- Short Communication - 19 3 (2006)
J. Fish Pathol., 19(3) 285 ~ 288 (2006)
285 Corresponding Author :
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Respiratory Burst Activity of Tilapia (Oreochromis mosambicus) Phagocytes to Edwardsiella tarda Ghosts
Se Ryun Kwon, Eun Hye Lee, Min Sun Kim, Yoon Kwon Nam and Ki Hong Kim Department of Aquatic Life Medicine, Department of Aquaculture,
Pukyong National University, Pusan 608-737, Korea
The respiratory burst activity of tilapia (Oreochromis mosambicus) phagocytes agaisnt Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated. Stimulation with E. tarda ghosts (ETG) showed markedly higher respiratory burst activity than that with formalin killed E. tarda (FKC) in phago- cytes. This result may suggest that ETG can induce effective cellular immune responses in fish.
Key words: Edwardsiella tarda, Ghosts, Respiratory burst activity, Phagocyte, Tilapia
The genetic inactivation of pathogenic Gram- negative bacteria by the controlled expression of cloned bacteriophage PhiX174 lysis gene E offers a promising new approach in non-living vaccine technology (Szostak et al., 1996; Eko et al., 1999).
Expression of plasmid-encoded gene E leads to the formation of a transmembrane tunnel structure through the cell envelope of Gram-negative bacte- ria, which consequently leading the loss of cyto- plasmic contents. The resultant bacterial ghosts have been known to retain the functional and anti- genic determinants of the envelope with their living counterparts, and are suggested as an alternative method for inactivation of bacteria without chemi- cal or physical stress which can reduce antigenicity.
Edwardsiella tarda, a Gram negative, motile, fla- gellated and rod-shaped bacterium, is the causative agent of edwardsiellosis and leads to extensive loss- es in many commercially important freshwater and marine fish (Thune et al., 1993; Plumb, 1999).
Although edwardsiellosis is still a common disease causing severe losses in many countries, effective
prophylactic or control measures are still needed.
Recently, we have generated E. tarda ghosts by gene E mediated lysis (Kwon et al., 2005), and have demonstrated significantly higher protection of fish immunized with E. tarda ghosts than that of fish immunized with formalin-killed E. tarda (Kwon et al., 2006).
Defense mechanism of phagocytes against invad- ing microorganisms is highly dependent on reactive oxygen species (ROS) generated during the respira- tory burst (Babior et al., 1973; Dahlgren and Karls- son, 1999; Neumann et al., 2001). During phagocy- tosis, the cells increase their oxygen consumption through the NADPH oxidase and generate various ROS that have strongly antimicrobial activities (Sharp and Secombes, 1993). In the present study, we investigated the potential of E. tarda ghosts in tilapia (Oreochromis mosambicus) to elicit cellular immune responses by analysis of phagocyte respi- ratory burst activity.
Juvenile tilapia, weighing 100 20 g, were obtained from the fish farm in Pukyong National Uni-
Corresponding Author : Ki Hong Kim, Tel : 051-620-6145, Fax : 051-628-7430, E-mail : [email protected]
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versity, Korea. Fish were stocked into three 100 aquaria, and were acclimated 2 weeks prior to the experiment.
E. tarda FSW910410 provided by National Fish- eries Research & Development Institute (Pusan, South Korea) and grown in Luria Broth (LB, Difco) at 27 . Transformed E. tarda was grown in LB containing 50 / ampicillin (Sigma). Incubation temperatures for repression and expression of lysis gene E in transformants were 27 and 42 , respectively. Growth and lysis of bacterial cultures were monitored by measuring the optical density at 600 nm (OD600).
Lyophilized E. tarda ghosts were produced as described previously (Kwon et al., 2005). Briefly, E. tarda harboring the lysis plasmid p PR-cI-Elysis was induced for lysis after growth under culture conditions by elevation of temperature from 27 to 42 . At the end of lysis, ghosts were harvested, washed and resuspended in PBS and then lyophilized. The efficiency of E-mediated killing of E. tarda was estimated by plating samples of appro- priate dilutions of lyophilized ETG on LB agar con- taining 50 / ampicillin and result was com- pared with those from samples obtained prior to onset of lysis. Results indicated a 100% killing effi- ciency as no colony-forming units were found on plates with lyophilized ETG preparations at any dilution.
E. tarda was grown for 24 h at 27 in tryptic soy broth (TSB, Sigma) containing 1.5% NaCl. For FKC preparation, formalin was added to 24 h cul- ture of bacterium to make the final concentration 0.5%. After 24 h incubation, cells were washed three times with phosphate buffered saline (PBS, pH 7.2) and resuspened in 10 PBS. The suspen- sions were streaked on tryptic soy agar containing 1.5% NaCl for checking sterility and stored at 4 until use.
Fish were anaesthetized with tricaine methanesul- fonate (MS222; Sigma). The head-kidney was extracted by ventral incision and transferred to min- imum essential medium (MEM; Sigma) supple- mented with 5% fetal calf serum (FCS; Sigma), heparin (10 units/ , Sigma), penicillin (100 / , Sigma) and streptomycin (100 U/ , Sigma). To get phagocytes, the cell suspensions obtained by forc- ing the organ through a nylon mesh were layered over a 34/51% Percoll density gradient. After cen- trifugation at 400 g for 30 min at 4 , the phago- cytes enriched interphase was collected and washed three times. Then, the cells were resuspended in culture medium, and dispensed into flat-bottomed 96-well plates. After 2 h at 20 , wells were washed with culture medium to remove non-adher- ent cells. The remained phagocytes were detached from the plates by incubating for 1 h at 4 . The cell viability was examined with tryphan blue exclusion and evaluated to be greater than 98%.
The number of phagocytes were adjusted to 1 106cells/ .
ROS produced from stimulated phagocytes was quantified using an automatic photoluminometer (Bio-Orbit 1251). Each test cuvette contained 0.7 luminol (Sigma), 0.4 cell suspension, and 0.3 ETG (1 108cells/ ) or FKC (1 108cells/
). Zymosan (Sigma) opsonized with tilapia serum was used as the positive control. The measurements were made for 2 h and the assay was carried out in triplicate. Respiratory burst activity data were ana- lyzed by the Student's t-test, and significant differ- ences were determined at P<0.05.
As shown in Fig. 1, phagocytes stimulated with ETG showed markedly higher respiratory burst activity than those stimulated with FKC. However, the peak value of chemiluminescent responses (CL) of phagocytes stimulated with ETG was significant- ly lower than that of phagocytes stimulated with
Se Ryun Kwon, Eun Hye Lee, Min Sun Kim, Yoon Kwon Nam and Ki Hong Kim
287
zymosan.
The markedly higher respiratory burst activity of phagocytes stimulated with ETG than with FKC suggests that fish phagocytes react to ETG more efficiently than to FKC. An efficient uptake by anti- gen presenting cells (APCs) is a prerequisite for directing T- and B-cells towards the desired immune responses. Therefore, the present result suggests that E. tarda ghosts might have a higher potential to induce specific immune responses in fish than E. tarda FKC.
Acknowledgements
This work was supported by the Korea Research Foundation Grant (KRF-2003-F00037).
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Respiratory burst activity of tilapia phagocytes to E. tarda ghosts
Fig. 1. Respiratory burst activity of tilapia (Oreochromis mosambicus) head kidney phagocytes exposed to zymosan, Edwardsiella tarda ghosts (ETG), and formalin-killed E.
tarda (FKC). The respiratory burst activity was measured by chemiluminescent response (CL). Results are mean of triplicate samples of 3 fish and bars represent standard error.
Different letters on the bar indicate statistically significant differences at P<0.05.
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Manuscript Received : November 13, 2006 Revision Accepted : December 8, 2006 Responsible Editorial Member : Myung-Joo Oh (Chonnam Univ.) Se Ryun Kwon, Eun Hye Lee, Min Sun Kim, Yoon Kwon Nam and Ki Hong Kim
