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Korean J Parasitol > Volume 27(2):1989 > Article

Original Article
Korean J Parasitol. 1989 Jun;27(2):87-100. Korean.
Published online Mar 20, 1994.  http://dx.doi.org/10.3347/kjp.1989.27.2.87
Copyright © 1989 by The Korean Society for Parasitology
Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells
Jong-Yil Chai,Soon-Hyung Lee,Woong-Heum Kim,* and Chong Ku Yun*
Department of Parasitology and Institute of Endemic Diseases, College of Medicine,Seoul National University, Seoul 110-460, Korea.
Abstract

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella oocysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.

Figures


Fig. 1
MDBK (Madin Darby bovine kidney) cell monolayer, obtained 2 days after subculture with 1×105 cells/ml suspended in EBSS-EMEM medium, Hematoxylin-acid fuchsin-orange G (HT-AF-OG) stain, ×100.


Fig. 2
A sporozoite of E. tenella (arrow) in the cytoplasm of MDBK cell surrounded by a parasitophorous vacuole of host origin, nearby the host cell nucleus(N). The sporozoite shows its nucleus at center and refractile vacuole at left terminal portion. One-day culture. HT-AF-OF stain, ×400.


Fig. 3
Ibid, another big sporozoite (arrow). The nucleus of sporozoite is seen in the center. One-day culture. HT-AF-OG stain, ×400.


Fig. 4
A round-up and an elongated form (trophozoite) of E. tenella in the cytoplasm of a MDBK cell, enveloped by surrounding parasitophorus vacuoles. One-day culture. HT-AF-OG stain, ×400.


Fig. 5
A round-up trophozoite of E. tenella, which is probably dead or under degeneration. No nucleus nor globule is recognizable. Two-day culture. HT-AF-OG stain, ×400.


Fig. 6
A very big trophozoite of E. tenella, under further development. Host cell nucleus(N). Two-day culture. HT-AF-OG stain, ×400.


Fig. 7
An immature(developing) schizont of E. tenella in the cytoplasm of a MDBK cell. Its nuclear chromatin is divided into 7 or more. The parasite is occupying almost all of the host cell cytoplasm. Two-day culture. HT-AF-OG stain, ×400.


Fig. 8
Two immature schizonts of E. tenella (arrows), first generation. The upper one has distinctly been divided into about 15 merozoites. Two-day culture. HT-AF-OG stain, ×400.


Fig. 9
Two immature schizonts of E. tenella (arrows), second generation. Numerous immature merozoites are seen at this stage. Host cell nucleus(N). Three-day culture. HT-AF-OG stain, ×400


Fig. 10
A mature schizoites (second generation) of E. tenella (arrows), containing numerous second generation merozoites. Three-day culture. HT-AF-OG stain, ×400.


Fig. 11
A fully mature schizont (large arrow) containing numerous mature merozoites (small arrows). The host cell is almost entirely occupied by the parasite and is about to rupture. Four-day culture. HT-AF-OG stain, ×400.


Fig. 12
Second generation merozoites (arrows) of E. tenella penetrating into new MDBK cells. Four-day culture. HT-AF-OG stain, ×400.


Fig. 13
3H-uracil uptake by E. tenella cultured in MDBK cell monolayer.


Fig. 14
3H-uracil uptake by E. tenella sporozoites preincubated in culture medium.

Tables


Table 1
3H-uracil uptake by E. tenella according to the dosage of sporozoite inoculum and time after inoculation into MDBK cell culture


Table 2
3H-uracil uptake by E. tenella sporozoites according to preincubation time


Table 3
3H-uracil uptake by E. tenella preincubated for 4 or 12 hours

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