Canine echinococcosis in the Alay Valley, southern Kyrgyzstan Freya van Kesteren Cestode Zoonoses Research Group, School of Environment and Life Sciences, University of Salford, UK Submitted in Partial Fulfilment of the Requirements of the Degree of Doctor of Philosophy, 6 January 2015 0 Contents 0 Contents ......................................................................................................................................... 1 1 Index of Tables ............................................................................................................................... 6 2 Index of Figures .............................................................................................................................. 7 Acknowledgements ................................................................................................................................ 8 3 Declaration ................................................................................................................................... 10 Abstract ................................................................................................................................................ 11 1 Chapter 1: Introduction ............................................................................................................... 12 1.1 General Introduction ............................................................................................................ 12 1.2 Taxonomy and species of Echinococcus ............................................................................... 12 1.2.1 E. vogeli and E. oligarthrus ........................................................................................... 13 1.2.2 E. equinus ..................................................................................................................... 14 1.2.3 E. ortleppi ..................................................................................................................... 14 1.2.4 E. felidis ........................................................................................................................ 14 1.2.5 E. shiquicus ................................................................................................................... 15 1.2.6 E. multilocularis ............................................................................................................ 15 1.2.7 E. granulosus ................................................................................................................ 16 1.2.8 E. canadensis ................................................................................................................ 16 1.3 Detection of Echinococcus spp. ............................................................................................ 17 1.3.1 Detecting Echinococcus spp. in intermediate hosts ..................................................... 18 1.3.2 Detecting Echinococcus spp. in final hosts ................................................................... 18 1.4 Cystic and alveolar echinococcosis ...................................................................................... 23 1.4.1 Transmission ................................................................................................................. 23 1.4.2 Pathology ...................................................................................................................... 24 1.4.3 Treatment ..................................................................................................................... 25 1.4.4 Prevention and control................................................................................................. 26 1.5 Impact of echinococcosis ..................................................................................................... 27 1.6 A neglected zoonotic disease ............................................................................................... 28 1.7 Echinococcus spp. in Central Asia ......................................................................................... 29 1.7.1 Background ................................................................................................................... 29 1.7.2 Re-emergence of echinococcosis in Central Asia ......................................................... 29 1.8 Aims and structure of this thesis .......................................................................................... 30 2 Chapter 2: General Methods and Materials ................................................................................ 33 2.1 Study Site: Alay Valley, Kyrgyzstan ....................................................................................... 33 2.2 Sample Collection ................................................................................................................. 35 1 2.3 Analysis of faecal samples .................................................................................................... 36 2.3.1 CoproELISA ................................................................................................................... 36 2.3.2 CoproPCR ...................................................................................................................... 38 2.4 Panel samples ....................................................................................................................... 43 2.4.1 Tissue panel samples .................................................................................................... 43 2.4.2 Faecal panel samples .................................................................................................... 44 2.5 Statistical analysis................................................................................................................. 46 3 Chapter 3: Development and optimization of a coproELISA for the detection of canine Echinococcus spp. infections ................................................................................................................ 48 3.1 Introduction ......................................................................................................................... 48 3.2 Methods ............................................................................................................................... 52 3.2.1 Rabbit immunization and selection of sera .................................................................. 52 3.2.2 Testing immunoreactivity of rabbit sera in ELISA ........................................................ 53 3.2.3 IgG purification from rabbit serum and conjugation ................................................... 55 3.3 Results .................................................................................................................................. 57 3.3.1 Assessment of isolated antibodies ............................................................................... 57 3.3.2 Assessment of Rabbit 91 antibodies ............................................................................ 58 3.3.3 Assessment of Rabbit 93 antibodies ............................................................................ 60 3.3.4 Assessment of Rabbit 5 antibodies .............................................................................. 60 3.3.5 Assessment of a combination of antibodies from Rabbit5 and Rabbit 91 in ELISA ..... 62 3.3.6 Calibration of R47 antibodies in ELISA ......................................................................... 64 3.3.7 Evaluation of a commercial Echinococcus spp. coproELISA kit .................................... 65 3.4 Discussion ............................................................................................................................. 66 3.5 Chapter 3 summary .............................................................................................................. 68 4 Chapter 4: Development of multiplex and uniplex PCR assays for the differentiation of E. granulosus sensu stricto, E. equinus, E. multilocularis and E. canadensis DNA in tissue and faecal samples ................................................................................................................................................ 70 4.1 Introduction ......................................................................................................................... 70 4.2 Methods ............................................................................................................................... 71 4.2.1 Primer design ............................................................................................................... 71 4.2.2 DNA extractions............................................................................................................ 72 4.2.3 E. granulosus sensu stricto, E. equinus and E. multilocularis PCR protocol ................. 73 4.2.4 E. canadensis PCR protocol .......................................................................................... 73 4.2.5 Multiplex PCR protocol................................................................................................. 74 4.2.6 Sequencing ................................................................................................................... 74 4.2.7 Evaluation of analytic PCR tissue DNA specificity ........................................................ 75 2 4.2.8 Evaluation of analytic PCR coproDNA specificity ......................................................... 75 4.2.9 Evaluation of analytic