Phylogenetic studies on Corynebacterium bovis isolated from bovine mammary glands.

Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.

Identification of corynebacterium bovis and other coryneforms isolated from bovine mammary glands.

Bovine mastitis remains the most economically important disease in dairy cows. Corynebacterium bovis, a lipid-requiring Corynebacterium spp., is frequently isolated from the milk of infected mammary glands of dairy cows and is associated with reduced milk production. A total of 212 coryneform bacteria isolated from the milk of dairy cows were obtained from mastitis reference laboratories in the United States and Canada. All isolates had been presumptively identified as Corynebacterium bovis based on colony morphology and growth in the presence of butterfat. Preliminary identification of the isolates was based on Gram stain, oxidase, catalase, and growth on unsupplemented trypticase soy agar (TSA), TSA supplemented with 5% sheep blood, and TSA supplemented with 1% Tween 80. Of the 212 isolates tested, 183 were identified 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 for identification to the species level by direct sequencing of the 16S rRNA gene, the Biolog system and the API Coryne system. Fifty strains were identified 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, respectively. The other coryneforms were identified as other Corynebacterium spp., Rhodococcus spp., or Microbacterium spp. These data indicate that the coryneform bacteria isolated from bovine mammary glands are a heterogeneous group of organisms. Routine identification of C. bovis should include Gram-stain, cell morphology, catalase production, nitrate reduction, stimulated growth on 1% Tween 80 supplemented media, and beta-galactosidase production as the minimum requirements.

Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model.

Actinobacillus pleuropneumoniae is a significant respiratory pathogen of swine causing a severe and often fatal fibrinous hemorrhagic bronchopneumonia with significant economic losses resulting from chronic as well as acute infections. This study describes the application of a signature-tagged mutagenesis (STM) system to identify in vivo critical genes of A. pleuropneumoniae. Twenty pools representing over 800 A. pleuropneumoniae mutants were screened in a natural-host porcine infection model and presumptive attenuated mutants were selected. The identity of the disrupted gene in each mutant was determined using an inverse PCR approach to amplify DNA sequences adjacent to the transposon insertion, followed by sequencing of the PCR product and comparison to bacterial databases. In vitro and in vivo competitive indices were determined for each unique mutant, and a total of 20 unique, attenuating gene disruptions were identified including insertions into homologues of genes involved in biosynthesis, virulence determinants, regulation, translation and unknown functions. Three of the genes required for virulence of A. pleuropneumoniae in this study were also identified in a previous STM study of Pasteurella multocida. Seven of the STM-derived mutants were also evaluated for their potential as live vaccine strains and provided good protection against homologous challenge.

Effects of acetaldehyde on glucose-6-phosphate dehydrogenase activity and mRNA levels in primary rat hepatocytes in culture.

Ethanol has been shown to induce the activity of glucose-6-phosphate dehydrogenase (G6PDH). To clarify the mechanism behind this induction, we examined the role of acetaldehyde (AA), the first product of ethanol metabolism. In primary adult rat hepatocytes maintained in chemically defined medium, we examined the effect of AA on G6PDH activity, mRNA levels and lipid synthesis. We observe a 40% increase in G6PDH activity and a similar increase in mRNA levels, following exposure to 100 microM AA. The increase in activity was found to be maximal at 24 h while mRNA levels increased over controls as early as 3 h. The induction in G6PDH by AA was found to occur at lower concentrations and earlier time points than those reported using ethanol. The role of insulin, a known inducer of G6PDH activity was studied alone and in combination with AA on both G6PDH activity and mRNA levels as well as lipid biosynthesis. Insulin (300 ng/ml) was found to increase G6PDH activity, mRNA levels and [14C]-acetate incorporation into lipid. It was also shown to have an additive effect with AA on G6PDH activity, suggesting their actions are mediated via different mechanistic pathways. No change in [14C]-acetate incorporation into lipid, however, was observed with acetaldehyde alone.