The Growth and Pathogenesis of the Intracellular Pathogen, Piscirickettsia salmonis

• Main Author: Makrinos, Daniel
• Format: Text
• Language: unknown
• Published: DigitalCommons@UMaine 2016
• Subjects: Piscirickettsia salmonis; agar; growth; intracellular; salmon; SRS; Aquaculture and Fisheries; Bacteriology; Cell Biology; Environmental Microbiology and Microbial Ecology; Immunity; Immunology of Infectious Disease; Immunopathology; Immunoprophylaxis and Therapy; Marine Biology; Medicinal Chemistry and Pharmaceutics; Microbial Physiology; Molecular Biology; Organismal Biological Physiology; Pathogenic Microbiology; Mak; Atlantic salmon
• Online Access: https://digitalcommons.library.umaine.edu/etd/2552; https://digitalcommons.library.umaine.edu/context/etd/article/3589/viewcontent/Daniel_L_Makrinos_Final_Thesis_P._salmonis_1_10_17.pdf

Piscirickettsia salmonis is the causative agent of piscirickettsiosis or salmon rickettsia syndrome (SRS). It is a Gram-negative intracellular bacterium that causes significant mortalities to salmonids, particularly in Chile. The initial isolation and identification of P. salmonis occurred in fish cell lines, therefore growth of P. salmonis is most often carried out in tissue culture. Three cell lines were analyzed in this study to determine which in vitro cellular environment was most suitable for additional research. Tissue culture has been used for the growth and cultivation of P. salmonis for nearly two decades, until the facultative nature of the pathogen was confirmed upon the development of blood and cysteine based agar. Since then, research has continued to drive the creation of novel agar and broth formulations in order to improve the efficacy of cultivation of P. salmonis in cell-free environments. In this thesis, several published agar and broth formulations were tested to understand how this intracellular bacterium grows in these environments. Cell-free media compositions were investigated by using previous formulations and atomic emission spectrophotometry in order to create novel media. This led to the development of MAK broth, MAK2 agar, and BCYE-SRS agar, which significantly improved overall cell-free growth. Furthermore, growth was evaluated using qPCR, SDS-PAGE gels, growth curves, cell counts, colony forming units, and TCID50 titration assays. Additionally, diagnosis of the pathogen was improved by the use of novel cell-free media, vital stains, and PCR assays. In order to study P. salmonis in vivo, two studies were conducted. The first used an intraperitoneal chamber model in Atlantic salmon to observe how the bacterium and host responded and intercommunicated among one another. A lethal dose trial was then carried out in freshwater to determine if P. salmonis grown in cell-free media was a viable option for future challenge trials. Overall, in vitro and in vivo studies provided an ...