The bacterial ecology of the sheep mammary gland Emma Monaghan Supervisors: Professor Laura Green and Dr Kevin Purdy Introduction Over 120 species of bacteria have been linked to intramammary infections in sheep This large number of species suggests that the sheep mammary gland could be a site where bacteria live as part of a microbial community Microbial communities are multi-species assemblages in which organisms live and interact in a shared environment One example of a microbial community is the natural colonisation of bacteria in the alimentary system Investigation of bacterial species within microbial communities improves our understanding of the interactions that occur between bacteria and disease For sheep intramammary infections, an improved understanding may produce alternative management strategies to prevent intramammary infections Preliminary results Study samples have been collected longitudinally within a lactation over eight weeks from each teat for ewes aged 1-10 years Each milk sample is processed using the techniques described in the methods section Figure One: PCR amplification using primers 27F/338r-GC of milk from ewes 1-3. PCR products labelled 1-8 and 9-14 are from the left half of the mammary gland and A-H from the right half. The symbols + and – indicate control samples Figure one shows amplification of a single specific product of 300bp in one round of PCR for each milk sample PCR products are then run on a DGGE gel for community-based analysis Figure Two: DGGE image of PCR products from PCR (1) Figure One. Numbers 1-8 correspond to samples from weeks 1-8 from the left half of the mammary gland and letters A-E correspond to milk samples from weeks 1-5 from the right half 3 1 2 1 2 3 4 5 6 7 8 + - A B C D 1 2 3 4 5 6 7 8 + - A B C D 1 2 3 4 5 6 7 + - A B C D E F G H + - 9 10 11 12 13 14 + - E F G + - + - E F G H + - + - Research hypotheses HYPOTHESES: 1. A natural microbial community forms in the sheep mammary gland 2. Perturbations in the community result in the development of disease To address these hypotheses I am: Investigating the bacterial genera in the microbial community of the mammary gland and how this changes over time and age of sheep Determining how the development of the microbial community and the colonising bacterial species within it, influence the health of the mammary gland using SCC as a measure of mammary gland health This research will provide a novel insight into bacterial colonisation of the mammary gland and its role in the maintenance of health Methods All methods are molecular-based whole community approaches that are culture-independent Methods include: (A) Extracting DNA from sheep milk collected on farm (B) Amplifying the bacterial DNA in a general bacterial PCR (C) Analysing the community diversity using denaturing gradient gel electrophoresis (DGGE) 1 2 3 4 5 6 7 8 + - A B C D E + These preliminary results suggest changes in the bacterial community both within and between the left and right halves of the mammary gland across the sampling period Conclusions and future work Multiple PCR primer sets should be tested and optimised for a single sample type to identify the most consistent approach Due to the small amounts of bacterial DNA present in sheep milk, PCRs need to be conducted in a single round to avoid false positive results DNA from sheep milk degrades rapidly with increasing freeze-thaw cycles so needs to be aliquoted and handled carefully Milk samples from three ewes in their first lactation will be processed to determine if there are changes in the bacterial community Selected samples will be pyrosequenced to identify the bacterial species present (A) (B) (C) For more information please visit my webpage: http://www2.warwick.ac.uk/fac/sci/lifesci/study/pg/research/currentstudents/lsrgbc : Contact e-mail: E.monaghan@warwick.ac.uk