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Cystoisospora belli: In vitro multiplication in mammalian cells
Experimental Parasitology 114 (2006) 189–192 www.elsevier.com/locate/yexpr
Cystoisospora belli: In vitro multiplication in mammalian cells M.B. Oliveira-Silva a,¤, E. Lages-Silva a, D.V. Resende a, A. Prata a, L.E. Ramirez a, J.K. Frenkel b a
Departamento Ciências Biológicas—Disciplina de Parasitologia, Universidade Federal do Triângulo Mineiro, Rua Frei Paulino, 30, 38025-180 Uberaba, MG, Brazil b University of New México, Albuquerque, NM; 1252 Vallecita Drive, Santa Fé, NM 87501, USA Received 18 October 2005; received in revised form 8 March 2006; accepted 9 March 2006 Available online 3 May 2006
Abstract Intracellular development of Cystoisospora belli was demonstrated in 4 diVerent mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12 h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Cystoisospora belli; Cell culture; In vitro multiplication; Isospora belli
1. Introduction The genus Cystoisospora was proposed because of the presence of unizoic tissue cysts in lymphoid tissues in rodents which function as intermediate hosts of Isospora felis and Isospora rivolta of cats (Frenkel, 1977). After a review of molecular aYnities of many Isospora species from birds and mammals, Barta et al. (2005) assign all tetrasporozoic, diplosporocystic oocysts from mammals without Stieda bodies in their sporocysts, to the genus Cystoisospora (Sarcocystidade), and all such oocysts from birds with Stieda bodies in their sporocysts to the genus Isospora. Cystoisospora belli is an obligate intracellular protozoan, believed to be homoxenous, which is responsible for human isosporiasis, a typically cosmopolitan infection, which is more frequent in tropical and subtropical regions. C. belli *
Corresponding author. Fax: +55 34 3318 5279. E-mail addresses: [email protected] (M.B. Oliveira-Silva), frenkeljk @earthlink.net (J.K. Frenkel). 0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.03.004
infection is reasonably attributed to the ingestion of the sporulated oocysts in water or contaminated food. All the endogenous reproduction of the parasite occurs in the epithelial cells of the small intestine (Lindsay et al., 1997). In several immunocompromised patients, a unizoic cyst has been observed in the lymph nodes, the spleen, the liver, and the lamina propria of the small intestine (Frenkel et al., 2003a,b; Michiels et al., 1994; Restrepo et al., 1987). Isosporiasis is characterized by diarrhea, steatorrhea, accompanied by abdominal pains, fever and loss of weight which may lead to dehydration and cachexia. The symptoms are more severe in children with immunity disorders, principally the acquired immuno-deWciency syndrome (AIDS). The rapid dissemination of the HIV virus gave rise to a considerable incidence of this intestinal pathogen, which was considered until the seventies (pre AIDS) to be a parasite of low frequency. (Lindsay et al., 1997). Although suppressed by sulfamethoxazole–trimetropim therapy, the infection is diYcult to eradicate. Recurrences are common and the chronic nature of the illness
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contributes to morbidity and mortality among these patients. (Frenkel et al., 2003a,b). Several Isospora species sensu latu have been studied in cell cultures, with evidence of penetration of the sporozoites into host cells and formation of meronts by endodyogeny (Fayer, 1972; Fayer and Mahrt, 1972; Fayer and Thompson, 1974; Guitiérrez and Arcay, 1987; Lindsay and Blagburn, 1987; Lindsay and Current, 1984), however, there were no data related to the development of C. belli in culture cells. In this study the ability of C. belli sporozoites to penetrate and multiply as merozoites in diVerent types of mammalian cells was evaluated.
ings in PBS at 1500 g for 10 min. After puriWcation, the oocysts were cleaned with 1% sodium hypochlorite solution during 10 min at 4 °C, washed in saline at 1500 g three times and counted a Neubauer chamber. Subsequently, the oocysts were diluted in a phosphate-buVer saline (PBS pH 7.2), containing sodium taurocholate 1.5%, and trypsin 0.5% and incubated at 37 °C for 30 min for excystation (Gold et al., 2001). The sporozoites obtained were diluted in Eagle’s minimal essential medium (MEM) supplemented 10% fetal bovine serum (FBS), to neutralize the trypsin, washed for 10 min at 1500 g and counted in the Neubauer chamber. The viability of the sporozoites was evaluated by means of their circular and sporadic movement.
2. Materials and methods 2.2. Cell lines 2.1. Parasites Cystoisospora belli oocysts were isolated from samples of feces of HIV/AIDS patients, which were attending the School Hospital of Universidade Federal doTriângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil. The oocysts were sporulated in 2.5% potassium dichromate (K2Cr2O7) at room temperature and puriWed according to the technique of Ortega-Mora et al., 1992; with modiWcations by Silva et al., 2006: the K2Cr2O7 was removed with a phosphate-buVer saline (PBS pH 7.2) containing Tween 20 to 2% (PBS–T20) in 1500 g for 10 min at 4 °C; the lipid portion was removed by means of washing in PBS–T20/ethyilether (2:1) at 1500 g for 10 min; the sediment, which contained oocysts, was added above a non-continuous gradient puriWcation of the 1.05 g/ml and 1.15 g/ml density sucrose and centrifuged at 1500g for 20 min at 4 °C; the oocyst layer bas identiWed between the 2 sucrose layer and was eliminated with 3 wash-
VERO cells (African green monkey kidney), MDBK (Madin-Darby Bovine Kidney), HCT-8 (Human Ileocecal Adenocarcinoma), and A-549 (Human Carcinoma Lung) were obtained from the ATCC (American Type Culture Collection) and maintained by successive passages in MEM (Gibco-BRL) culture medium, supplemented with 2 mM glutamine, penicillin(10,000 UI/ml)/streptomycin (10,000 m/ml) solution (Sigma–Aldrich), 0.1 mM non-essential amino acids, amphotericin B (400 l), and fetal bovine serum (FBS) at 10%. The incubation system used was an open one, with maintenance of the cells in an atmosphere containing 5% of CO2 at 37 °C, in 12.5 cm2 culture Xasks. 2.3. “In vitro” infection The cell lines used in the experiment were treated with a solution of trypsin/EDTA 0,05%, neutralized with
Fig. 1. Fresh culture of VERO and HCT-8 cells infected by C. belli: (A) VERO cells with sporozoites in vacuoles 12 h after infection and (B) immature meront 48 h after infection (full arrow) and free merozoite (empty arrow). (C) HCT-8 cells demonstrating division of merozoites in vacuole (full arrow) and (D) immature meronts (full arrow) and escape of merozoites (empty arrow) 72 h after infection. 400£.
M.B. Oliveira-Silva et al. / Experimental Parasitology 114 (2006) 189–192
MEM medium with 10% FBS for 10 min at 1500 g and 3 £ 104 cells/well were transferred to chambers (Chamber slides -LAB-TEK Brand products). After the formation of the cell monolayer, 1.5 £ 105 sporozoites were added to the cell cultures and these were maintained at 37 °C in a CO2 incubator for 12 h. After this time, oocysts, which had not excysted, and extracellular sporozoites, were removed from the cultures by means of three washings, and 1 ml of MEM/FBS was added to each well. The kinetics of the infection was evaluated every 24 h, over 4 days. The monolayers were Wxed with an alcohol (100%)-acetic-acid solution (v/v) during 30 min. The slides were stained with Giemsa and examined under a light microscope at 400£ and 1000£ magniWcation. 3. Results Cystoisospora belli sporozoites invaded all the cell types between 4 and 12 h after exposure (Figs. 1A and 2A), positioning themselves near the nucleus of the host cell and in the majority of the cells in the interior of the vacuoles.
191
Extracellular motile parasites (sporozoites) were observed in the culture medium, immediately after inoculation, and at each examination of the cell cultures under an inverted microscope for up to 24 h. Multiplication by endodyogeny was observed 24 h after exposure, producing paired merozoites (Figs. 2A, B, and E). Free merozoites were observed in the culture medium (Figs. 1B and D) and penetrating other cells. VERO cells showed a better development of merogony with multiple parasites per cell and a greater production of merozoites all in large parasitophorous vacuoles (Figs. 1A, 2C, and D). This was also observed to a lesser degree in the HCT-8 cells (Figs. 1C and D). On the other hand, in MDBK and HCT8 cells, the vacuole was less evident and some non-motile merozoites were observed in the cytoplasm. Motile merozoites in the interior of the parasitophorous vacuoles were observed in all the cell types, which when Wxed and stained with Giemsa were 0.924 m (§0.189) in length and 0.319 m (§ 0.053) in width. After several cycles of penetration and multiplication in the cells, the mobility of zoites diminished, and they died.
Fig. 2. DiVerent mammalian cells lines infected with C. belli in Giemsa stain. (A) MDBK cells 24 h after infection, (arrow) meronts with 2 merozoites; (B) A-549 cells 24 h after infection, (arrow) meronts with 2 merozoites; VERO cells infection (C–F) 12 h after infection (C) with large vacuole (arrow), 24 h (D) multiple infection with large vacuoles (arrow), and merozoites under division by endodyogeny (E) (arrow); 48 h meronts containing 2 and 4 merozoites (F). Bar D 1 .
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During the 4 days of this experiment, there was no formation of the sexual stages (gamonts and oocysts) in any of the cell types used. 4. Discussion It is believed that C. belli is a parasite speciWc to man, since no other hosts are now to be susceptible to it. Its sporozoites invade epithelial cells of the human small intestine and although this speciWcity occurs in vivo, the invasion in vitro of four other cell types observed in this study, suggests that there are adhesion receptors involved in the penetration of diVerent cell lines and hosts. Although C. belli sporozoites and merozoites had penetrated in all the cell lines studies, their ability to invade and multiply varied; it was greatest with VERO cells, where multiple infections by sporozoites and greater numbers of merozoites were observed. The size of the inoculum has direct eVects on the quantity of intracellular parasites that develop (Fayer and Hammond, 1967 and Hermosilla et al., 2002). These authors,using Eimeria bovis observed multiple infections in embryonic bovine cell cultures when the inoculum was higher than 2 £ 107 sporozoites/ml. By using only 3 £ 104 C. belli sporozoites we found multiple infections in VERO and HCT-8 cells. Fayer and Mahrt, 1972; made similar observations when they inoculated Isospora canis sporozoites in canine and bovine cells. In this study, the size of the parasitophorous vacuole was diVerent in the various types of cells. In VERO cells a large parasitophorous vacuole developed around the parasite after invasion, while in the other cells, the vacuoles were small and indistinct. VERO cells may be useful in the study of the parasitophorous vacuole and in tests of the sensitivity or resistance to anticoccidial drugs, particularly when lines of human intestinal cells are not available. During our 4 days observation period, we did not see gametocytes, which, could develop in the diVerent cell lines after a long observation times. Acknowledgment The authors thank FAPEMIG for Wnancial support. References Barta, J.R., Schrenzel, M.D., Carreno, R., Rideout, B.A., 2005. The genus Atoxoplasma (Garnham 1950) as a junior objective synonym of the genus Isospora (Schneider 1881) species infecting birds and resurrection
of Cystoisospora (Frenkel 1977) as the correct genus for Isospora species infecting mammals. The Journal of Patrasitology 9, 726–727. Fayer, R., Hammond, D.M., 1967. Development of Wrst-generation Schizonts of Eimeria bovis in cultured bovine cells. Journal of Protozoology 14, 764–772. Fayer, R., 1972. Cultivation of feline Isospora rivoltain mammalian cells. The Journal of Parasitology 58, 1207–1208. Fayer, R., Mahrt, J.L., 1972. Development of Isospora canis (Protozoa; Sporozoa) in cell culture. Zeitschrift fur Parasitenkunde 38, 313–318. Fayer, R., Thompson, D.E., 1974. Isospora felis: development in cultured cells with some cytological observations. The Journal of Parasitology 60, 160–168. Frenkel, J.K., 1977. Besnoitia wallacei of cats and rodents: with a reclassiWcation of other cyst-forming isosporid coccidia. The Journal of Parasitology 63, 611–628. Frenkel, J.K., Silva, M.B.O., Saldanha, J., Silva, M.L., Filho, V.D.C., Barata, C.H., Silva, E.L., Ramirez, L.E., Prata, A., 2003a. Isospora belli infection: observation of unicellular cysts in mesenteric lymphoid tissues of a Brazilian patient with aids and animal inoculation. The Journal of Eukaryotic Microbiology 50, 682–684. Frenkel, J.K., Silva, M.B.O., Saldanha, J., Silva, M.L, Filho, V.D.C., Barata, C.H, Silva, E.L., Ramirez, L.E., Prata, A., 2003b. Presença extra-intestinal de Cistos Unizóicos de Isospora belli em Paciente com SIDA. Relato de Caso. Revista da Sociedade Brasileira de Medicina Tropical 36, 409–412. Gold, D., Stein, B., Tzipori, S., 2001. The utilization of taurocholate in excystation of Cryptosporidium parvum and infection of tissue culture. The Journal of Parasitology 87, 997–1000. Guitiérrez, F., Arcay, L., 1987. Cultivo de Cystoisospora felis (Isospora felis (Wasielewski, 1904, Wenyo), 1923) en la Membrana Corioalantoidea de Embrion de Pollo. Acta Cient´Wca Venezolana 38, 474–483. Hermosilla, C., Barbisch, B., Heise, A., Kowalik, S., Zahner, H., 2002. Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointesinal, Madin-Darby bovine kidney (MDBK) and African green monkey (VERO) cells. Parasitology Research 88, 301–307. Lindsay, D.S., Blagburn, B.L., 1987. Development of Isospora suis from pigs in primary porcine and bovine cell cultures. Veterinary Parasitology 24, 301–304. Lindsay, D.S., Current, W.L., 1984. Complete development of Isospora suis of swine in chicken embryos. Journal of Protozoology 31, 152–155. Lindsay, D.S., Dubey, J.P., Blagburn, B.L., 1997. Biology of Isospora spp from humans, nonhuman primates, and domestic animals. Clinical Microbiology Review 10, 19–34. Michiels, J.F., Hofman, P., Bernard, E., Saint Paul, M.C., Boissy, C., Mondain, V., LeFichoux, Y., Loubiere, R., 1994. Intestinal and extraintestinal Isospora belli infection in an aids patient. Pathology Research Practices 190, 1089–1093. Ortega-Mora, L.M., Troncoso, J.M., Rojo-Vasquez, F.A., Gómez-Bautista, M., 1992. Evaluation of an improved method to purify Cryptosporidium parvum oocysts. Research and Reviews in Parasitology 52, 127–130. Restrepo, C., Macher, A.M., Radany, E.H., 1987. Disseminated extraintestinal isosporiasis in a patient with acquired immune-deWciency syndrome. American Journal of Clinical Pathology 87, 536–542. Silva, M.B.O., Lima, J.D., Vieira, L.S., Vitor, R.W.A., 2006. Humoral response and IgG avidity in goat kids experimentally infected with Cryptosporidium parvum. Revista Brasileira de Parasitologia Veterinária, in press.
Cystoisospora belli: In vitro multiplication in mammalian cells M.B. Oliveira-Silva a,¤, E. Lages-Silva a, D.V. Resende a, A. Prata a, L.E. Ramirez a, J.K. Frenkel b a
Departamento Ciências Biológicas—Disciplina de Parasitologia, Universidade Federal do Triângulo Mineiro, Rua Frei Paulino, 30, 38025-180 Uberaba, MG, Brazil b University of New México, Albuquerque, NM; 1252 Vallecita Drive, Santa Fé, NM 87501, USA Received 18 October 2005; received in revised form 8 March 2006; accepted 9 March 2006 Available online 3 May 2006
Abstract Intracellular development of Cystoisospora belli was demonstrated in 4 diVerent mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12 h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Cystoisospora belli; Cell culture; In vitro multiplication; Isospora belli
1. Introduction The genus Cystoisospora was proposed because of the presence of unizoic tissue cysts in lymphoid tissues in rodents which function as intermediate hosts of Isospora felis and Isospora rivolta of cats (Frenkel, 1977). After a review of molecular aYnities of many Isospora species from birds and mammals, Barta et al. (2005) assign all tetrasporozoic, diplosporocystic oocysts from mammals without Stieda bodies in their sporocysts, to the genus Cystoisospora (Sarcocystidade), and all such oocysts from birds with Stieda bodies in their sporocysts to the genus Isospora. Cystoisospora belli is an obligate intracellular protozoan, believed to be homoxenous, which is responsible for human isosporiasis, a typically cosmopolitan infection, which is more frequent in tropical and subtropical regions. C. belli *
Corresponding author. Fax: +55 34 3318 5279. E-mail addresses: [email protected] (M.B. Oliveira-Silva), frenkeljk @earthlink.net (J.K. Frenkel). 0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.03.004
infection is reasonably attributed to the ingestion of the sporulated oocysts in water or contaminated food. All the endogenous reproduction of the parasite occurs in the epithelial cells of the small intestine (Lindsay et al., 1997). In several immunocompromised patients, a unizoic cyst has been observed in the lymph nodes, the spleen, the liver, and the lamina propria of the small intestine (Frenkel et al., 2003a,b; Michiels et al., 1994; Restrepo et al., 1987). Isosporiasis is characterized by diarrhea, steatorrhea, accompanied by abdominal pains, fever and loss of weight which may lead to dehydration and cachexia. The symptoms are more severe in children with immunity disorders, principally the acquired immuno-deWciency syndrome (AIDS). The rapid dissemination of the HIV virus gave rise to a considerable incidence of this intestinal pathogen, which was considered until the seventies (pre AIDS) to be a parasite of low frequency. (Lindsay et al., 1997). Although suppressed by sulfamethoxazole–trimetropim therapy, the infection is diYcult to eradicate. Recurrences are common and the chronic nature of the illness
190
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contributes to morbidity and mortality among these patients. (Frenkel et al., 2003a,b). Several Isospora species sensu latu have been studied in cell cultures, with evidence of penetration of the sporozoites into host cells and formation of meronts by endodyogeny (Fayer, 1972; Fayer and Mahrt, 1972; Fayer and Thompson, 1974; Guitiérrez and Arcay, 1987; Lindsay and Blagburn, 1987; Lindsay and Current, 1984), however, there were no data related to the development of C. belli in culture cells. In this study the ability of C. belli sporozoites to penetrate and multiply as merozoites in diVerent types of mammalian cells was evaluated.
ings in PBS at 1500 g for 10 min. After puriWcation, the oocysts were cleaned with 1% sodium hypochlorite solution during 10 min at 4 °C, washed in saline at 1500 g three times and counted a Neubauer chamber. Subsequently, the oocysts were diluted in a phosphate-buVer saline (PBS pH 7.2), containing sodium taurocholate 1.5%, and trypsin 0.5% and incubated at 37 °C for 30 min for excystation (Gold et al., 2001). The sporozoites obtained were diluted in Eagle’s minimal essential medium (MEM) supplemented 10% fetal bovine serum (FBS), to neutralize the trypsin, washed for 10 min at 1500 g and counted in the Neubauer chamber. The viability of the sporozoites was evaluated by means of their circular and sporadic movement.
2. Materials and methods 2.2. Cell lines 2.1. Parasites Cystoisospora belli oocysts were isolated from samples of feces of HIV/AIDS patients, which were attending the School Hospital of Universidade Federal doTriângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil. The oocysts were sporulated in 2.5% potassium dichromate (K2Cr2O7) at room temperature and puriWed according to the technique of Ortega-Mora et al., 1992; with modiWcations by Silva et al., 2006: the K2Cr2O7 was removed with a phosphate-buVer saline (PBS pH 7.2) containing Tween 20 to 2% (PBS–T20) in 1500 g for 10 min at 4 °C; the lipid portion was removed by means of washing in PBS–T20/ethyilether (2:1) at 1500 g for 10 min; the sediment, which contained oocysts, was added above a non-continuous gradient puriWcation of the 1.05 g/ml and 1.15 g/ml density sucrose and centrifuged at 1500g for 20 min at 4 °C; the oocyst layer bas identiWed between the 2 sucrose layer and was eliminated with 3 wash-
VERO cells (African green monkey kidney), MDBK (Madin-Darby Bovine Kidney), HCT-8 (Human Ileocecal Adenocarcinoma), and A-549 (Human Carcinoma Lung) were obtained from the ATCC (American Type Culture Collection) and maintained by successive passages in MEM (Gibco-BRL) culture medium, supplemented with 2 mM glutamine, penicillin(10,000 UI/ml)/streptomycin (10,000 m/ml) solution (Sigma–Aldrich), 0.1 mM non-essential amino acids, amphotericin B (400 l), and fetal bovine serum (FBS) at 10%. The incubation system used was an open one, with maintenance of the cells in an atmosphere containing 5% of CO2 at 37 °C, in 12.5 cm2 culture Xasks. 2.3. “In vitro” infection The cell lines used in the experiment were treated with a solution of trypsin/EDTA 0,05%, neutralized with
Fig. 1. Fresh culture of VERO and HCT-8 cells infected by C. belli: (A) VERO cells with sporozoites in vacuoles 12 h after infection and (B) immature meront 48 h after infection (full arrow) and free merozoite (empty arrow). (C) HCT-8 cells demonstrating division of merozoites in vacuole (full arrow) and (D) immature meronts (full arrow) and escape of merozoites (empty arrow) 72 h after infection. 400£.
M.B. Oliveira-Silva et al. / Experimental Parasitology 114 (2006) 189–192
MEM medium with 10% FBS for 10 min at 1500 g and 3 £ 104 cells/well were transferred to chambers (Chamber slides -LAB-TEK Brand products). After the formation of the cell monolayer, 1.5 £ 105 sporozoites were added to the cell cultures and these were maintained at 37 °C in a CO2 incubator for 12 h. After this time, oocysts, which had not excysted, and extracellular sporozoites, were removed from the cultures by means of three washings, and 1 ml of MEM/FBS was added to each well. The kinetics of the infection was evaluated every 24 h, over 4 days. The monolayers were Wxed with an alcohol (100%)-acetic-acid solution (v/v) during 30 min. The slides were stained with Giemsa and examined under a light microscope at 400£ and 1000£ magniWcation. 3. Results Cystoisospora belli sporozoites invaded all the cell types between 4 and 12 h after exposure (Figs. 1A and 2A), positioning themselves near the nucleus of the host cell and in the majority of the cells in the interior of the vacuoles.
191
Extracellular motile parasites (sporozoites) were observed in the culture medium, immediately after inoculation, and at each examination of the cell cultures under an inverted microscope for up to 24 h. Multiplication by endodyogeny was observed 24 h after exposure, producing paired merozoites (Figs. 2A, B, and E). Free merozoites were observed in the culture medium (Figs. 1B and D) and penetrating other cells. VERO cells showed a better development of merogony with multiple parasites per cell and a greater production of merozoites all in large parasitophorous vacuoles (Figs. 1A, 2C, and D). This was also observed to a lesser degree in the HCT-8 cells (Figs. 1C and D). On the other hand, in MDBK and HCT8 cells, the vacuole was less evident and some non-motile merozoites were observed in the cytoplasm. Motile merozoites in the interior of the parasitophorous vacuoles were observed in all the cell types, which when Wxed and stained with Giemsa were 0.924 m (§0.189) in length and 0.319 m (§ 0.053) in width. After several cycles of penetration and multiplication in the cells, the mobility of zoites diminished, and they died.
Fig. 2. DiVerent mammalian cells lines infected with C. belli in Giemsa stain. (A) MDBK cells 24 h after infection, (arrow) meronts with 2 merozoites; (B) A-549 cells 24 h after infection, (arrow) meronts with 2 merozoites; VERO cells infection (C–F) 12 h after infection (C) with large vacuole (arrow), 24 h (D) multiple infection with large vacuoles (arrow), and merozoites under division by endodyogeny (E) (arrow); 48 h meronts containing 2 and 4 merozoites (F). Bar D 1 .
192
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During the 4 days of this experiment, there was no formation of the sexual stages (gamonts and oocysts) in any of the cell types used. 4. Discussion It is believed that C. belli is a parasite speciWc to man, since no other hosts are now to be susceptible to it. Its sporozoites invade epithelial cells of the human small intestine and although this speciWcity occurs in vivo, the invasion in vitro of four other cell types observed in this study, suggests that there are adhesion receptors involved in the penetration of diVerent cell lines and hosts. Although C. belli sporozoites and merozoites had penetrated in all the cell lines studies, their ability to invade and multiply varied; it was greatest with VERO cells, where multiple infections by sporozoites and greater numbers of merozoites were observed. The size of the inoculum has direct eVects on the quantity of intracellular parasites that develop (Fayer and Hammond, 1967 and Hermosilla et al., 2002). These authors,using Eimeria bovis observed multiple infections in embryonic bovine cell cultures when the inoculum was higher than 2 £ 107 sporozoites/ml. By using only 3 £ 104 C. belli sporozoites we found multiple infections in VERO and HCT-8 cells. Fayer and Mahrt, 1972; made similar observations when they inoculated Isospora canis sporozoites in canine and bovine cells. In this study, the size of the parasitophorous vacuole was diVerent in the various types of cells. In VERO cells a large parasitophorous vacuole developed around the parasite after invasion, while in the other cells, the vacuoles were small and indistinct. VERO cells may be useful in the study of the parasitophorous vacuole and in tests of the sensitivity or resistance to anticoccidial drugs, particularly when lines of human intestinal cells are not available. During our 4 days observation period, we did not see gametocytes, which, could develop in the diVerent cell lines after a long observation times. Acknowledgment The authors thank FAPEMIG for Wnancial support. References Barta, J.R., Schrenzel, M.D., Carreno, R., Rideout, B.A., 2005. The genus Atoxoplasma (Garnham 1950) as a junior objective synonym of the genus Isospora (Schneider 1881) species infecting birds and resurrection
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