Identification of Corynebacterium bovis and other coryneforms isolated from bovine mammary glands

Bovine mastitis remains the most economically important disease in dairy co ws. Corynebacterium bovis, a lipid-requiring Corynebacterium spp., is frequ ently isolated from the milk of infected mammary glands of dairy cows and i s associated with reduced milk production. A total of 212 coryneform bacter ia isolated from the milk of dairy cows were obtained from mastitis referen ce laboratories in the United States and Canada. All isolates had been pres umptively identified as Corynebacterium bovis based on colony morphology an d growth in the presence of butterfat. Preliminary identification of the is olates was based on Gram stain, oxidase, catalase, and growth on unsuppleme nted trypticase soy agar (TSA), TSA supplemented with 5% sheep blood, and T SA supplemented with 1% Tween 80. Of the 212 isolates tested, 183 were iden tified as Corynebacterium spp. based on preliminary characteristics. Of the strains misidentified, one was identified as a yeast, two as Bacillus spp. , 11 as Enterobacteriaceae, 18 as staphylococci, one as a Streptococcus spp ., and one as an Enterococcus spp. Eighty-seven coryneforms were selected f or identification to the species level by direct sequencing of the 16S rRNA gene, the Biolog system and the API Coryne system. Fifty strains were iden tified as C. bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne systems correctly identified 54.0 and 88.0% of these strains, respec tively. The other coryneforms were identified as other Corynebacterium spp. , Rhodococcus spp., or Microbacterium spp. These data indicate that the cor yneform bacteria isolated from bovine mammary glands are a heterogeneous gr oup of organisms. Routine identification of C. bovis should include Gram-st ain, cell morphology, catalase production, nitrate reduction, stimulated gr owth on 1% Tween 80 supplemented media, and beta-galactosidase production a s the minimum requirements.