Transcriptomic and genomic evidence for Streptococcus agalactiae adaptation to the bovine environment.

BACKGROUND: Streptococcus agalactiae is a major cause of bovine mastitis, which is the dominant health disorder affecting milk production within the dairy industry and is responsible for substantial financial losses to the industry worldwide. However, there is considerable evidence for host adaptation (ecotypes) within S. agalactiae, with both bovine and human sourced isolates showing a high degree of distinctiveness, suggesting differing ability to cause mastitis. Here, we (i) generate RNAseq data from three S. agalactiae isolates (two putative bovine adapted and one human) and (ii) compare publicly available whole genome shotgun sequence data from an additional 202 isolates, obtained from six host species, to elucidate possible genetic factors/adaptations likely important for S. agalactiae growth and survival in the bovine mammary gland. RESULTS: Tests for differential expression showed distinct expression profiles for the three isolates when grown in bovine milk. A key finding for the two putatively bovine adapted isolates was the up regulation of a lactose metabolism operon (Lac.2) that was strongly correlated with the bovine environment (all 36 bovine sourced isolates on GenBank possessed the operon, in contrast to only 8/151 human sourced isolates). Multi locus sequence typing of all genome sequences and phylogenetic analysis using conserved operon genes from 44 S. agalactiae isolates and 16 additional Streptococcus species provided strong evidence for acquisition of the operon via multiple lateral gene transfer events, with all Streptococcus species known to be major causes of mastitis, identified as possible donors. Furthermore, lactose fermentation tests were only positive for isolates possessing Lac.2. Combined, these findings suggest that lactose metabolism is likely an important adaptation to the bovine environment. Additional up regulation in the bovine adapted isolates included genes involved in copper homeostasis, metabolism of purine, pyrimidine, glycerol and glucose, and possibly aminoglycoside antibiotic resistance. CONCLUSION: We detected several genetic factors likely important in S. agalactiae's adaptation to the bovine environment, in particular lactose metabolism. Of concern is the up regulation of a putative antibiotic resistance gene (GCN5-related N-acetyltransferase) that might reflect an adaptation to the use of aminoglycoside antibiotics within this environment.

Evaluation of an alternative dosing regimen of a J-5 mastitis vaccine against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows.

The objective of the study was to evaluate the efficacy of an alternative vaccination regimen of a J-5 bacterin against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows. The parameters analyzed to assess the effect of vaccination were milk yield, milk conductivity, somatic cell count, J-5-specific serum IgG titers, and clinical signs. Twenty multiparous Holstein cows from the Cornell teaching and research dairy herd were paired by days in milk and were randomly selected to receive either the alternative off-label regimen of commercial J-5 bacterin or act as nonvaccinated controls. Cows received a first dose of bacterin 15 d before dry off, a second dose with the same product at the day of dry off, and the third dose 2 wk after dry off. The cows in both groups were challenged 10 d before the expected calving date. Serum IgG (total, IgG1 and IgG2) levels were higher in vaccinates compared with control cows. Eighty-five percent of challenged quarters became infected between both groups of animals. Eight of the 10 vaccinated and 9 of the 10 control cows showed signs of clinical mastitis postfreshening. A non-severe clinical mastitis was observed 24 to 48 h postparturition, characterized by flakes or clots in milk and mild swelling or pain. Off-label vaccination did reduce the clinical severity of clinical mastitis in the vaccinated group of cows as evidenced by reduced California mastitis test score, fewer flakes and lower overall clinical mastitis score. No significant differences between vaccinated and control groups were detected for rectal temperature. In conclusion, the alternative off-label vaccination scheme used in our study and evaluated in a novel E. coli challenge model did not prevent new intramammary infections but reduced clinical severity of experimentally induced E. coli mastitis.

Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens beta 2-toxin.

Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. beta2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of beta2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 A resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a = b = 103.71, c = 193.48 A, alpha = beta = 90, gamma = 120 degrees using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles omega = 90.0, phi = 210.3 degrees.