UNIVERSITY OF NAIROBI MOLECULAR EPIDEMIOLOGY OF ECHINOCOCCUS AND TAENIA SPECIES IN DOGS FROM CYSTIC ECHINOCOCCOSIS ENDEMIC AREAS IN KENYA ERASTUS MULINGE KAKUNDI (B.Sc., M.Sc.) I80/92641/2013 A thesis submitted in Fulfilment of the Requirements for Award of the Degree of Doctor of Philosophy in Applied Parasitology of the University of Nairobi School of Biological Sciences 2020 i DECLARATION I declare that this thesis is my original work and has not been submitted elsewhere for examination or award of a degree. Where other people’s work has been used, this has been properly acknowledged and referenced in accordance with the University of Nairobi’s requirements Signature: ……………………………………………………….. Date: …………………………….… Erastus Mulinge Kakundi I80/92641/2013 School of Biological Sciences University of Nairobi This thesis is submitted for examination with our approval as research supervisors Signature Date 1. Dr. David Odongo …………………… …………………… School of Biological Sciences University of Nairobi Nairobi, Kenya 2. Prof. Japhet Magambo ………………….... …………………… Meru University of Science & Technology Meru, Kenya 3. Dr. Thomas Romig …………………… ……………………. University of Hohenheim Parasitology unit Stuttgart, Germany 4. Prof. Sammy Njenga …………………….. ………………….. Eastern and Southern Africa Centre of International Parasite Control (ESACIPAC), Kenya Medical Research Institute Nairobi, Kenya ii ABSTRACT Dogs are reservoirs and hosts for several parasites which cause zoonoses of public health significance worldwide. All four hookworm species of canines are zoonotic and cause infections ranging from transient skin irritations to prolonged ‘creeping eruptions’, eosinophilic enteritis and patent intestinal infections. Cestodes belonging to the Taeniidae family are of medical and veterinary importance in resource poor communities and cause cystic echinococcosis (CE), cysticercosis and coenurosis in livestock and humans. There is scanty data on the prevalence of intestinal parasites of dogs and their impact on public health in Kenya. Furthermore, the role of domestic dogs in maintaining the transmission cycles and environmental contamination with infective stages of these parasites remains unknown in Kenya. This study aimed at establishing the prevalence and distribution of Echinococcus granulosus sensu lato (s. l.), Taenia spp. and other zoonotic intestinal parasites in dogs from CE endemic areas of Kenya. Dog faecal samples were collected from the environment in Turkana, Meru, Isiolo and Narok (Maasai Mara) counties and microscopically examined for the detection of intestinal parasites using McMaster and zinc chloride flotation-sieving technique. Genotyping of hookworms, Echinococcus and Taenia spp. was performed by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and or DNA sequencing. Echinococcus and Taenia spp. haplotypes were determined by sequencing and analysis of cytochrome C oxidase subunit 1 gene (cox1) and NADH dehydrogenase subunit 1 (nad1) genes respectively. Of the 1,621 faecal samples, 701 (43.24%) contained at least one parasite. Eleven parasites genera/families were identified namely hookworms, Taeniidae, Spirometra spp., Coccidia, Toxocara spp., Trichuris spp., Toxascaris leonina, Dipylidium caninum, Anoplocephala spp., Mesostephanus spp. and Uncinaria stenocephala (in order of frequency). Hookworms were the most common parasites detected followed by taeniids. Microscopy detected hookworm eggs in 490/1621 (30.23%, 95% CI 27.99–32.53) faecal samples. The prevalence of hookworms was high in counties receiving higher rainfall (Narok 46.80%, Meru 44.88%) and low in those with a more arid climate (Isiolo 19.73%, Turkana 11.83%). In a subset of 70 hookworm eggs positive faecal samples Ancylostoma caninum (n = 59), A. braziliense (n = 10) and A. cf. duodenale (n = 1) were identified. Eleven percent (178/1,621) of faecal samples had taeniid eggs, among them 4.4% (71/1,621) were Echinococcus spp. eggs. Area-wise, the faecal prevalence of Echinococcus spp. was 9.2% (48/524) in Turkana, 4.0% (20/500) in Maasai Mara, 0.7% (2/294) in Isiolo and 0.3% iii (1/303) in Meru. Four Echinococcus spp. were identified with E. granulosus sensu stricto (s. s.) being the dominant Echinococcus taxon, followed by E. canadensis (G6/7) that was detected in 51 and 23 faecal samples, respectively. E. ortleppi and E. felidis were rare and only detected in 5 and 2 faecal samples respectively. Overall 79/1621 (4.9%) faecal samples contained eggs of Taenia or Hydatigera (8.0% in Turkana, 4.8% in Isiolo, 3.8% in Maasai Mara and 1.3% in Meru). Taenia hydatigena and T. multiceps were the most frequent, found in 36 and 15 samples, respectively. Other eggs detected in the faecal samples belonged to T. serialis (sensu lato), T. madoquae (the first record in domestic dogs), T. ovis, T. saginata and Hydatigera taeniaeformis. Polymorphism of nad1 sequences revealed 22 and 8 haplotypes of T. hydatigena and T. multiceps, respectively, but only a few haplotypes showed wide geographical distribution for both species. This study identified zoonotic parasites in dogs that pose potential public health risk to humans. Canine hookworm species and genetic variability of T. hydatigena and T. multiceps are reported for the first time in Kenya. The data confirms differences in diversity and abundance of Echinococcus and Taenia spp. between regions of Kenya and demonstrates that domestic dogs play a role in linking the domestic and sylvatic cycles of Echinococcus and Taenia spp. The unusual detection of A. cf. duodenale, T. saginata and E. felidis in dog faeces indicates coprophagy as a common behaviour of dogs. These findings emphasize the need for control measures such as enforcing laws for restraining stray dogs, dog population management, regular deworming of dogs, proper disposal of slaughter offal and dog faeces and public health educational awareness programmes. iv DEDICATION I wish to dedicate this thesis to my late father Rev. Canon Julius Kakundi Kiilu, who has been my inspiration through-out all my education levels. v ACKNOWLEDGEMENTS First and foremost I wish to thank the almighty God for His everlasting love, care, protection and good health during the study period. I wish to sincerely thank all my supervisors for their tirelessly effort, encouragement, guidance and mentoring they offered me throughout the period of study. I wish to appreciate Prof. Japhet Magambo for the support he offered to me specifically during field sampling, always providing a car and funds required for field work. I sincerely thank Prof. Sammy Njenga, for providing laboratory space and occasionally materials from his laboratory. I wish to appreciate Dr. David Odongo for the academic guidance, encouragement and finding time to visit in the laboratory at KEMRI. To Dr. Thomas Romig thank you for first of all entrusting me with the work on echinococcosis in dogs and also for providing the support I needed for this work, for offering me the opportunity to spend two months of laboratory work at the parasitology unit, University of Hohenheim, as well as providing funds for sequencing all the samples analysed in this study. I wish to thank Prof. Peter Kern for sponsoring my tuition fees at the University of Nairobi and funding the all the activities of this study including cost of attending World Congress of Echinococcosis in 2011, 2013 and 2017. I also wish to acknowledge most sincerely the kind support offered by Mr. Eberhard Zeyhle, who always was involved in all the field work I conducted for three years together with my fellow PhD student Dorothy Kagendo. I wish to appreciate the wealth of experience in parasitology, specifically in cystic echinococcosis which I learnt from Mr. Eberhard Zeyhle. I also sincerely thank my colleague at KEMRI, Dr. Cecilia Mbae for entrusting me with all the laboratory work for Cystic Echinococcosis sub-Saharan Africa Research initiative (CESSARi) Kenya, her constant support and encouragement during the whole period of study, and for ensuring that all laboratory materials and consumables needed for this project were procured in good time. I wish to appreciate the support I got from the current and former Centre for vi Microbiology Research (CMR) directors; Dr. Willie Sang and Prof. Sam Kariuki respectively. To all my other colleagues in CMR-headquarters unit Tabitha, Joyce, Bridget, Agnes, Mike and the entire fraternity of CMR, KEMRI. More specifically thank Zilla Wakio for assisting in organizing the questionnaire data and Collins Okoyo for helping in data analysis. I sincerely acknowledge the cooperation of KEMRI colleagues at ESACIPAC Henry Kanyi, Bernard Chieng and Sylvie Araka for making it easier for me to access the molecular biology laboratory. I wish to say thank you to CESSARi consortium members including Dr. Marion Wassermann, Dr. Francis Addy, and Dr. Dennis Ebi for the time they afforded me and guidance in many aspects of this study. I sincerely thank Prof. Rebecca Traub for her guidance and providing positive control for canine hookworm study. I appreciate the guidance and support I received from Prof. Samuel Githigia and Mr. Richard Otieno during laboratory analysis at the department of Veterinary Pathology, Microbiology and Pathology, University of Nairobi. I also wish to acknowledge Timothy Mutuma and all other undergraduate students attached at the CMR – KEMRI, parasitology laboratory, who offered technical support. I am greatly indebted to the people of Turkana, Meru, Isiolo and Maasai Mara where this study was carried and for