Date of Award


Degree Name

Doctor of Philosophy


Biological Sciences

First Advisor

Dr. Silvia Rossbach

Second Advisor

Dr. Robert C. Eisenberg

Third Advisor

Dr. Leonard Ginsberg

Fourth Advisor

Dr. Charles W. Ford


Coryneform bacteria are frequently isolated from bovine mastitis and these infections are associated with economic losses. Corynehacterium bovis, a lipid requiring species, has been the most frequently isolated coryneform from the milk of infected bovine mammary glands. However, the taxonomic status of this organism is uncertain. In the current study, a polyphasic approach was used to identify coryneform bacteria isolated from bovine mastitis and determ ine the phylogenetic relationships among the identified species. A total of 212 coryneform bacteria isolated from bovine mastitis was obtained from mastitis reference laboratories in the United States and Canada. Presumptive identification based upon G ram -stain, oxidase, catalase, and Tween 80 stimulated growth classified 183 isolates as Corynehacterium species. Eighty-seven strains were selected for species level identification by 16S rRNA gene sequencing, the Biolog system and the API Coryne system . Fifty strains were identified as Corynebacterium bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne system s identified 54.0 and 88.0% of these strains, respectively. Antimicrobial susceptibility testing o f 46 C. bovis and 14 C. amylocolatum strains determined these organisms were susceptible to am picillin, oxacillin, cephalothin, ceftiofur, a com bination of penicillin and novobiocin, erythromycin, clindamycin, pirlimycin, tetracycline, florfenicol, enrofloxacin, sarafloxacin, danofloxacin, and prem afloxacin but not tilmicosin. Finally, phylogenetic studies were perform ed by direct sequencing of the 16S ribosom al RNA and phylogenetic analyses perform ed. All strains identified as C. bovis examined clustered with the reference strains indicating that C. bovis is a well defined species within the genus Corynehacterium. Rep-PCR o f these strains indicated that only minor genetic variation exists within strains of C. bovis. Corynehacterium bovis ATCC 13722 was determ ined to be most closely related to Brevibacterium helvolum. Based on phylogenetic analyses, this organism was placed in the genus Brevibacterium as Brevibacterium neaveae sp. nov. Results of this study confirm that the coryneform s isolated from bovine mammary glands are a heterogeneous group of organisms. Furthermore, direct sequencing of the 16S rRNA gene appears to be the most accurate method for identification of Corynehacterium species isolated from bovine mastitis.

Access Setting

Dissertation-Open Access