Experimental Parasitology 114 (2006) 189–192

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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 2006Available online 3 May 2006

Abstract

Intracellular development of Cystoisospora belli was demonstrated in 4 diVerent mammalian cell lines. Human ileocecal adenocarci-noma (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 cel-lular types between 4 and 12 h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed inVERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoiteswere observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. Nooocysts 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 thepresence of unizoic tissue cysts in lymphoid tissues inrodents which function as intermediate hosts of Isosporafelis and Isospora rivolta of cats (Frenkel, 1977). After areview of molecular aYnities of many Isospora species frombirds and mammals, Barta et al. (2005) assign all tetrasp-orozoic, diplosporocystic oocysts from mammals withoutStieda bodies in their sporocysts, to the genus Cystoisos-pora (Sarcocystidade), and all such oocysts from birds withStieda bodies in their sporocysts to the genus Isospora.

Cystoisospora belli is an obligate intracellular protozoan,believed to be homoxenous, which is responsible for humanisosporiasis, a typically cosmopolitan infection, which ismore frequent in tropical and subtropical regions. C. belli

* Corresponding author. Fax: +55 34 3318 5279.E-mail addresses: mbosilva@yahoo.com.br (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 thesporulated oocysts in water or contaminated food. All theendogenous reproduction of the parasite occurs in the epi-thelial cells of the small intestine (Lindsay et al., 1997). Inseveral immunocompromised patients, a unizoic cyst hasbeen observed in the lymph nodes, the spleen, the liver, andthe 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 weightwhich may lead to dehydration and cachexia. The symp-toms are more severe in children with immunity disorders,principally the acquired immuno-deWciency syndrome(AIDS). The rapid dissemination of the HIV virus gave riseto a considerable incidence of this intestinal pathogen,which was considered until the seventies (pre AIDS) to be aparasite of low frequency. (Lindsay et al., 1997).

Although suppressed by sulfamethoxazole–trimetropimtherapy, the infection is diYcult to eradicate. Recurrencesare common and the chronic nature of the illness

190 M.B. Oliveira-Silva et al. / Experimental Parasitology 114 (2006) 189–192

contributes to morbidity and mortality among thesepatients. (Frenkel et al., 2003a,b).

Several Isospora species sensu latu have been studied incell cultures, with evidence of penetration of the sporozoitesinto host cells and formation of meronts by endodyogeny(Fayer, 1972; Fayer and Mahrt, 1972; Fayer and Thomp-son, 1974; Guitiérrez and Arcay, 1987; Lindsay and Blag-burn, 1987; Lindsay and Current, 1984), however, therewere no data related to the development of C. belli in cul-ture cells. In this study the ability of C. belli sporozoites topenetrate and multiply as merozoites in diVerent types ofmammalian cells was evaluated.

2. Materials and methods

2.1. Parasites

Cystoisospora belli oocysts were isolated from samples offeces of HIV/AIDS patients, which were attending theSchool Hospital of Universidade Federal doTriânguloMineiro (UFTM), Uberaba, Minas Gerais, Brazil. Theoocysts were sporulated in 2.5% potassium dichromate(K2Cr2O7) at room temperature and puriWed according tothe technique of Ortega-Mora et al., 1992; with modiWcationsby Silva et al., 2006: the K2Cr2O7 was removed with a phos-phate-buVer saline (PBS pH 7.2) containing Tween 20 to 2%(PBS–T20) in 1500g for 10 min at 4 °C; the lipid portion wasremoved by means of washing in PBS–T20/ethyilether (2:1)at 1500g for 10min; the sediment, which contained oocysts,was added above a non-continuous gradient puriWcation ofthe 1.05g/ml and 1.15g/ml density sucrose and centrifuged at1500g for 20 min at 4 °C; the oocyst layer bas identiWedbetween the 2 sucrose layer and was eliminated with 3 wash-

ings in PBS at 1500 g for 10min. After puriWcation, theoocysts were cleaned with 1% sodium hypochlorite solutionduring 10 min at 4 °C, washed in saline at 1500 g three timesand counted a Neubauer chamber. Subsequently, the oocystswere diluted in a phosphate-buVer saline (PBS pH 7.2), con-taining sodium taurocholate 1.5%, and trypsin 0.5% andincubated at 37 °C for 30 min for excystation (Gold et al.,2001). The sporozoites obtained were diluted in Eagle’s mini-mal essential medium (MEM) supplemented 10% fetalbovine serum (FBS), to neutralize the trypsin, washed for10 min at 1500 g and counted in the Neubauer chamber. Theviability of the sporozoites was evaluated by means of theircircular and sporadic movement.

2.2. Cell lines

VERO cells (African green monkey kidney), MDBK(Madin-Darby Bovine Kidney), HCT-8 (Human IleocecalAdenocarcinoma), and A-549 (Human Carcinoma Lung)were obtained from the ATCC (American Type Culture Col-lection) and maintained by successive passages in MEM(Gibco-BRL) culture medium, supplemented with 2 mM glu-tamine, 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) at10%. The incubation system used was an open one, withmaintenance of the cells in an atmosphere containing 5% ofCO2 at 37 °C, in 12.5 cm2 culture Xasks.

2.3. “In vitro” infection

The cell lines used in the experiment were treated witha 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) immaturemeront 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) 189–192 191

MEM medium with 10% FBS for 10 min at 1500 gand 3 £ 104 cells/well were transferred to chambers(Chamber slides -LAB-TEK Brand products). After theformation of the cell monolayer, 1.5 £ 105 sporozoiteswere added to the cell cultures and these were main-tained at 37 °C in a CO2 incubator for 12 h. After thistime, oocysts, which had not excysted, and extracellularsporozoites, were removed from the cultures by means ofthree washings, and 1 ml of MEM/FBS was addedto each well. The kinetics of the infection was evaluatedevery 24 h, over 4 days. The monolayers were Wxedwith an alcohol (100%)-acetic-acid solution (v/v)during 30 min. The slides were stained with Giemsa andexamined under a light microscope at 400£ and 1000£magniWcation.

3. Results

Cystoisospora belli sporozoites invaded all the cell typesbetween 4 and 12 h after exposure (Figs. 1A and 2A), posi-tioning themselves near the nucleus of the host cell and inthe majority of the cells in the interior of the vacuoles.

Extracellular motile parasites (sporozoites) were observedin the culture medium, immediately after inoculation, andat each examination of the cell cultures under an invertedmicroscope for up to 24 h.

Multiplication by endodyogeny was observed 24 hafter exposure, producing paired merozoites (Figs. 2A,B, and E). Free merozoites were observed in the culturemedium (Figs. 1B and D) and penetrating other cells.VERO cells showed a better development of merogonywith multiple parasites per cell and a greater productionof merozoites all in large parasitophorous vacuoles(Figs. 1A, 2C, and D). This was also observed to a lesserdegree in the HCT-8 cells (Figs. 1C and D). On the otherhand, in MDBK and HCT8 cells, the vacuole was lessevident and some non-motile merozoites were observedin the cytoplasm. Motile merozoites in the interior of theparasitophorous vacuoles were observed in all the celltypes, which when Wxed and stained with Giemsa were0.924 �m (§0.189) in length and 0.319 �m (§ 0.053) inwidth. After several cycles of penetration and multiplica-tion in the cells, the mobility of zoites diminished, andthey 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 M.B. Oliveira-Silva et al. / Experimental Parasitology 114 (2006) 189–192

During the 4 days of this experiment, there was no for-mation of the sexual stages (gamonts and oocysts) in any ofthe 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 spo-rozoites invade epithelial cells of the human small intestineand although this speciWcity occurs in vivo, the invasionin vitro of four other cell types observed in this study, sug-gests that there are adhesion receptors involved in the pene-tration of diVerent cell lines and hosts. Although C. bellisporozoites and merozoites had penetrated in all the celllines studies, their ability to invade and multiply varied; itwas greatest with VERO cells, where multiple infections bysporozoites and greater numbers of merozoites wereobserved.

The size of the inoculum has direct eVects on the quan-tity of intracellular parasites that develop (Fayer and Ham-mond, 1967 and Hermosilla et al., 2002). Theseauthors,using Eimeria bovis observed multiple infections inembryonic bovine cell cultures when the inoculum washigher than 2£ 107 sporozoites/ml. By using only 3£ 104 C.belli sporozoites we found multiple infections in VERO andHCT-8 cells. Fayer and Mahrt, 1972; made similar observa-tions when they inoculated Isospora canis sporozoites incanine and bovine cells.

In this study, the size of the parasitophorous vacuolewas diVerent in the various types of cells. In VERO cells alarge parasitophorous vacuole developed around the para-site after invasion, while in the other cells, the vacuoles weresmall and indistinct. VERO cells may be useful in the studyof the parasitophorous vacuole and in tests of the sensitiv-ity or resistance to anticoccidial drugs, particularly whenlines of human intestinal cells are not available.

During our 4 days observation period, we did not seegametocytes, which, could develop in the diVerent cell linesafter 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 genusAtoxoplasma (Garnham 1950) as a junior objective synonym of thegenus Isospora (Schneider 1881) species infecting birds and resurrection

of Cystoisospora (Frenkel 1977) as the correct genus for Isospora speciesinfecting mammals. The Journal of Patrasitology 9, 726–727.

Fayer, R., Hammond, D.M., 1967. Development of Wrst-generationSchizonts of Eimeria bovis in cultured bovine cells. Journal of Protozo-ology 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 culturedcells with some cytological observations. The Journal of Parasitology60, 160–168.

Frenkel, J.K., 1977. Besnoitia wallacei of cats and rodents: with a reclassiW-cation of other cyst-forming isosporid coccidia. The Journal of Parasi-tology 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 belliinfection: observation of unicellular cysts in mesenteric lymphoid tis-sues of a Brazilian patient with aids and animal inoculation. The Jour-nal 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. Presençaextra-intestinal de Cistos Unizóicos de Isospora belli em Paciente comSIDA. Relato de Caso. Revista da Sociedade Brasileira de MedicinaTropical 36, 409–412.

Gold, D., Stein, B., Tzipori, S., 2001. The utilization of taurocholate inexcystation 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 Corioalantoideade 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 frompigs in primary porcine and bovine cell cultures. Veterinary Parasitol-ogy 24, 301–304.

Lindsay, D.S., Current, W.L., 1984. Complete development of Isospora suisof swine in chicken embryos. Journal of Protozoology 31, 152–155.

Lindsay, D.S., Dubey, J.P., Blagburn, B.L., 1997. Biology of Isospora sppfrom humans, nonhuman primates, and domestic animals. ClinicalMicrobiology Review 10, 19–34.

Michiels, J.F., Hofman, P., Bernard, E., Saint Paul, M.C., Boissy, C., Mon-dain, V., LeFichoux, Y., Loubiere, R., 1994. Intestinal and extraintesti-nal Isospora belli infection in an aids patient. Pathology ResearchPractices 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 Cryptosporidiumparvum oocysts. Research and Reviews in Parasitology 52, 127–130.

Restrepo, C., Macher, A.M., Radany, E.H., 1987. Disseminated extraintes-tinal isosporiasis in a patient with acquired immune-deWciency syn-drome. American Journal of Clinical Pathology 87, 536–542.

Silva, M.B.O., Lima, J.D., Vieira, L.S., Vitor, R.W.A., 2006. Humoralresponse and IgG avidity in goat kids experimentally infected withCryptosporidium parvum. Revista Brasileira de Parasitologia Veteriná-ria, in press.