Search

Background Pathogens dependent upon vectors for transmission to new hosts undergo environment specific changes in gene transcription dependent on whether they are replicating in the vector or the mammalian host. Differential gene transcription, especially of potential vaccine candidates, is of interest in Anaplasma marginale, the tick-borne causative agent of bovine anaplasmosis. Methods RNA-seq technology allowed a comprehensive analysis of the transcriptional status of A. marginale genes in two conditions: bovine host blood and tick derived cell culture, a model for the tick vector. Quantitative PCR was used to assess transcription of a set of genes in A. marginale infected tick midguts and salivary glands at two time points during the transmission cycle. Results Genes belonging to fourteen pathways or component groups were found to be differentially transcribed in A. marginale in the bovine host versus the tick vector. One of the most significantly altered groups was composed of surface proteins. Of the 56 genes included in the surface protein group, eight were up regulated and 26 were down regulated. The down regulated surface protein encoding genes include several that are well studied due to their immunogenicity and function. Quantitative PCR of a set of genes demonstrated that transcription in tick cell culture most closely approximates transcription in salivary glands of recently infected ticks. Conclusions The ISE6 tick cell culture line is an acceptable model for early infection in tick salivary glands, and reveals disproportionate down regulation of surface protein genes in the tick. Transcriptional profiling in other cell lines may help us simulate additional microenvironments. Understanding vector-specific alteration of gene transcription, especially of surface protein encoding genes, may aid in the development of vaccines or transmission blocking therapies.

We have adapted a methylene blue staining assay to measure proliferation of equine satellite cell clones in a 96-well format. This technique allows rapid and accurate measurement of proliferating satellite cells which is a considerable enhancement over manual counting methods. Methylene blue is incorporated into the nuclei and intracellular matrix of satellite cells and then released into the aqueous phase. Absorbance of stained cells is read at 620 nm and correlates with increasing numbers of cells (range tested from 1 × 103 to 4 × 104). This method was used to determine the response of equine satellite cells to FGF, both human recombinant and bovine, and to IGF-1. This format is very efficient in measuring and comparing the proliferation of equine satellite cells. However, fusion of cells to form multinucleated myotubes cannot be assayed using this method because it lacks the sensitivity and specificity to differentiate multinucleated from mononucleated cells, and to detect expression of myogenic proteins. The assay could be accurately applied from 0 to 144 hours, before significant fusion and differentiation takes place. Using this assay will reduce analysis time to quantitate the proliferation response of equine satellite cells to different growth factors.