The objectives of this work were: 1) to complete a whole genome sequence of a Staphylococcus chromogenes isolate that is known to persist in the mammary gland of a cow, 2) to define a genetic target that could easily and reliably be used to differentiate Staphylococcus agnetis and Staphylococcus hyicus and to use this new assay along with pulsed-field gel electrophoresis (PFGE) strain typing to characterize a collection of non-Staphylococcus aureus coagulase positive staphylococci, 3) to determine if the recently described S. aureus Genotype B was present among previously characterized isolates from cases of bovine intramammary infection in the USA and to compare PFGE to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for strain typing of S. aureus, 4) to further the understanding of the association between body site colonization and intramammary infection before and after calving in heifers using molecular methods of speciation and strain typing, and 5) to describe the prevalence and distribution of Staphylococcus species on the teat and inguinal skin of dairy heifers throughout the heifer lifecycle using molecular speciation methods. A draft genome sequence of S. chromogenes MU 970, a strain that was isolated from the right rear quarter of a Holstein cow for 16 consecutive months, was completed. Whole genome sequences for S. chromogenes, S. hyicus, and S. agnetis were then used to construct a multiplex PCR used to differentiate S. hyicus, S. agnetis, and S. aureus. The multiplex PCR was then used to characterize a collection of isolates and found that S. agnetis was much more prevalent than S. hyicus. Furthermore, S. agnetis can cause persistent infections and can potentially be contagious. Next, RS-PCR plus toxin gene identification was explored as a possible strain typing method, however it was found to be less discriminatory than PFGE. Hence, PFGE was used as the strain typing method for the remainder of the dissertation research. The final two studies were conducted to characterize body site colonization with staphylococcal species and to explore the relationship of prepartum body site colonization to intramammary infections, both pre- and post-calving. MALDI-TOF was explored as a speciation method in these studies and was found to be accurate, rapid, and inexpensive. Overall, S. chromogenes was the most common species identified in prepartum mammary secretions and post-calving milk samples. Through PFGE strain typing, it was determined that many different strains of each species can be found on one heifer. Finally, it was determined that colonization of the inguinal region and teat skin was associated with age for S. agnetis, S. chromogenes, Staphylococcus devriesei, Staphylococcus equorum, Staphylococus haemolyticus, Staphylococcus lentus, Staphylococcus sciuri, Staphylococcus vitulinus, and Staphylococcus xylosus. Overall, these studies demonstrate that the staphylococcal species and subspecies associated with IMI are extremely abundant and diverse on dairy farms.
Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing