Characterisation of Streptococcus uberis from bovine milk

Mastitis is the most important and expensive disease of the dairy industry. Streptococcus uberis is an important environmental pathogen causing bovine mastitis. This thesis investigated (i) the epidemiology of clinical bovine mastitis, (ii) factors affecting biofilm formation of S. uberis, and (iii) the virulence factors of S. uberis. Sixty S. uberis isolates from 27 cows with recurrent clinical mastitis had 34 different DNA restriction patterns, indicating a wide variety of S. uberis strains causing clinical mastitis. There were examples of the same strain being isolated from different cows in the same herd, suggesting either transmission at milking or acquisition from a common environmental source. It was not uncommon for the same PFGE type to be isolated from the same udder quarter on more than one occasion, probably because the organisms persisted and re-emergedto cause clinical mastitis later. Weak biofilm production occurred in most isolates from cows with or without mastitis. The addition of milk markedly increased the amount of biofilm formation of S. uberis isolates. No specific milk component milk enhanced or reduced biofilm formation. Iron limitation led to a concentration-dependent reduction in biofilm formation by the clinical isolates but not isolates from apparently uninfected cows (with low cell counts). The indigenous flora of raw milk appears to contribute to biofilm formation since limited amounts of biofilm were produced when indigenous flora were removed from milk. Microarray analysis and construction of a dendogram showed a clear differentiation between clinical isolates and isolates from apparently uninfected cows. The first cluster in the dendogram contained seven isolates; among them just one isolate from a cow with clinical mastitis. These isolates are unlikely candidates for possessing S. uberis virulence-associated factors. The second cluster was mixed, consisting of seven isolates; four from cows with clinical mastitis and three from cows with low milk cell counts. These S. uberis isolates may represent opportunistic pathogens, possibly able to cause infections in particular groups of cows with increased susceptibility to infection, for example heifers or older animals. The third, fourth and fifth clusters contained 15 isolates from cows with clinical infection, but no isolates from cows with low milk cell counts. Analysis of selected sequences that correlated with disease status identified several sequences found in metabolic genes in streptococcal genomes. The correlation between the distributions of these DNA sequences and disease status could indicate their essential role in the growth or survival of S. uberis in diverse environments rather than their roles as virulence factors. Two further probes (W3-30 and W2-38) matched with a putative transposase of S. salivarius, suggesting the presence of an insertion sequence gained through horizontal gene transfer from other Streptococcus spp. The sequence W3-21 was highly homologous to a hypothetical protein in Enterococcus faecalis, and is unlikely to not represent a S. uberis virulence factor; however, the sequence of probe W2-17 had no significant match in the database and therefore could represent a novel virulence marker gene.