- Email: [email protected]
Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells
Experimental Parasitology 122 (2009) 192–195
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells Múcio F.B. Ribeiro a,*, Camila V. Bastos a, Maria Mercês C. Vasconcelos a, Lygia M.F. Passos b,c a
Departamento de Parasitologia, ICB-UFMG, Belo Horizonte, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, Minas Gerais, Brazil Institute for Comparative Tropical Medicine and Parasitology, Ludwig Maximilian University, Munich, Leopoldstr. 5, 80802 Munich, Germany c Departamento de Medicina Veterinária Preventiva, Escola de Veterinária – UFMG, Belo Horizonte, Minas Gerais, Brazil b
a r t i c l e
i n f o
Article history: Received 9 July 2008 Received in revised form 6 October 2008 Accepted 16 March 2009 Available online 24 March 2009 Keywords: Babesia bigemina IDE8 cells In vitro culture
a b s t r a c t This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Babesia bigemina is a tick-transmitted intraerythrocytic protozoa that infects cattle, resulting in economic loss in most tropical and subtropical regions in the world. A common vector is the one-host tick Rhipicephalus ( Boophilus) microplus (Alonso et al., 1992). The development of a continuous system to cultivate B. bigemina in vitro in bovine erythrocytes (Vega et al., 1985) represented a great achievement in research on babesiosis. However, in endemic areas for bovine babesiosis, the maintenance of bovine donors for cells and serum, free from Babesia sp. and other arthropod-transmitted infections is very difficult and limits the implementation of conventional in vitro culture systems in these areas. On the other hand, attempts to establish and to multiply Babesia species in tick cells, with the aim to produce antigens, have been unsuccessful (Kurtti et al., 1983). The IDE8 cell line was established from embryonic Ixodes scapularis ticks (Munderloh et al., 1994), providing a new tool for the establishment of some parasites and several rickettsial organisms in vitro (Munderloh et al., 1996a,b; Bell-Sakyi et al., 2000; Kocan et al., 1998; Zweygarth et al., 2006). The aim of this paper is to describe the invasive process and the multiplication of B. bigemina sporokinetes in an IDE8 cell line.
2.1. IDE8 culture conditions
* Corresponding author. Fax: +55 3134092970. E-mail address: [email protected] (M.F.B. Ribeiro). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.03.011
The IDE8 cell line was cultured with permission given by Dr. U.G. Munderloh (University of Minnesota, USA) at the Department of Preventive Veterinary Medicine, UFMG, Brazil, following standard procedures for maintenance (Munderloh et al., 1994), in 25 cm2 flasks (TPP, Brazil), at 30 °C (±2 °C) with 5 ml of supplemented L-15B culture medium. Medium changes were carried out weekly and subcultures every 15 days. 2.2. Inoculum One hundred and ten R. microplus engorged females, larger than 4.5 mm in length, were collected from four naturally B. bigeminainfected calves of 2–3 months of age living in a farm located in Pedro Leopoldo, Minas Gerais, Brazil. The ticks were washed, blotted dry, and disinfected with Germekil (Johnson, Brazil), for 30 min at room temperature. After several washes in sterile distillated water, the ticks were individually placed into polystyrene plates and were incubated at 27 °C and relative humidity over 83%. After an 8-day incubation period, smears were prepared from a section at a distal region of one leg of each engorged female. Smears were stained with Giemsa, and examined microscopically under oil immersion. Hemolymph collected from infected ticks after 10 days of incubation provided material for infecting IDE8 cells. The cuticula was again sterilized, as previously described. Each tick was held with sterile forceps and the leg cut with a sterile scalpel blade. The
M.F.B. Ribeiro et al. / Experimental Parasitology 122 (2009) 192–195
193
Fig. 1. Light microscopy of developmental stages of Babesia bigemina in Ixodes scapularis tick cell line (IDE8); Giemsa staining, Bar 10 lm. (A) Inoculum of sporokinetes. (B) Sporokinete adherence and orientation to the target cell. (C) Sporokinete inside a cell; note the absence of a parasitophorous vacuole. (D and E) Modification of the sporokinete; note the bluish cytoplasm and the round form of the parasite. (F) Masses of uninucleated sporokinetes.
hemolymph was collected using a capillary tube to gather the draining fluid. Hemolymph from 10 ticks were pooled in a tube containing 200 ll of culture medium, which constitute the inoculum to infect one culture flask containing an on growing IDE8 cell monolayer. After infection, the culture flask was monitored daily by examination of cytocentrifuge smears made from 50 ll aliquots taken from the culture suspension. Smears were fixed twice with methanol (for 10 min), stained with an 8% Giemsa solution for 30 min and examined under oil immersion at 1000. Maintenance of infected cultures was carried out as described for IDE8 cells. In order to identify the species of Babesia present in the inoculum, DNA was extracted from the hemolymph and from IDE8 cells from day 6 post-infection, using a Puregene D-5500A DNA extraction kit (Gentra Systems Inc., Minneapolis, MN, USA), following the protocol described by Smeenk et al. (2000). Genomic DNA was amplified by duplex PCR (Quintão-Silva et al., 2007). Positive controls contained Babesia-DNA templates extracted from ticks obtained from animals experimentally infected with either Babesia bovis or B. bigemina, while negative PCR controls did not contain DNA.
3. Results Among the 110 engorged females examined, 18 (16.4%) were infected with B. bigemina sporokinetes, from which 10 showed more than 20 sporokinetes in the smear and were selected to provide the inoculum. The final pooled hemolymph inoculum (200 ll) had six sporokinetes per microscopic field (Fig. 1A). During the first 2 days after inoculation, some parasites were seen in the culture medium. These forms measured approximately 10.3 lm (ranging from 9.5 to 12.7 lm) in length, had an apical complex strongly stained, and a spherical nucleus located in the center. At this stage, the parasite was seen close to the cells, suggesting attachment to the cells (Fig. 1B). After 72 h of inoculation, most sporokinetes were inside a cell, in direct contact with the cytoplasm, and constantly close to the nucleus (Fig. 1C). From day 4 to day 5 post-inoculation the parasites increased in size and some of them were no longer elongated but round-shaped forms, measuring 11.6–21.1 lm in length and 3.7–20.4 lm in width. Their cytoplasm stained blue in Giemsa-stained smears with a distinct central nuclear material (Fig. 1D and E).
194
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Fig. 2. Light microscopy of developmental stages of Babesia bigemina in Ixodes scapularis tick cell line (IDE8); Giemsa staining, Bar 10 lm. (G and H) Masses of uninucleated sporokinetes. (I and J) Extracellular ring form adhered to the cell. (K and L) Intra- and extracellular degeneration of sporokinetes.
From day 6 to day 8 post-infection the cells presented masses of single uninucleated sporokinetes that appeared to resemble processes of radial budding from differentiated multiple-fission bodies (Figs. 1F and 2G, H). The sporokinetes were either ring forms containing a central nucleus within a pale blue cytoplasm, with some single-shaped, or they tended to be elongated forms, measuring 3–4 lm in length and 1–2 lm in width (Fig. 2I). These ring forms were observed free in culture medium or in close proximity to the cells, being engulfed or surrounded by the tick cell membrane (Fig. 2J). On day 8 onwards, these ring forms were observed either inside the cells (Fig. 2K), or free in the culture medium; however, they had dilated nucleous suggesting degeneration (Fig. 2H). The duplex PCR method allowed the identification of B. bigemina present in the hemolymph of engorged female ticks and in the infected IDE8 cells (Fig. 3).
hemocytes, and oocytes, where successive cycles of further schizogony take place (Mehlhorn and Schein, 1984). The IDE8 cell line, derived from embryonic cells of I. scapularis, has characteristically very long, often branching, pseudopodial cells, resembling either neural cells (Munderloh et al., 1994) or haemocytes. The successful infection of these cells with B. bigemina
4. Discussion During the life cycle of B. bigemina parasites, the sporokinetes are formed in epithelial gut cells of engorged females of R. (B.) microplus ticks. When released into the hemolymph, they initiate secondary cycles of schizogony in a wide variety of cell types and tissues, including gut, malpighian tubules cell, muscle fibers,
Fig. 3. Silver-stained polyacrylamide gel electrophoresis of products from the duplex PCR. Lane 1 shows DNA marker ladder; lane 2 is a sample from infected IDE8 cells showing the presence of B. bigemina DNA; lane 3 is a control sample showing the presence of both B. bigemina and B. bovis; lane 4 is a control sample showing the presence of B. bigemina DNA in hemolymph of engorged ticks.
M.F.B. Ribeiro et al. / Experimental Parasitology 122 (2009) 192–195
demonstrated that the sporokinetes have low specificity for the tick cell type. First, the parasite was in contact to the cell surface; this was followed of phagocytosis by the cells, suggesting the presence of compatible receptors on the membrane of IDE8 cells. In erythrocytes one of these receptors has been identified as sialic acid (Gaffar et al., 2003), which is also present in I. ricinus cells (Vancová et al., 2006). This fact suggests that IDE8 cells may also contain sialic acid acting as a receptor for phagocytosis of Babesia sporokinetes. Inside the cells, the sporokinetes remained in direct contact with the cytoplasm of the IDE8 cells with no evidence of vacuole formation. Friedhoff and Scholtyseck (1969) found that intracellular B. bigemina kinetes were not surrounded by host provided membranes and caused considerable disruption of fine structure of the host cell cytoplasm. The intracellular forms observed later demonstrated that the parasite developed a process of schizogony inside the IDE8 cells, producing several sporokinetes with distinct morphology. However, the round forms observed after the initial multiplication were phagocyted by new IDE8 cells but were not able to undergo further schizogony and degenerated. Gaffar et al. (2003) reported that B. bovis intraerythrocytic merozoites were able to penetrate and multiply into human erythrocytes, although the parasitemia reduces gradually. It is believed that human erythrocytes are efficiently invaded; however, the parasite development is blocked in later stages, as egress from the host cell after intracellular maturation might be inefficient. In the present paper, we report for the first time the in vitro multiplication of B. bigemina sporokinetes in IDE8 cells, with a parasite survival period of 8 days inside the cells. In vitro short viability of Babesia sporokinetes inside other tick embryonic cells have been previously reported in the literature. Kurtti et al. (1983) observed that B. caballi sporokinetes persisted in embrionary cells of Anocentor nitens up to 5 days. Mosqueda et al. (2003) also reported the invasion of B. bigemina kinetes into in vitro cultured R. microplus embryonic cells and their survival for up to 10 days. Examination of Giemsa-stained smears from hemolymph showed that only few hemocytes were present in the inoculum and none of them were infected with Babesia. Thus, the possibility that Babesia-infected hemocytes could be present in the inoculum is remote. It is clear that the IDE8 cells system should be optimized to support countinous in vitro maintanance of B. bigemina parasites; however these results are promising and open a new approach for the establishment of alternative Babesia in vitro systems that do not require vertebrate host donors. In addition, in vitro infections using a tick cell system open a new window for a better understanding of biological and molecular features, as well as the development of tick stages of Babesia parasites and their relationship with invertebrate host cells, which are not possible with the erythrocyte-based system available.
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Acknowledgments This work received financial support from FAPEMIG (Grant EDT 2198/03). The authors thank Dr. U.G. Munderloh (University of Minnesota, USA) for permission to use the IDE8 cell line and CNPq for awarding scholarships. References Alonso, M., Arellano-Sota, C., Cereser, V.H., Cordoves, C.O., Guglielmone, A.A., Kessler, R., Mangold, A.J., Nari, A., Patarroyo, J.H., Solari, M.A., Veja, C.A., 1992. Epidemiology of bovine anaplasmosis and babesiosis in Latin America and the Caribbean. Revue Scientifique et Technique Office International des Epizoties 11, 713–733. Bell-Sakyi, L., Paxton, E.A., Munderloh, U.G., Sumption, K.J., 2000. Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line. Journal of Clinical Microbiology 38, 1238–1240. Friedhoff, K., Scholtyseck, E., 1969. Fine structure of the merozoites of Babesia bigemina in the ovary of Boophilus microplus and Boophilus decoloratus. Zeitschrift fur Parasitenkunde 32, 266–283. Gaffar, F.R., Franssen, F.F., de Vries, E., 2003. Babesia bovis merozoites invade human, ovine, equine, porcine and caprine erythrocytes by a sialic acid-dependent mechanism followed by developmental arrest after a single round of cell fission. International Journal for Parasitology 33, 1595–1603. Kocan, K.M., Munderloh, U.G., Ewing, S.A., 1998. Development of the ebony isolate of Ehrlichia canis in culture Ixodes scapularis cells. In: Proceedings of the 79th Conference of Research Workers in Animal Diseases, Chicago, USA. Kurtti, T.J., Munderloh, U.G., Stiller, D., 1983. The interaction of Babesia caballi kinetes with tick cells. Journal of Invertebrate Pathology 42, 334–343. Mehlhorn, H., Schein, E., 1984. The piroplams life cycle and sexual stages. Advances in Parasitology 23, 37–103. Mosqueda, J., Ramos, J.A., Salto, M., Cossio-Bayugar, R., 2003. In vitro infection of Babesia bigemina kinetes to Boophilus microplus embryonic cells. In: Fifth International Seminar in Animal Parasitology. Merida, Yucatan, Mexico, 2003, pp. 240–247. Munderloh, U.G., Liu, Y., Wang, M., Chen, C., Kurtti, T.J., 1994. Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. Journal of Parasitology 80, 533–543. Munderloh, G.U., Blouin, E.F., Kocan, K.M., Edwards, W.L., Kurtti, T.J., 1996a. Establishment of the tick (Acari: Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) in tick cell culture. Journal of Medical Entomology 33, 656–664. Munderloh, U.G., Madigan, J.E.M., Dumler, J.S., Goodman, J.L., Hayes, S.F., Barlough, F.E., Nelson, C.M., Kurtti, T.J., 1996b. Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture. Journal of Clinical Microbiology 34, 664–670. Quintão-Silva, M.G., Melo, M.N., Ribeiro, M.F.B., 2007. Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus. Zoonoses Public Health 54, 147–151. Smeenk, I., Kelly, P.J., Wray, K., Musuka, G., Tress, A.J., Jongejan, F., 2000. Babesia bovis and Babesia bigemina DNA detected in cattle and ticks from Zimbabwe by polymerase chain reaction. Journal of the South African Veterinary Association 71, 21–24. Vancová, M., Zacharovová, K., Grubhoffer, L., Nebesárová, J., 2006. Ultrastructure and lectin characterization of granular salivary cells from Ixodes ricinus females. Journal of Parasitology 92, 431–440. Vega, C.A., Buening, G., Green, T., Carson, C.A., 1985. In vitro cultivation of Babesia bigemina. American Journal of Veterinary Research 46, 416–420. Zweygarth, E., Josemans, A.I., Spickett, A.M., Steyn, H.C., Putterill, J., Troskie, P.C., Mtshali, M.S., Bell-Sakyi, L., Shkap, V., Fish, L., Kocan, K.M., Blouin, E.F., 2006. In vitro cultivation of a South African isolate of Anaplasma sp. in tick cell cultures. Onderstepoort Journal of Veterinary Research 73, 251–255.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells Múcio F.B. Ribeiro a,*, Camila V. Bastos a, Maria Mercês C. Vasconcelos a, Lygia M.F. Passos b,c a
Departamento de Parasitologia, ICB-UFMG, Belo Horizonte, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, Minas Gerais, Brazil Institute for Comparative Tropical Medicine and Parasitology, Ludwig Maximilian University, Munich, Leopoldstr. 5, 80802 Munich, Germany c Departamento de Medicina Veterinária Preventiva, Escola de Veterinária – UFMG, Belo Horizonte, Minas Gerais, Brazil b
a r t i c l e
i n f o
Article history: Received 9 July 2008 Received in revised form 6 October 2008 Accepted 16 March 2009 Available online 24 March 2009 Keywords: Babesia bigemina IDE8 cells In vitro culture
a b s t r a c t This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Babesia bigemina is a tick-transmitted intraerythrocytic protozoa that infects cattle, resulting in economic loss in most tropical and subtropical regions in the world. A common vector is the one-host tick Rhipicephalus ( Boophilus) microplus (Alonso et al., 1992). The development of a continuous system to cultivate B. bigemina in vitro in bovine erythrocytes (Vega et al., 1985) represented a great achievement in research on babesiosis. However, in endemic areas for bovine babesiosis, the maintenance of bovine donors for cells and serum, free from Babesia sp. and other arthropod-transmitted infections is very difficult and limits the implementation of conventional in vitro culture systems in these areas. On the other hand, attempts to establish and to multiply Babesia species in tick cells, with the aim to produce antigens, have been unsuccessful (Kurtti et al., 1983). The IDE8 cell line was established from embryonic Ixodes scapularis ticks (Munderloh et al., 1994), providing a new tool for the establishment of some parasites and several rickettsial organisms in vitro (Munderloh et al., 1996a,b; Bell-Sakyi et al., 2000; Kocan et al., 1998; Zweygarth et al., 2006). The aim of this paper is to describe the invasive process and the multiplication of B. bigemina sporokinetes in an IDE8 cell line.
2.1. IDE8 culture conditions
* Corresponding author. Fax: +55 3134092970. E-mail address: [email protected] (M.F.B. Ribeiro). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.03.011
The IDE8 cell line was cultured with permission given by Dr. U.G. Munderloh (University of Minnesota, USA) at the Department of Preventive Veterinary Medicine, UFMG, Brazil, following standard procedures for maintenance (Munderloh et al., 1994), in 25 cm2 flasks (TPP, Brazil), at 30 °C (±2 °C) with 5 ml of supplemented L-15B culture medium. Medium changes were carried out weekly and subcultures every 15 days. 2.2. Inoculum One hundred and ten R. microplus engorged females, larger than 4.5 mm in length, were collected from four naturally B. bigeminainfected calves of 2–3 months of age living in a farm located in Pedro Leopoldo, Minas Gerais, Brazil. The ticks were washed, blotted dry, and disinfected with Germekil (Johnson, Brazil), for 30 min at room temperature. After several washes in sterile distillated water, the ticks were individually placed into polystyrene plates and were incubated at 27 °C and relative humidity over 83%. After an 8-day incubation period, smears were prepared from a section at a distal region of one leg of each engorged female. Smears were stained with Giemsa, and examined microscopically under oil immersion. Hemolymph collected from infected ticks after 10 days of incubation provided material for infecting IDE8 cells. The cuticula was again sterilized, as previously described. Each tick was held with sterile forceps and the leg cut with a sterile scalpel blade. The
M.F.B. Ribeiro et al. / Experimental Parasitology 122 (2009) 192–195
193
Fig. 1. Light microscopy of developmental stages of Babesia bigemina in Ixodes scapularis tick cell line (IDE8); Giemsa staining, Bar 10 lm. (A) Inoculum of sporokinetes. (B) Sporokinete adherence and orientation to the target cell. (C) Sporokinete inside a cell; note the absence of a parasitophorous vacuole. (D and E) Modification of the sporokinete; note the bluish cytoplasm and the round form of the parasite. (F) Masses of uninucleated sporokinetes.
hemolymph was collected using a capillary tube to gather the draining fluid. Hemolymph from 10 ticks were pooled in a tube containing 200 ll of culture medium, which constitute the inoculum to infect one culture flask containing an on growing IDE8 cell monolayer. After infection, the culture flask was monitored daily by examination of cytocentrifuge smears made from 50 ll aliquots taken from the culture suspension. Smears were fixed twice with methanol (for 10 min), stained with an 8% Giemsa solution for 30 min and examined under oil immersion at 1000. Maintenance of infected cultures was carried out as described for IDE8 cells. In order to identify the species of Babesia present in the inoculum, DNA was extracted from the hemolymph and from IDE8 cells from day 6 post-infection, using a Puregene D-5500A DNA extraction kit (Gentra Systems Inc., Minneapolis, MN, USA), following the protocol described by Smeenk et al. (2000). Genomic DNA was amplified by duplex PCR (Quintão-Silva et al., 2007). Positive controls contained Babesia-DNA templates extracted from ticks obtained from animals experimentally infected with either Babesia bovis or B. bigemina, while negative PCR controls did not contain DNA.
3. Results Among the 110 engorged females examined, 18 (16.4%) were infected with B. bigemina sporokinetes, from which 10 showed more than 20 sporokinetes in the smear and were selected to provide the inoculum. The final pooled hemolymph inoculum (200 ll) had six sporokinetes per microscopic field (Fig. 1A). During the first 2 days after inoculation, some parasites were seen in the culture medium. These forms measured approximately 10.3 lm (ranging from 9.5 to 12.7 lm) in length, had an apical complex strongly stained, and a spherical nucleus located in the center. At this stage, the parasite was seen close to the cells, suggesting attachment to the cells (Fig. 1B). After 72 h of inoculation, most sporokinetes were inside a cell, in direct contact with the cytoplasm, and constantly close to the nucleus (Fig. 1C). From day 4 to day 5 post-inoculation the parasites increased in size and some of them were no longer elongated but round-shaped forms, measuring 11.6–21.1 lm in length and 3.7–20.4 lm in width. Their cytoplasm stained blue in Giemsa-stained smears with a distinct central nuclear material (Fig. 1D and E).
194
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Fig. 2. Light microscopy of developmental stages of Babesia bigemina in Ixodes scapularis tick cell line (IDE8); Giemsa staining, Bar 10 lm. (G and H) Masses of uninucleated sporokinetes. (I and J) Extracellular ring form adhered to the cell. (K and L) Intra- and extracellular degeneration of sporokinetes.
From day 6 to day 8 post-infection the cells presented masses of single uninucleated sporokinetes that appeared to resemble processes of radial budding from differentiated multiple-fission bodies (Figs. 1F and 2G, H). The sporokinetes were either ring forms containing a central nucleus within a pale blue cytoplasm, with some single-shaped, or they tended to be elongated forms, measuring 3–4 lm in length and 1–2 lm in width (Fig. 2I). These ring forms were observed free in culture medium or in close proximity to the cells, being engulfed or surrounded by the tick cell membrane (Fig. 2J). On day 8 onwards, these ring forms were observed either inside the cells (Fig. 2K), or free in the culture medium; however, they had dilated nucleous suggesting degeneration (Fig. 2H). The duplex PCR method allowed the identification of B. bigemina present in the hemolymph of engorged female ticks and in the infected IDE8 cells (Fig. 3).
hemocytes, and oocytes, where successive cycles of further schizogony take place (Mehlhorn and Schein, 1984). The IDE8 cell line, derived from embryonic cells of I. scapularis, has characteristically very long, often branching, pseudopodial cells, resembling either neural cells (Munderloh et al., 1994) or haemocytes. The successful infection of these cells with B. bigemina
4. Discussion During the life cycle of B. bigemina parasites, the sporokinetes are formed in epithelial gut cells of engorged females of R. (B.) microplus ticks. When released into the hemolymph, they initiate secondary cycles of schizogony in a wide variety of cell types and tissues, including gut, malpighian tubules cell, muscle fibers,
Fig. 3. Silver-stained polyacrylamide gel electrophoresis of products from the duplex PCR. Lane 1 shows DNA marker ladder; lane 2 is a sample from infected IDE8 cells showing the presence of B. bigemina DNA; lane 3 is a control sample showing the presence of both B. bigemina and B. bovis; lane 4 is a control sample showing the presence of B. bigemina DNA in hemolymph of engorged ticks.
M.F.B. Ribeiro et al. / Experimental Parasitology 122 (2009) 192–195
demonstrated that the sporokinetes have low specificity for the tick cell type. First, the parasite was in contact to the cell surface; this was followed of phagocytosis by the cells, suggesting the presence of compatible receptors on the membrane of IDE8 cells. In erythrocytes one of these receptors has been identified as sialic acid (Gaffar et al., 2003), which is also present in I. ricinus cells (Vancová et al., 2006). This fact suggests that IDE8 cells may also contain sialic acid acting as a receptor for phagocytosis of Babesia sporokinetes. Inside the cells, the sporokinetes remained in direct contact with the cytoplasm of the IDE8 cells with no evidence of vacuole formation. Friedhoff and Scholtyseck (1969) found that intracellular B. bigemina kinetes were not surrounded by host provided membranes and caused considerable disruption of fine structure of the host cell cytoplasm. The intracellular forms observed later demonstrated that the parasite developed a process of schizogony inside the IDE8 cells, producing several sporokinetes with distinct morphology. However, the round forms observed after the initial multiplication were phagocyted by new IDE8 cells but were not able to undergo further schizogony and degenerated. Gaffar et al. (2003) reported that B. bovis intraerythrocytic merozoites were able to penetrate and multiply into human erythrocytes, although the parasitemia reduces gradually. It is believed that human erythrocytes are efficiently invaded; however, the parasite development is blocked in later stages, as egress from the host cell after intracellular maturation might be inefficient. In the present paper, we report for the first time the in vitro multiplication of B. bigemina sporokinetes in IDE8 cells, with a parasite survival period of 8 days inside the cells. In vitro short viability of Babesia sporokinetes inside other tick embryonic cells have been previously reported in the literature. Kurtti et al. (1983) observed that B. caballi sporokinetes persisted in embrionary cells of Anocentor nitens up to 5 days. Mosqueda et al. (2003) also reported the invasion of B. bigemina kinetes into in vitro cultured R. microplus embryonic cells and their survival for up to 10 days. Examination of Giemsa-stained smears from hemolymph showed that only few hemocytes were present in the inoculum and none of them were infected with Babesia. Thus, the possibility that Babesia-infected hemocytes could be present in the inoculum is remote. It is clear that the IDE8 cells system should be optimized to support countinous in vitro maintanance of B. bigemina parasites; however these results are promising and open a new approach for the establishment of alternative Babesia in vitro systems that do not require vertebrate host donors. In addition, in vitro infections using a tick cell system open a new window for a better understanding of biological and molecular features, as well as the development of tick stages of Babesia parasites and their relationship with invertebrate host cells, which are not possible with the erythrocyte-based system available.
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