CHARACTERIZATION OF A GREEN FLUORESCENT PROTEIN EXPRESSING
Hammac, Gena Kenitra
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Anaplasma marginale is a tick-transmitted, obligate intraerythrocytic pathogen of cattle, causing anaplasmosis characterized by anemia, decreased milk and meat production, and occasionally death. Animals that survive are persistently infected and serve as a reservoir for tick transmission. Biological transmission via ixodid ticks is the most significant means of transmission, with Dermacentor andersoni being the most common vector in the Pacific Northwest. Molecular studies have been technically challenging as A. marginale is not receptive to genetic manipulation. The creation of a green fluorescent protein expressing mutant of the St. Maries strain of A. marginale (AmStM-GFP) by non-targeted recombination has provided new ways to address questions regarding protection and superinfection.There is no universally accepted vaccine for anaplasmosis and experimentally only partial protection is provided by specific immunodominant or subdominant proteins. We used cell culture based AmStM-GFP as a live vaccine to test for safety and protection upon homologous challenge as compared to the live A. marginale ssp centrale vaccine strain used in Israel, Australia and South America. AmStM-GFP was as safe as A. centrale, and provided superior clinical protection. This study is a proof of concept for cell culture based live vaccines.Superinfection of a persistently infected animal with a second strain of the same pathogen is thought to occur due to the presence of unique alleles in the superinfecting strain to which the host's immune system has not been primed. This has been demonstrated for the intracellular bovine pathogen, Anaplasma marginale, as a second strain with a single unique allele of an immunodominant surface protein is able to superinfect. AmStM-GFP was used as a tool to determine if a strain with an identical repertoire of surface protein alleles as the primary infection can establish superinfection. AmStM-GFP was not detected in animals following challenge, and there was a lack of correlation between specific antibody and specific variants identified in the transmission ticks.AmStM-GFP was also tested against a cell culture based wild type strain (AmStM) using RNA-seq data for global transcriptional analysis. As a spin-off of this study, we compared AmStM in tick derived cell culture with AmStM from bovine blood to identify differential transcription between the two environments. RNA-seq revealed overrepresentation of surface proteins in the differentially transcribed genes. We tested as set of genes by quantitative PCR in A. marginale collected from tick tissues at two time points to assess how well tick cell culture serves as a model for the tick vector. We identified surface remodeling of A.marginale in the tick vector, and found that tick cell culture most closely approximates the tick salivary gland following acquisition feed.