The diversity of the microbial community of the udders of sheep

The relationship between age of sheep and the microbial community structure in their udders will be investigated to test the hypotheses: 1. With increasing lactation number, the species and strains of bacteria colonising the udder increase 2. The bacterial load of pathogens and the ratio of pathogenic to benign strains of microbes affect weight gain in lambs, a marker for milk production 3. Pathogen load and presence of specific virulence markers in sheep with either clinical or sub-clinical mastitis is related to sheep age (number of lactations). This will contextualise the processes by which udders of ruminants become infected, the importance of the balance between benign and pathogenic strains and begin to understand the dynamics of the interactions between the host and its microbes, whether good or bad. To address Hypothesis 1, 10 - 30 sheep (200 in total) from one farm of each year group will be sampled 48 hrs after lambing. We will record the identity, age (from teeth), breed, body condition, number and birth weight of lambs and physical appearance and texture of the udder and teat. Ewes will be body condition scored and lambs weighed at 1, 3, 5, and 7 weeks. The udder bacterial community will be investigated using culture and molecular-based whole community approaches. Milk samples will be split into two aliquots, one frozen for molecular analysis and the other chilled for culturing. Chilled samples will be cultured according to standard practice. Isolates from these plates will be characterised using biochemical tests and selected isolates will be characterised more fully using molecular diagnostic tests. Isolates will be stored at -80 in 10% glycerol. The whole bacterial community in the milk will be analysed using a general bacterial PCR approach. DNA isolated from the milk samples will be amplified using general bacterial PCR primers and the community diversity determined using a fingerprinting technique such as DGGE. Using the fingerprinting analysis selected samples will be deep-sequenced using long-read amplicon 454 pyrosequencing. Such an analysis will provide complete coverage of the bacterial community in all of the sampled udders of sheep at different ages and so allow an analysis of how the microbial community changes as sheep age and whether specific populations are linked to reduced milk production and the healthy development of lambs. The impact of the community diversity of bacteria and presence of specific genotypes on lamb growth having adjusted for birth weight, litter size, ewe body condition and any other circumstances that might have affected growth e.g. an episode of diarrhoea in the lamb will be used to assess the association between infected udders and milk production. This type of in-depth analysis has not been done before. The presence and number of virulence determinants, such as the coagulase genes in a detected staphylococcal community, will be analysed using qPCR. The ratio of pathogenic to benign strains will be determined with reference to the bacterial community structure and statistical analyses used to determine relationships and patterns in the data. Hypothesis 3 will be tackled primarily using isolated strains grown from the sampled milk. The presence of virulence factors in isolates will be analysed with a particular focus on determining whether there are other physiological or genetic factors that can be determined to gain a deeper understanding of the process from infection to disease in ruminant mastitis. An overall aim will be to determine whether microbial colonisation of the udder is inevitable, always detrimental or potentially beneficial if pathogen strain load is limited. We will test results for hypothesis 3 by manipulating the udder of sheep in vivo by introducing intramammary treatment five sheep of different ages and assessing the impact of this compared with controls not given such treatment.