei but not T. vivax showing significant interactions involving Sodalis. Although it has previously been concluded that presence of Sodalis increases susceptibility to trypanosomes, the results presented here suggest a more complicated relationship, which may be biased by differences in the distribution and intrinsic factors of tsetse flies, as well as which trypanosome species are considered. In chapter 4 trypanosome status was studied in relation to blood meal sources, feeding status and feeding patterns of G. pallidipes (which was the predominant fly species collected for this study) as determined by sequencing the mitochondrial cytochrome B gene using DNA extracted from abdomen samples. African buffalo and African elephants were the main sources of blood meals but antelopes, warthogs, humans, giraffes and hyenas were also identified. Feeding on multiple hosts was common in flies sampled from the Shimba Hills but most flies from Nguruman had fed on single host species. Based on Multiple Correspondence Analysis (MCA), host-feeding patterns showed a correlation with site of sample collection and Sodalis status, while trypanosome status was correlated with sex and age of the flies, suggesting that recent host-feeding patterns from blood meal analysis cannot predict trypanosome status. In conclusion, the complexity of interactions found suggests that strategies of tsetse fly control should be specific to particular epidemic areas. Future studies should include laboratory experiments that use local colonies of tsetse flies, local strains of trypanosomes and local S. glossinidius under controlled environmental conditions to tease out the factors that affect vector competence and the relative influence of external environmental factors on the dynamics of these interactions. iii List of Contents Abstract .................................................................................. i List of Contents ........................................................................... iii List of Tables ............................................................................... ix List of Figures ............................................................................ xiii Acknowledgements ..................................................................... xviii Author’s Declaration .................................................................... xix Abbreviations and symbols ............................................................. xx Chapter 1 Introduction ................................................................. 1 1.1 Biology of Trypanosoma spp. ................................................... 1 1.1.1 Taxonomy of trypanosomes ................................................ 1 1.1.2 Life cycle of African trypanosomes ....................................... 2 1.1.3 Importance of African trypanosomiasis ................................... 7 1.2 Trypanosome diagnosis ......................................................... 11 1.3 Prevalence of Animal African trypanosomes ................................ 14 1.4 Trypanosome control and drug treatment ................................... 17 1.5 Biology of Glossina spp. ........................................................ 19 1.5.1 Taxonomy of tsetse flies................................................... 19 1.5.2 Distribution and habitat of tsetse flies .................................. 20 1.5.3 Life cycle of tsetse flies ................................................... 23 1.5.4 Age determination .......................................................... 24 1.5.5 Sodalis glossinidius ......................................................... 25 1.5.6 The feeding process and behaviour of tsetse flies ..................... 27 1.5.7 Hosts ranges of tsetse flies ................................................ 30 1.5.8 Blood meal analysis in tsetse flies ....................................... 32 1.6 Trypanosoma spp. infection in tsetse flies .................................. 33 1.6.1 Determinants of trypanosome infection in tsetse flies ............... 33 iv 1.6.2 Trypanosome identification based on PCR method .................... 43 1.6.3 Trypanosome prevalence in tsetse flies ................................. 48 1.6.4 Tsetse-fly control ........................................................... 51 1.7 Aims of this thesis ............................................................... 54 1.8 Chapter objectives .............................................................. 57 Chapter 2 Tsetse fly distribution and trypanosome prevalence in tsetse flies ................................................................................ 59 2.1 Abstract ........................................................................... 59 2.2 Introduction ...................................................................... 60 2.3 Materials and methods .........................................................