Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: During this period the microarrays for Mannheimia haemolytica were redesigned by Nimblegen. The microarrays now carry 72,000 features in a 4-plex format. In collaboration with a research group at Cambridge University in the United Kingdom, RNA has been prepared from Mannheimia haemolytica cells grown on bovine tissue explants in tissue culture. cDNA has been created from this RNA and it will soon be submitted to Nimblegen to probe the new arrays. PARTICIPANTS: Dr. Sarah Highlander has acted as supervisor of the project. She supervises a Masters' level technician who is performing molecular biology aspects of the project. Dr. Mark Ackermann is responsible for coordination and supervision of animal experiments. Dr. Dan Tucker is a collaborator who has provided cDNA from bacteria growing in the presence of bovine explants. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Two major changes in approach have occurred. First, we have changed to a new microarry format. This occurred because Nimblegen changed formats and no longer produced the arrays that we used for bacterial cells grown in vitro. Second, with the help of a collaborator, we added an experiment to examine the effect of bovine explant tissue on the expression of Mannheimia haemolytica genes. We believe that these data will create a sort of bridge between the in vitro and in vivo grown bacteria.
Impacts
None at this time.
Publications
- No publications reported this period
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Progress 09/30/06 to 09/30/07
Outputs
During this period we have had microarrays constructed with four oligonucleotides per gene. These were constructed and optimized by Nimblegen. RNA has been prepared from Mannheimia haemolytica cells grown with and without iron, with and without paraquat, to induce oxidative stress and at 30 versus 39 degrees Celsius. RNAs were prepared in triplicate. The RNAs were biotin labelled and hybridized to the arrays. Data were normalized and were compared using the MeV_4.0.1 program. There were numerous statistical differences between the iron replete and iron deplete cells and at 30 versus 39 degrees. No significant differences were observed between the cells grown with and without paraquat.
Impacts
These experiments are hoped to reveal how Mannheimia haemolytica responds to differences in the host and to potentially reveal new virulence mechanisms. Knowledge of these mechanisms could lead to new treatments for shipping fever pneumonia, which costs the feedlot industry in the US over one billion dollars each year.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Mannheimia haemolytica is a commensal of the ruminant upper respiratory tract and is the primary bacterial pathogen in polymicrobic pneumonia called bovine respiratory disease complex (BRDC). BRDC causes losses to the US feedlot industry of nearly one billion dollars each year. Viral infection is often a predisposing factor to the development of BRDC because respiratory viruses, such as bovine herpesvirus-1 (BHV-1), impair primary immune responses and induce cytokine responses that can lead to superinfection by commensal upper respiratory bacteria like M. haemolytica. We are interested in examining M. haemolytica gene expression in the bovine host and would like to compare transcription, by microarray analysis, in the upper and lower respiratory tracts in virally and non-virally challenged animals. The draft sequence of M. haemolytica has been annotated and analyzed. The genome contains 2838 predicted open reading frames. These sequences have been used to create a
whole genome oligonucleotide microarray. M. haemolytica RNAs from cells growing under a variety of in vitro conditions (temperature, iron concentration, oxidative stress) have been prepared and will be used to interrogate the arrays. Once the in vitro data have been collected we will begin experiments aimed at isolating bacterial RNAs from the lungs of M. haemolytica-infected calves.
Impacts
Microarray analysis should reveal known and novel bacterial genes that are differentially expressed in the host. It may also reveal differences between upper respiratory and lower respiratory gene expression, and may indicate whether viral-bacterial interactions are direct or immune-mediated. Genes identified by this project will be subjects for future functional characterization.
Publications
- No publications reported this period
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