title: Improved purification of Piscirickettsia salmonis using Percoll gradients.
abstract: Viable preparations of intact Piscirickettsia salmonis, purified from host cell material, are necessary for studying characteristics associated with this bacterium. However, purification of the organism is difficult due to its obligate intracellular nature. A simple and effective method for isolating whole P. salmonis, which is quick and easy to perform, but still maintains the viability and antigenicity of the bacterium is described. P. salmonis was purified by differential pelleting and density gradient centrifugation using 30%, 40%, or 50% (v/v) Percoll gradients. Following fractionation, a band with a density of 1.056-1.080 was found to be composed entirely of rickettsiae, confirmed by fluorescent antibody technique (IFAT). Purity of the P. salmonis from different stages of the purification process was assessed by light and transmission electron microscopy, and the viability of yields determined from a plaque assay and a tissue culture infective dose (TCID(50) ml(-1)). P. salmonis recovered from the 30% Percoll gradient appeared to retain their intracellular structure better than P. salmonis obtained from the 40% and 50% Percoll gradients, and appeared to have a greater viability. Differences were seen between P. salmonis-infected CHSE-214 cells and purified P. salmonis when compared by SDS-PAGE and Western blotting, and less host cell contamination was present in preparations obtained from the 30% Percoll gradient. Finally ten different P. salmonis isolates obtained from three different geographical locations and four different fish species, were purified using the 30% Percoll gradient. When the morphology of these was compared by transmission electron microscopy (TEM), they appeared similar in size and appearance, although isolate R980769 was highly pleomorphic and isolate R-29 was larger than the other isolates examined.
authors.affiliation: Akdeniz Su Urunleri Arastirma, Uretim ve Egitim Enstitusu, Mediterranean Aquaculture Research Institute, Ministry of Agriculture and Rural Affairs, Antalya, Turkey.
authors.forename: S A
authors.initials: SA
authors.lastname: Yuksel
authors.forename: K D
authors.initials: KD
authors.lastname: Thompson
authors.forename: A E
authors.initials: AE
authors.lastname: Ellis
authors.forename: A
authors.initials: A
authors.lastname: Adams
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial; D015153:Blotting, Western; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods*; D005399:Fishes / Q000382:microbiology*; D019084:Fluorescent Antibody Technique, Indirect; D046529:Microscopy, Electron, Transmission; D044147:Piscirickettsiaceae / Q000302:isolation & purification* / Q000648:ultrastructure; D011205:Povidone / Q000737:chemistry*; D012474:Salmon; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D000907:Antibodies, Bacterial; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone