A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.
KEY WORDS: Rickettsia-like organism · Crassostrea ariakensis · Antigenic characteristics · Purification
Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [149][150][151][152][153][154] 2005 Populations of the oysters Crassostrea ariakensis suffered heavy losses during recent winters and springs in China. A potential pathogen, a rickettsialeslike organism, was discovered in the oyster (Wu & Pan 2000, Sun & Wu 2004. To better understand the significance of these molluscan RLOs in terms of pathogenicity, epidemiological investigation and taxonomy, we should characterize them at the molecular level.An important step in characterization of RLO proteins is the purification of the organism from infected tissues. Intracellular bacteria are usually incompletely released from cells, and more than half of the total infectious particles may remain associated with intact cells or cell ulcer debris (Moulder 1985). Some protocols have been established for vertebrate Rickettsiales, which can be cultured in the yolk sac of chicken embryos (Dasch & Weiss 1977, Williams et al. 1981 or grown in tissue culture (Weiss et al. 1975). Several techniques have been used for purifying various Rickettsiales (Tamura et al. 1982), specifically, isopycnic density gradient centrifugation in Percoll or in diatrizoate meglumine and diatrizoate sodium (DMDS, commercially known as renografin or hypaque). A Percoll density gradient centrifugation technique, modified from Tamura et al. (1982), was used by Kuzyk et al. (1996) to purify Piscirickettsia salmonis from cultured cells. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, rickettsia-like organisms must be purified from infected marine mollusk tissues. Applying centrifugation tec...