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Decoquinate induces tissue cyst formation by the RH strain of Toxoplasma gondii
Veterinary Parasitology 77 Ž1998. 75–81
Decoquinate induces tissue cyst formation by the RH strain of Toxoplasma gondii David S. Lindsay ) , Maria A. Toivio-Kinnucan, Byron L. Blagburn Department of Pathobiology, College of Veterinary Medicine, Auburn UniÕersity, Auburn, AL 36849-5519, USA Received 11 September 1997; accepted 10 December 1997
Abstract Decoquinate is an anticoccidial agent that inhibits respiration in the parasites mitochondrion. We examined human foreskin fibroblast cell cultures infected with the normally tissue cyst-less RH strain of Toxoplasma gondii and treated with decoquinate for evidence of tissue cyst induction and formation. Transmission electron microscopy observations demonstrated tissue cysts in decoquinate-treated cultures on days 3, 4, 5, and 6 after inoculation. Tissue cysts contained a tissue cyst wall that enclosed stages that resembled tachyzoites and stages that were structurally bradyzoites. Similar treatment of human foreskin fibroblast cells infected with tachyzoites of the TS-4 temperature-sensitive mutant of the RH strain did not result in production of tissue cysts. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Decoquinate; Tissue cyst; RH strain; Toxoplasma gondii
1. Introduction Toxoplasmic encephalitis is a life threatening condition observed in many AIDS patients ŽLuft and Remington, 1988.. The development of toxoplasmic encephalitis is associated with reactivation Žrupture. of the latent tissue cyst with release of bradyzoites and subsequent destruction of brain tissue by actively dividing tachyzoites ŽFrenkel and Escajadillo, 1987.. Reactivation of bradyzoites in tissue cysts is also the cause of retinochoriditis observed in congenitally infected humans. Because of this importance much attention has been given to the production of Toxoplasma gondii tissue cysts in )
Corresponding author. DBMP, CMMID, Virginia-Maryland Regional College of Veterinary Medicine, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA. 0304-4017r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 0 1 7 Ž 9 8 . 0 0 0 9 9 - 5
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D.S. Lindsay et al.r Veterinary Parasitology 77 (1998) 75–81
cell culture systems since the late 1980’s Žsee Dubey et al., 1998.. Prior to the early 1990’s, it was believed that most strains of T. gondii did not produce tissue cysts in vitro, however it was demonstrated that this was not the case and that most strains examined would produce a small number of tissue cysts in vitro with no manipulation ŽLindsay et al., 1993a.. Several groups of workers have demonstrated that agents which induce stress will cause an increase in this background production of tissue cysts of several of these tissue cyst forming strains of T. gondii in vitro Žsee Dubey et al., 1998.. Decoquinate is a quinolone anticoccidial approved for use in the prevention of intestinal coccidiosis in goats and cattle. It inhibits coccidial respiration by interfering with electron transport in mitochondria ŽWang, 1975, 1976; Fry and Williams, 1984.. The precise site of action appears to be within the bc 1 complex where electrons are transferred from ubiquinone to cytochrome C. Decoquinate and the related antimalarialranti-Toxoplasma agent atovaquone have excellent activity against T. gondii in cell cultures ŽPfefferkorn et al., 1993. and atovaquone will cause an increase in tissue cyst production of the PLK strain of T. gondii in Vero cell cultures ŽTomavo and Boothroyd, 1995.. We have previously demonstrated that treatment of cells infected with the NC-1 strain of Neospora caninum with 0.1 m grml decoquinate for 5 min completely kills all parasites ŽLindsay et al., 1997.. In conducting comparative studies with the RH strain of T. gondii, we observed parasite survival and apparent tissue cyst formation using light microscopy. We conducted the present study to determine if decoquinate treatment did indeed cause tissue cyst production by the normal tissue cyst-less RH strain and extended the study to the TS-4 mutant strain ŽPfefferkorn and Pfefferkorn, 1976. of the RH strain which does not persist in the tissues of animals and is considered tissue cyst-less ŽWaldeland and Frenkel, 1983; Elwell and Frenkel, 1984a,b; Lindsay et al., 1993b; Pinckney et al., 1994, 1995..
2. Materials and methods 2.1. Maintenance of tachyzoites and cell cultures Tachyzoites of our cloned line ŽLindsay et al., 1995. of the RH strain of T. gondii and the TS-4 temperature-sensitive mutant of the RH strain were grown in monolayers of human foreskin fibroblast cells ŽHS68 cells, ATTC CRL 1635, Rockville, MD. grown in 25 cm2 plastic cell culture flasks. The growth media consisted of 10% Žvrv. fetal bovine serum in RPMI 1640 medium supplemented with 100 unitsrml penicillin G, 100 m grml streptomycin sulfate, 1 mM sodium pyruvate, and 5 = 10y2 mM 2-mercaptoethanol. The HS68 cells were maintained in and decoquinate studies were conducted in medium identical to the growth medium but which had the fetal bovine serum concentration lowered to 2% Žvrv.. Cell cultures were grown at 378C in a humidified atmosphere consisting of 5% CO 2 –95% air. Cells harboring the TS-4 strain were infected and maintained at 32.58C while those harboring the RH strain were infected and maintained at 378C.
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2.2. Tissue cyst induction by decoquinate Decoquinate Žlot 7916Z4, Rhone-Poulenc Animal Nutrition, N.A., Atlanta, GA. was dissolved in 90% methanolr0.1 N sodium hydroxide solution and diluted in cell culture maintenance medium prior to use in these studies. The concentration of this vehicle used Ž0.009% methanolr0.00001 N sodium hydroxide. was not toxic for host HS68 cells ŽLindsay et al., 1997.. Monolayers of HS68 cells were inoculated with 2 = 10 6 tachyzoites of the RH or TS-4 strains. All cultures were now kept at 378C. Two hours postinoculation ŽPI. the medium was removed and the intracellular tachyzoites were treated with 0.1 m grml Ž0.24 m m. decoquinate for 5 min. The decoquinate containing medium was removed and the monolayers washed 5 times in Hanks’ balanced salt solution. Decoquinate free
Fig. 1. Transmission electron micrograph of a tissue cyst of the RH strain of T. gondii in a human foreskin fibroblast cell. Bar s1.0 m m. Note the tissue cyst wall Žarrows., the identifiable bradyzoite ŽB. and the tachyzoite-like stage ŽT..
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D.S. Lindsay et al.r Veterinary Parasitology 77 (1998) 75–81
maintenance medium was added and the cell cultures were incubated at 378C until examined. Monolayers were processed for transmission electron microscopy ŽTEM. on days 3, 4, 5, and 6 PI for the RH strain and 5 and 11 days PI for the TS-4 strain. The monolayers were scraped from the plastic growth surface using a cell scraper and the cells pelleted by centrifugation a 1000 g for 5 min. The cell pellet was fixed in 3% Žvrv. glutaraldehyde in Millonig buffer ŽpH 7.3. for approximately 24 h at 48C. Cell pellets were postfixed in 1% Žwrv. osmium tetroxide, dehydrated in an ascending series of ethanols, embedded in plastic, sectioned and stained with uranyl acetate and lead citrate. Sections were examined using a Philips 301 transmission electron microscope operating at 60 kV.
3. Results T. gondii tissue cysts were observed in RH infected decoquinate-treated cultures on all examination periods. The number of tissue cyst present could not be determined. We estimate that it was between 5 and 15% of the infected cells. No tissue cysts were observed in TS-4 strain infected decoquinate-treated cultures.
Fig. 2. Transmission electron micrographs of tissue cysts of the RH strain of T. gondii in a human foreskin fibroblast cells. Bar s1.0 m m. Note the tissue cyst wall Žarrows., the identifiable bradyzoite ŽB., the tachyzoite-like stage ŽT. and the meront ŽM. that is dividing by endodyogeny. Arrowhead points to internal membrane of a developing zoite.
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Fig. 3. Transmission electron micrographs of tissue cysts of the RH strain of T. gondii in a human foreskin fibroblast cells. Bar s1.0 m m. Portion of tissue cyst from Fig. 1. Note the tissue cyst wall Žarrow. and the degenerated ŽD. organism. A portion of the bradyzoite Žarrowhead. is also present.
The host cell mitochondria were often present adjacent to the parasitophorous vacuole of developing tissue cysts and the tissue cyst wall of tissue cysts. The tissue cysts contained a tissue cyst wall that enclosed developing stages that usually resembled tachyzoites ŽFigs. 1 and 2.. Tachyzoite-like stages contained rhoptries that had a spongy appearance, few amylopectin granules, and a centrally located nucleus. Division of the tachyzoite-like stages by endodyogeny was observed frequently in the tissue cysts ŽFig. 2.. Some of the tachyzoite-like stages contained more amylopectin granules than are normal for tachyzoites indicating they were transitional stages. Stages that were structurally consistent with bradyzoites were present in most tissue cysts ŽFigs. 1–3.. The bradyzoites contained electron dense rhoptries, many amylopectin granules, and a posteriorly located nucleus. There were usually fewer bradyzoites in tissue cysts than the tachyzoite-like stages. Degenerative zoites were also often present in the tissue cysts ŽFig. 3..
4. Discussion We have not observed tissue cysts in cultures infected with our lines of the RH or TS-4 strain previously ŽLindsay et al., 1993a,c. using TEM. It has previously been reported that other mitochondrial inhibitors Žoligomycin, antimycin A, atovaquone, rotenone, myxothiazol, carbonyl cyanide m-chlorophenyl-hydrazone. will also induce tachyzoiterbradyzoite stage conversion ŽBohne et al., 1994; Tomavo and Boothroyd, 1995. of T. gondii strains that spontaneously produce tissue cysts in vitro. Results of these studies were based on immunostaining using bradyzoitespecific antibodies or by western blotting. Our study conclusively demonstrated tissue cyst production by the normally tissue cyst-less RH strain using TEM examination of decoquinate-treated cultures. Studies using host cells with nonfunctional mitochondria indicate that the mitochondrial inhibitors are effecting the T. gondii mitochondrion and not the host cell mitochondria ŽBohne et al., 1994; Tomavo and Boothroyd, 1995.. Spontaneous tissue cyst formation by the RH strain in vitro has been reported by Hogan et al. Ž1960. in retinoblastoma cells using TEM. Nagineni et al. Ž1996. reported
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D.S. Lindsay et al.r Veterinary Parasitology 77 (1998) 75–81
cyst-like bodies in human retinal pigment epithelial cells infected with the RH strain. However, they examined only material stained with a Wrights–Giemsa type stain and the absolute identification of tissue cysts is not possible using this technique ŽDubey et al., 1998.. We have not observed spontaneous tissue cyst formation for our cloned line of the RH strain. Soete ˆ et al. Ž1994. used three methods of induction to obtain RH strain tissue cysts in Vero and human foreskin fibroblast cells. Tissue cysts were seen in pH 8-treated cells, cells exposed to a temperature shift of 37–428C or sodium arsenate-treated cells. Soete ˆ et al. Ž1994. used immunofluorescence, TEM and western blotting to demonstrate tissue cysts. DeChamps et al. Ž1997. used pH 8 treatment and TEM to demonstrate RH tissue cysts in human fibroblast cell cultures. Interestingly, the organisms in their TEM photograph show only tachyzoite-like stages in the confines of a tissue cyst wall. Our observation that both tachyzoite-like stages and bradyzoites could be observed in the same tissue cyst is similar to observations by Bohne et al. Ž1993. and Soete ˆ et al. Ž1993. for the NTE and Prugniaud strains of T. gondii, respectively. These researchers used stage-specific antibodies to demonstrate mixed populations of tachyzoites and bradyzoites in host cells. These observations reflect the asynchronous development of T. gondii. Our inability to demonstrate tissue cysts of the TS-4 mutant strain of T. gondii further documents the tissue cyst-less nature of this strain. Comparative studies using the TS-4 strain with other tissue cyst forming strains should provide valuable information on the process of tissue cystogenesis in T. gondii.
References Bohne, W., Heeseman, J., Gross, U., 1993. Coexistence of heterogeneous populations of Toxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody. Parasitol. Res. 79, 485–487. Bohne, W., Heeseman, J., Gross, U., 1994. Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion. Infect. Immun. 62, 1761–1767. DeChamps, C., Imbert-Bernard, C., Belmeguenal, A., Ricard, J., Pelloux, H., Brambilla, E., Ambroise-Thomas, P., 1997. Toxoplasma gondii: In vivo and in vitro cystogenesis of the virulent RH strain. J. Parasitol. 83, 152–155. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structure of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites, and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267–299. Elwell, M.R., Frenkel, J.K., 1984a. Acute toxoplasmosis in hamsters and mice: measurement of pathogenicity by fever and weight loss. Am. J. Vet. Res. 45, 2663–2667. Elwell, M.R., Frenkel, J.K., 1984b. Immunity to toxoplasmosis in hamsters. Am. J. Vet. Res. 45, 2668–2674. Frenkel, J.K., Escajadillo, A., 1987. Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am. J. Trop. Med. Hyg. 36, 517–522. Fry, M., Williams, R.B., 1984. Effects of decoquinate and clopidol on electron transport in mitochondria of Eimeria tenella ŽApicomplexa: Coccidia.. Biochem. Pharmacol. 33, 229–240. Hogan, M.J., Yoneda, C., Feeney, L., Zweigart, P., Lewis, A., 1960. Morphology and culture of Toxoplasma. Arch. Ophthalmol. 64, 655–667. Lindsay, D.S., Toivio-Kinnucan, M.A., Blagburn, B.L., 1993a. Ultrastructural determination of cystogenesis of various Toxoplasma gondii isolates in cell cultures. J. Parasitol. 79, 289–292.
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Lindsay, D.S., Blagburn, B.L., Dubey, J.P., 1993b. Safety and results of challenge of weaned pigs given a temperature-sensitive strain of Toxoplasma gondii. J. Parasitol. 79, 71–76. Lindsay, D.S., Mitchler, R.R., Toivio-Kinnucan, M.A., Upton, S.J., Dubey, J.P., Blagburn, J.P., 1993c. Association of host cell mitochondria with developing Toxoplasma gondii issue cysts. Am. J. Vet. Res. 54, 1663–1667. Lindsay, D.S., Rippey, N.S., Toivio-Kinnucan, M.A., Blagburn, B.L., 1995. Ultrastructural effects of diclazuril against Toxoplasma gondii and investigation of a diclazuril resistant mutant. J. Parasitol. 81, 459–466. Lindsay, D.S., Butler, J.M., Blagburn, B.L., 1997. Efficacy of decoquinate against Neospora caninum tachyzoites in cell cultures. Vet. Parasitol. 68, 35–40. Luft, B.J., Remington, J.S., 1988. Toxoplasmic encephalitis. J. Infect. Dis. 157, 1–6. Nagineni, C.N., Pardhasaradhi, K., Martins, M.C., Detrick, B., Hooks, J.J., 1996. Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells. Infect. Immun. 64, 4188–4196. Pfefferkorn, E.R., Pfefferkorn, L.C., 1976. Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants. Exp. Parasitol. 39, 365–376. Pfefferkorn, E.R., Borotz, S.E., Nothnagel, R.F., 1993. Mutants of Toxoplasma gondii resistant to atovaquone or decoquinate. J. Parasitol. 79, 559–564. Pinckney, R.D., Lindsay, D.S., Blagburn, B.L., Boosinger, T.R., McLaughlin, S.A., Dubey, J.P., 1994. Evaluation of the safety and efficacy of vaccination of nursing pigs with living tachyzoites of two strains of Toxoplasma gondii. J. Parasitol. 80, 438–448. Pinckney, R.D., Lindsay, D.S., Blagburn, B.L., 1995. Further characterization of the TS-4 temperature-sensitive mutant of Toxoplasma gondii in mice. J. Parasitol. 81, 118–121. Soete, ˆ M., Fortier, B., Camus, D., Dubremetz, J.F., 1993. Toxoplasma gondii: kinetics of bradyzoite-tachyzoite interconversion in vitro. Exp. Parasitol. 76, 259–264. Soete, ˆ M., Camus, D., Dubremetz, J.F., 1994. Experimental induction of bradyzoite-specific antigen expression and cyst formation by the RH strain of Toxoplasma gondii in vitro. Exp. Parasitol. 78, 361–370. Tomavo, S., Boothroyd, J.C., 1995. Interconnection between organellar functions development and drug resistance in the protozoan parasite, Toxoplasma gondii. Int. J. Parasitol. 25, 1293–1299. Waldeland, H., Frenkel, J.K., 1983. Live and killed vaccines against toxoplasmosis in mice. J. Parasitol. 69, 60–65. Wang, C.C., 1975. Studies of the mitochondria from Eimeria tenella and inhibition of electron transport by quinolone coccidiostats. Biochem. Biophys. Acta 396, 210–219. Wang, C.C., 1976. Inhibition of the respiration of Eimeria tenella by quinolone coccidiostats. Biochem. Pharmacol. 25, 343–349.
Decoquinate induces tissue cyst formation by the RH strain of Toxoplasma gondii David S. Lindsay ) , Maria A. Toivio-Kinnucan, Byron L. Blagburn Department of Pathobiology, College of Veterinary Medicine, Auburn UniÕersity, Auburn, AL 36849-5519, USA Received 11 September 1997; accepted 10 December 1997
Abstract Decoquinate is an anticoccidial agent that inhibits respiration in the parasites mitochondrion. We examined human foreskin fibroblast cell cultures infected with the normally tissue cyst-less RH strain of Toxoplasma gondii and treated with decoquinate for evidence of tissue cyst induction and formation. Transmission electron microscopy observations demonstrated tissue cysts in decoquinate-treated cultures on days 3, 4, 5, and 6 after inoculation. Tissue cysts contained a tissue cyst wall that enclosed stages that resembled tachyzoites and stages that were structurally bradyzoites. Similar treatment of human foreskin fibroblast cells infected with tachyzoites of the TS-4 temperature-sensitive mutant of the RH strain did not result in production of tissue cysts. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Decoquinate; Tissue cyst; RH strain; Toxoplasma gondii
1. Introduction Toxoplasmic encephalitis is a life threatening condition observed in many AIDS patients ŽLuft and Remington, 1988.. The development of toxoplasmic encephalitis is associated with reactivation Žrupture. of the latent tissue cyst with release of bradyzoites and subsequent destruction of brain tissue by actively dividing tachyzoites ŽFrenkel and Escajadillo, 1987.. Reactivation of bradyzoites in tissue cysts is also the cause of retinochoriditis observed in congenitally infected humans. Because of this importance much attention has been given to the production of Toxoplasma gondii tissue cysts in )
Corresponding author. DBMP, CMMID, Virginia-Maryland Regional College of Veterinary Medicine, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA. 0304-4017r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 0 1 7 Ž 9 8 . 0 0 0 9 9 - 5
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D.S. Lindsay et al.r Veterinary Parasitology 77 (1998) 75–81
cell culture systems since the late 1980’s Žsee Dubey et al., 1998.. Prior to the early 1990’s, it was believed that most strains of T. gondii did not produce tissue cysts in vitro, however it was demonstrated that this was not the case and that most strains examined would produce a small number of tissue cysts in vitro with no manipulation ŽLindsay et al., 1993a.. Several groups of workers have demonstrated that agents which induce stress will cause an increase in this background production of tissue cysts of several of these tissue cyst forming strains of T. gondii in vitro Žsee Dubey et al., 1998.. Decoquinate is a quinolone anticoccidial approved for use in the prevention of intestinal coccidiosis in goats and cattle. It inhibits coccidial respiration by interfering with electron transport in mitochondria ŽWang, 1975, 1976; Fry and Williams, 1984.. The precise site of action appears to be within the bc 1 complex where electrons are transferred from ubiquinone to cytochrome C. Decoquinate and the related antimalarialranti-Toxoplasma agent atovaquone have excellent activity against T. gondii in cell cultures ŽPfefferkorn et al., 1993. and atovaquone will cause an increase in tissue cyst production of the PLK strain of T. gondii in Vero cell cultures ŽTomavo and Boothroyd, 1995.. We have previously demonstrated that treatment of cells infected with the NC-1 strain of Neospora caninum with 0.1 m grml decoquinate for 5 min completely kills all parasites ŽLindsay et al., 1997.. In conducting comparative studies with the RH strain of T. gondii, we observed parasite survival and apparent tissue cyst formation using light microscopy. We conducted the present study to determine if decoquinate treatment did indeed cause tissue cyst production by the normal tissue cyst-less RH strain and extended the study to the TS-4 mutant strain ŽPfefferkorn and Pfefferkorn, 1976. of the RH strain which does not persist in the tissues of animals and is considered tissue cyst-less ŽWaldeland and Frenkel, 1983; Elwell and Frenkel, 1984a,b; Lindsay et al., 1993b; Pinckney et al., 1994, 1995..
2. Materials and methods 2.1. Maintenance of tachyzoites and cell cultures Tachyzoites of our cloned line ŽLindsay et al., 1995. of the RH strain of T. gondii and the TS-4 temperature-sensitive mutant of the RH strain were grown in monolayers of human foreskin fibroblast cells ŽHS68 cells, ATTC CRL 1635, Rockville, MD. grown in 25 cm2 plastic cell culture flasks. The growth media consisted of 10% Žvrv. fetal bovine serum in RPMI 1640 medium supplemented with 100 unitsrml penicillin G, 100 m grml streptomycin sulfate, 1 mM sodium pyruvate, and 5 = 10y2 mM 2-mercaptoethanol. The HS68 cells were maintained in and decoquinate studies were conducted in medium identical to the growth medium but which had the fetal bovine serum concentration lowered to 2% Žvrv.. Cell cultures were grown at 378C in a humidified atmosphere consisting of 5% CO 2 –95% air. Cells harboring the TS-4 strain were infected and maintained at 32.58C while those harboring the RH strain were infected and maintained at 378C.
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2.2. Tissue cyst induction by decoquinate Decoquinate Žlot 7916Z4, Rhone-Poulenc Animal Nutrition, N.A., Atlanta, GA. was dissolved in 90% methanolr0.1 N sodium hydroxide solution and diluted in cell culture maintenance medium prior to use in these studies. The concentration of this vehicle used Ž0.009% methanolr0.00001 N sodium hydroxide. was not toxic for host HS68 cells ŽLindsay et al., 1997.. Monolayers of HS68 cells were inoculated with 2 = 10 6 tachyzoites of the RH or TS-4 strains. All cultures were now kept at 378C. Two hours postinoculation ŽPI. the medium was removed and the intracellular tachyzoites were treated with 0.1 m grml Ž0.24 m m. decoquinate for 5 min. The decoquinate containing medium was removed and the monolayers washed 5 times in Hanks’ balanced salt solution. Decoquinate free
Fig. 1. Transmission electron micrograph of a tissue cyst of the RH strain of T. gondii in a human foreskin fibroblast cell. Bar s1.0 m m. Note the tissue cyst wall Žarrows., the identifiable bradyzoite ŽB. and the tachyzoite-like stage ŽT..
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maintenance medium was added and the cell cultures were incubated at 378C until examined. Monolayers were processed for transmission electron microscopy ŽTEM. on days 3, 4, 5, and 6 PI for the RH strain and 5 and 11 days PI for the TS-4 strain. The monolayers were scraped from the plastic growth surface using a cell scraper and the cells pelleted by centrifugation a 1000 g for 5 min. The cell pellet was fixed in 3% Žvrv. glutaraldehyde in Millonig buffer ŽpH 7.3. for approximately 24 h at 48C. Cell pellets were postfixed in 1% Žwrv. osmium tetroxide, dehydrated in an ascending series of ethanols, embedded in plastic, sectioned and stained with uranyl acetate and lead citrate. Sections were examined using a Philips 301 transmission electron microscope operating at 60 kV.
3. Results T. gondii tissue cysts were observed in RH infected decoquinate-treated cultures on all examination periods. The number of tissue cyst present could not be determined. We estimate that it was between 5 and 15% of the infected cells. No tissue cysts were observed in TS-4 strain infected decoquinate-treated cultures.
Fig. 2. Transmission electron micrographs of tissue cysts of the RH strain of T. gondii in a human foreskin fibroblast cells. Bar s1.0 m m. Note the tissue cyst wall Žarrows., the identifiable bradyzoite ŽB., the tachyzoite-like stage ŽT. and the meront ŽM. that is dividing by endodyogeny. Arrowhead points to internal membrane of a developing zoite.
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Fig. 3. Transmission electron micrographs of tissue cysts of the RH strain of T. gondii in a human foreskin fibroblast cells. Bar s1.0 m m. Portion of tissue cyst from Fig. 1. Note the tissue cyst wall Žarrow. and the degenerated ŽD. organism. A portion of the bradyzoite Žarrowhead. is also present.
The host cell mitochondria were often present adjacent to the parasitophorous vacuole of developing tissue cysts and the tissue cyst wall of tissue cysts. The tissue cysts contained a tissue cyst wall that enclosed developing stages that usually resembled tachyzoites ŽFigs. 1 and 2.. Tachyzoite-like stages contained rhoptries that had a spongy appearance, few amylopectin granules, and a centrally located nucleus. Division of the tachyzoite-like stages by endodyogeny was observed frequently in the tissue cysts ŽFig. 2.. Some of the tachyzoite-like stages contained more amylopectin granules than are normal for tachyzoites indicating they were transitional stages. Stages that were structurally consistent with bradyzoites were present in most tissue cysts ŽFigs. 1–3.. The bradyzoites contained electron dense rhoptries, many amylopectin granules, and a posteriorly located nucleus. There were usually fewer bradyzoites in tissue cysts than the tachyzoite-like stages. Degenerative zoites were also often present in the tissue cysts ŽFig. 3..
4. Discussion We have not observed tissue cysts in cultures infected with our lines of the RH or TS-4 strain previously ŽLindsay et al., 1993a,c. using TEM. It has previously been reported that other mitochondrial inhibitors Žoligomycin, antimycin A, atovaquone, rotenone, myxothiazol, carbonyl cyanide m-chlorophenyl-hydrazone. will also induce tachyzoiterbradyzoite stage conversion ŽBohne et al., 1994; Tomavo and Boothroyd, 1995. of T. gondii strains that spontaneously produce tissue cysts in vitro. Results of these studies were based on immunostaining using bradyzoitespecific antibodies or by western blotting. Our study conclusively demonstrated tissue cyst production by the normally tissue cyst-less RH strain using TEM examination of decoquinate-treated cultures. Studies using host cells with nonfunctional mitochondria indicate that the mitochondrial inhibitors are effecting the T. gondii mitochondrion and not the host cell mitochondria ŽBohne et al., 1994; Tomavo and Boothroyd, 1995.. Spontaneous tissue cyst formation by the RH strain in vitro has been reported by Hogan et al. Ž1960. in retinoblastoma cells using TEM. Nagineni et al. Ž1996. reported
80
D.S. Lindsay et al.r Veterinary Parasitology 77 (1998) 75–81
cyst-like bodies in human retinal pigment epithelial cells infected with the RH strain. However, they examined only material stained with a Wrights–Giemsa type stain and the absolute identification of tissue cysts is not possible using this technique ŽDubey et al., 1998.. We have not observed spontaneous tissue cyst formation for our cloned line of the RH strain. Soete ˆ et al. Ž1994. used three methods of induction to obtain RH strain tissue cysts in Vero and human foreskin fibroblast cells. Tissue cysts were seen in pH 8-treated cells, cells exposed to a temperature shift of 37–428C or sodium arsenate-treated cells. Soete ˆ et al. Ž1994. used immunofluorescence, TEM and western blotting to demonstrate tissue cysts. DeChamps et al. Ž1997. used pH 8 treatment and TEM to demonstrate RH tissue cysts in human fibroblast cell cultures. Interestingly, the organisms in their TEM photograph show only tachyzoite-like stages in the confines of a tissue cyst wall. Our observation that both tachyzoite-like stages and bradyzoites could be observed in the same tissue cyst is similar to observations by Bohne et al. Ž1993. and Soete ˆ et al. Ž1993. for the NTE and Prugniaud strains of T. gondii, respectively. These researchers used stage-specific antibodies to demonstrate mixed populations of tachyzoites and bradyzoites in host cells. These observations reflect the asynchronous development of T. gondii. Our inability to demonstrate tissue cysts of the TS-4 mutant strain of T. gondii further documents the tissue cyst-less nature of this strain. Comparative studies using the TS-4 strain with other tissue cyst forming strains should provide valuable information on the process of tissue cystogenesis in T. gondii.
References Bohne, W., Heeseman, J., Gross, U., 1993. Coexistence of heterogeneous populations of Toxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody. Parasitol. Res. 79, 485–487. Bohne, W., Heeseman, J., Gross, U., 1994. Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion. Infect. Immun. 62, 1761–1767. DeChamps, C., Imbert-Bernard, C., Belmeguenal, A., Ricard, J., Pelloux, H., Brambilla, E., Ambroise-Thomas, P., 1997. Toxoplasma gondii: In vivo and in vitro cystogenesis of the virulent RH strain. J. Parasitol. 83, 152–155. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structure of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites, and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267–299. Elwell, M.R., Frenkel, J.K., 1984a. Acute toxoplasmosis in hamsters and mice: measurement of pathogenicity by fever and weight loss. Am. J. Vet. Res. 45, 2663–2667. Elwell, M.R., Frenkel, J.K., 1984b. Immunity to toxoplasmosis in hamsters. Am. J. Vet. Res. 45, 2668–2674. Frenkel, J.K., Escajadillo, A., 1987. Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am. J. Trop. Med. Hyg. 36, 517–522. Fry, M., Williams, R.B., 1984. Effects of decoquinate and clopidol on electron transport in mitochondria of Eimeria tenella ŽApicomplexa: Coccidia.. Biochem. Pharmacol. 33, 229–240. Hogan, M.J., Yoneda, C., Feeney, L., Zweigart, P., Lewis, A., 1960. Morphology and culture of Toxoplasma. Arch. Ophthalmol. 64, 655–667. Lindsay, D.S., Toivio-Kinnucan, M.A., Blagburn, B.L., 1993a. Ultrastructural determination of cystogenesis of various Toxoplasma gondii isolates in cell cultures. J. Parasitol. 79, 289–292.
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