The bacterial ecology of the sheep mammary gland

1. The bacterial ecology of the sheep mammary gland • Emma Monaghan • Supervisors: Professor Laura Green and Dr Kevin Purdy • 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 Introduction 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 + - + - • 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 Research hypotheses 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