| Gene expression by M. haemolytica in the natural bovine host during infection was examined. RT-PCR analysis of bacterial RNA derived from pneumonic lung of infected calves revealed the expression of important virulence-associated genes. Transcripts for lktA, gcp, nmaA and narP were routinely detected. Transcripts for the gene encoding Lkt (one of the most important virulence factors in pathogenesis) were detected in the majority of the samples. Transcripts for the majority of the genes were detected in samples obtained from animals with high pneumonic damage. qRT-PCR analysis of the same samples did not reveal any noticeable pattern due to large inter-animal variations. To account for this, a time-course study was designed to analyze gene expression profiles at different time points within the same host. RNA samples were derived from pharyngeal swabs at 6 and 12 h post infection and lung washings at necropsy. Expression of key virulence-associated genes, such as lktA, gcp and tbpB, was higher in all in vivo samples compared to in vitro culture. Changes in the level of expression of most genes were observed during the course of study from 6 to 12 h. Expression of lktA and gapA increased while expression of fbpA, gs60, nmaA and tbpB decreased over time. Gene expression profiles in the lung versus the pharynx also differed, with most genes (fbpA, tbpB, nmaA, gs60, lktA and narP) showing higher expression in lung washings. Microarray analysis of lung washings from 6 days post-challenge revealed 44 genes that were differentially expressed by more than 8-fold. Lower levels of expression of genes involved in energy production and conversion, amino acid transport and metabolism and cell envelope biogenesis was observed, indicating a possible slowdown in growth. Most genes with higher expression are novel since their respective functions are currently unknown, including a putative lipoprotein. This study revealed that expression of some important virulence-associated genes, which have been postulated to have a role in pathogenesis, are high during early infection. Results from this study provided the evidence gene expression in vivo is dynamic during the course of infection, as wen as at different sites of infection. |
