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Characterising the Microbial Communities Associated with the Water Distribution System of a Broiler Farm and Their Role In Characterising the microbial communities associated with the water distribution system of a broiler farm and their role in Campylobacter infection PAZ ARANEGA BOU Submitted in partial fulfilment of the requirements of the Degree of Doctor of Philosophy, May 2017 University of Salford, School of Environment and Life Sciences List of contents LIST OF FIGURES……………………………………………………………………………..…………………………………VIII LIST OF TABLES……………………………………………………………………………………………………………………..X LIST OF ABBREVIATIONS …………………………………………………………………………………………………...XIII ACKNOWLEDGEMENTS ……………………………………………………………………………………….…………….XV ABSTRACT………………………………………………………………………………………………………….…………….XVII CHAPTER 1: General introduction…………………………………………….………………………………...1 1.1The genus Campylobacter…………………………………………………………………………………..……….1 1.2 Epidemiology of campylobacteriosis in humans ............................................................ 2 1.2.1 Prevalence and disease trends ................................................................................. 2 1.2.2 Symptoms and treatment ........................................................................................ 5 1.2.3 C. jejuni pathogenesis .............................................................................................. 6 1.2.4 Attribution of human campylobacteriosis ............................................................... 8 1.2.4.1 Farm and domestic animals as a reservoir ...................................................... 12 1.2.4.2 The environment and wildlife as a reservoir ................................................... 14 1.2.5 Campylobacter along the food chain ..................................................................... 16 1.3 Campylobacter ecology in broiler chickens .................................................................. 18 1.3.1 Prevalence and trends of Campylobacter colonisation of chickens ...................... 20 1.3.2 Colonization and transmission ............................................................................... 21 1.3.3 Sources of infection ................................................................................................ 22 1.4 Survival strategies of Campylobacter spp. in the environment ................................... 28 1.4.1 Viable but nonculturable (VBNC) state .................................................................. 28 II 1.4.2 Biofilm formation ................................................................................................... 30 1.4.3 Interactions with other microorganisms................................................................ 36 1.4.3.1 Interactions of Campylobacter with other bacteria ........................................ 36 1.4.3.2 Interactions with eukaryotic organisms .......................................................... 40 1.5 Key questions and general hypotheses ........................................................................ 43 CHAPTER 2: Materials and methods…………………………………………………………………….……45 2.1 Strains and culture conditions ...................................................................................... 45 2.1.1 Bacterial culture ..................................................................................................... 45 2.1.2 Amoebae culture .................................................................................................... 47 2.1.3 Enrichment and Selective media for isolation of microorganisms. ....................... 47 2.2 Sample sites and sample collection .............................................................................. 49 2.2.1 Large-scale model drinking water distribution system (DWDS) ............................ 49 2.2.2 Commercial broiler farm in the UK ........................................................................ 49 2.2.2.1 Farm description .............................................................................................. 49 2.2.2.2 Biofilm and nipple drinker sampling................................................................ 52 2.2.2.3 Bulk water sampling ........................................................................................ 53 2.2.2.4. Sample collection for Campylobacter isolation .............................................. 54 2.2.3 Small-scale semi-intensive chicken farms in Uganda ............................................ 54 2.3 Culture-based screening and isolation of microorganisms from water and farm samples ............................................................................................................................................. 55 2.3.1 Screening for the presence of viable Campylobacter species in the large model DWDS............................................................................................................................... 55 2.3.2 Screening for the presence of viable Campylobacter species on the commercial broiler farm in UK ............................................................................................................ 55 III 2.3.3 Screening for the presence of viable Campylobacter species on the small-scale semi- intensive chicken farms in Uganda ................................................................................. 56 2.3.4 Isolation of Pseudomonas species from the large model DWDS ........................... 56 2.3.5 Isolation of Pseudomonas species from the DWDS of a large commercial broiler farm ......................................................................................................................................... 57 2.3.6 Gram Staining ......................................................................................................... 57 2.3.7 Preparation of stocks ............................................................................................. 58 2.3.8 Detection of Amoebae from a large DWDS model ................................................ 59 2.4 Molecular methods for detection / identification ........................................................ 59 2.4.1 DNA extractions ..................................................................................................... 59 2.4.1.1 DNA extraction from bacterial isolates ........................................................... 59 2.4.1.2 DNA extraction from farm samples for 16S and 18S community profiling ..... 60 2.4.1.2.1 Nipple drinker processing………………………………………………………………….60 2.4.1.2.2 Bulk water processing……………………………………………………………………….60 2.4.1.2.3 DNA extraction………………………………………………………………………………….61 2.4.1.3 DNA extraction from frozen Campylobacter enrichment samples ................. 62 2.4.1.4 DNA extraction from faeces ............................................................................ 63 2.4.1.5 DNA Quality Analysis ....................................................................................... 63 2.4.2 PCR ...................................................................................................................... 63 2.4.2.1 Preparation of Boil preps ................................................................................. 63 2.4.2.2 PCR primers and conditions ............................................................................ 63 2.4.3 Quantitative (q)PCR ................................................................................................ 67 2.4.4 Gel electrophoresis ............................................................................................... 67 2.4.5 Strain sequencing ................................................................................................... 68 2.4.6 Whole-genome sequencing of H. pullorum ........................................................... 68 2.4.6.1 Library preparation and sequencing ............................................................... 68 2.4.6.2 Primer cross-reactivity with the H. pullorum genome .................................... 69 IV 2.5 Microbial community profiling .................................................................................. 69 2.5.1 16S and 18S library preparation ............................................................................. 69 2.5.2 Bioinformatics ........................................................................................................ 74 2.5.3 Statistical analyses ................................................................................................. 75 2.6 Interaction assays ......................................................................................................... 75 2.6.1 Co-culture bacterial growth assays ........................................................................ 75 2.6.2 Gentamicin protection assay ................................................................................. 76 CHAPTER 3: Longitudinal and spatial microbial community profiling of the drinking water system (DWS) of a commercial broiler farm during a rearing cycle………………………..…..79 3.1 Introduction .................................................................................................................. 79 3.2 Changes in temperature and flow rate and administration of prophylaxis and vaccinations occur on the farm DWS during a typical rearing cycle .................................. 84 3.3 DNA extraction and library preparation efficiency differ for samples from different environmental niches ......................................................................................................... 88 3.4 Sequencing results represent all samples but show differing sequencing
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