Ultrastructure of Babesia equi trophozoites isolated in Minas Gerais, Brazil

Ultra-estrutura de trofozoítos de Babesia equi isolados em Minas Gerais, Brasil

ARTIGO DE REVISÃO

Ultrastructure of Babesia equi trophozoites isolated in Minas Gerais, Brazil

Ultra-estrutura de trofozoítos de Babesia equi isolados em Minas Gerais, Brasil

Antônio M. Guimarães^I; José D. Lima^II; Múcio F. B. Ribeiro^II

^IDepartamento de Medicina Veterinária, Universidade Federal de Lavras, Cx. Postal 37, Lavras, MG 37200-000, Brasil. E-mail: [e-mail]

^IIDepartamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, Cx. Postal 486, Belo Horizonte, MG 31270-901, Brasil

ABSTRACT

A transmission electron microscope study was carried out on Babesia equi obtained from a splenectomized horse, from the municipality of Santa Luzia, Minas Gerais, Brazil. The isolate was inoculated into two splenectomized foals (1.05 x 10¹⁰ parasitized erythrocytes by B. equi). Trophozoites have a single membrane in direct contact with the cytoplasm of the red blood cells, a prominent nucleus, well-developed rough and smooth endoplasmic reticulum, numerous free ribosomes and small food vacuoles. B. equi trophozoites have a cytostome and a long tubular feeding structure in direct contact with the blood plasma.

Index terms:Babesia equi, ultrastructure, trophozoites, feeding mechanism.

RESUMO

Neste estudo de microscopia eletrônica de transmissão utilizou-se um isolado de Babesia equi obtido a partir de um eqüino esplenectomizado, oriundo do município de Santa Luzia, Minas Gerais, Brasil. O isolado foi inoculado em dois potros esplenectomizados (1,05 x 10¹⁰ eritrócitos parasitados com B. equi). Os trofozoítos apresentaram uma membrana simples em contato direto com o citoplasma das hemácias, núcleo proeminente, retículo endoplasmático liso e rugoso bem desenvolvidos, numerosos ribosomos livres e pequenos vacúolos alimentares. Em trofozoítos de B. equi foi observado citostoma e uma longa estrutura tubular de alimentação em contato direto com o plasma sangüíneo.

Termos de indexação:Babesia equi, ultra-estrutura, trofozoítos, mecanismos de alimentação.

INTRODUCTION

Equine babesiosis is an infectious disease of horses and other equids caused by the protozoan hemoparasites Babesia equi and Babesia caballi (De Waal 1992). B. equi infects 90% of the world equine population (Schein 1988), and is transmitted naturally by ticks of the genera Hyalomma, Dermacentor and Rhipicephalus (Friedhoff 1988), and experimentally by Boophilus microplus (Knowles et al. 1992, Guimarães et al. 1998a,b). B. equi was first isolated in Brazil by Ribeiro & Lima (1989), from naturally infected horses to produce an antigen for serological purposes. Although the disease is probably endemic in Brazil, only a small number of studies were published, all of them based on clinical and serological surveys (Guimarães et al. 1997, Heuchert et al. 1999, Kerber et al. 1999, Ribeiro et al. 1999).

Intracellular parasites depend on the host cell to obtain the majority of their nutrients during development. In Plasmodium spp the parasites ingest hemoglobin through a cytostome (Aikawa et al. 1966) and digestion is through proteolytic enzymes in digestive vesicles (Slomianny et al. 1983). Electron-dense hemozoin pigments in food vacuoles comprise the final product of hemoglobin digestion (Aikawa 1971). The feeding mechanism of Babesia spp remains incompletely described, and the formation of food vacuoles has been the subject of some controversy (Langreth & Trager 1976, Rudzinska 1976). Electron microscope studies on B. equi trophozoites have demonstrated a cytostome and a tubular food vacuole (Simpson et al. 1967, Frerichs & Holbrook 1974, Simpson & Neal 1980) involved in the formation of food vacuoles, similar to the observed in some species of Theileria (Higuchi et al. 1984, Conrad et al. 1985, Fawcett et al. 1987).

This paper describes a transmission electron microscope (TEM) study of Babesia equi trophozoites with emphasis on the structures involved in nutrient intake.

MATERIALS AND METHODS

The strain of Babesia equi (BE/MG) used in this study was isolated following splenectomy of a naturally infected 17-month old crossbred foal from Santa Luzia town, State of Minas Gerais, Brazil. The foal had been medicated previously with diminazene diaceturate; two applications of 6 mg/kg, at intervals of 24 hours. This was to eliminate possible infection with Babesia caballi. The foal was kept free of ticks by spraying weekly with deltametrine. It developed relapse of B. equi 22 days following splenectomy. Thirteen days later, smears of peripheral blood stained with Giemsa demonstrated that 44% of the red cells were infected. Subsequently, blood was collect by jugular venipuncture using EDTA as anticoagulant and preserved in liquid nitrogen using dimethylsulphoxide.

Two 12 and 15-month old crossbred, splenectomized foals were used for experimental infection. They had been maintained free of ticks since birth by weekly spraying with deltametrine. They were inoculated intravenously with 1.05 x 10¹⁰B. equi (BE/MG) infected erythrocytes. After achieving 40% of the red cells infected, peripheral blood was collected as described above.

After centrifugation at 1,400g for 5 minutes, plasma and leucocyte coat were removed. Erythrocytes were fixed by the addition of 500 µl of sediment to 5 ml of a fixative containing 2.5% glutaraldehyde in sodium cacodylate buffer 0.1M, pH 7.2, for 24 hours at 4ºC. Fixed cells were then centrifuged at 1,400g for 5 minutes, washed in cacodylate buffer and post-fixed in 2% osmium tetroxide in 0.1M cacodylate buffered, for 2 hours at 4ºC and subsequently set in 2% agar. After three washes in PBS with 17.8% sucrose, the samples were stained ''en bloc'' with uranyl acetate at 0.5% (w/v) in aqueous solution with 13.3% sucrose, at 4º C for 14 hours. The samples were then washed three times in aqueous solution with 13.3% sucrose, dehydrated in increasing concentrations of ethyl alcohol, and embedded in epon-araldite. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope Zeiss EM 10A.

RESULTS

Trophozoites of Babesia equi were round shaped with 1.3 µm diameter, covered by single membrane in direct contact with the cytoplasm of the erythrocyte (Fig. 1). There was observed a prominent nucleus (0.8 mm in diameter) with a double membrane, well-developed rough and smooth endoplasmic reticulum, vacuoles and numerous free ribosomes (Fig. 2).

Trophozoites presented a single cytostome composed of a single electron-dense concentric ring (terminal fringes), with an internal diameter of 0.1 mm and located on the periphery of the parasite (Fig. 3). The material inside the cavity of the cytostome was of the same density as the cytoplasm of the erythrocyte. Small intracytoplasmic food vacuoles were observed next to the cytostome. Food vacuoles were eventually observed, surrounded by a single cytoplasmic membrane and containing material of similar density to hemoglobin.

Trophozoites located very close to the erythrocyte membrane presented a tubular feeding structure, which emerged from the interior of the parasite and extended to the blood plasma (Fig. 4). This tubular feeding structure was formed by the concomitant invagination of the plasma membranes of the erythrocyte and the parasite (Fig. 5). These membranes were in intimate contact throughout the invagination, and were separated by a central lumen measuring 60 hm, containing a small quantity of ingested material. The invaginations of the plasma membrane of the trophozoites enclosed small quantities of the cell hemoglobin. The ingested material appeared to be continuous with and had the same intensity of color as the rest of the host erythrocyte cytoplasm.

DISCUSSION

The results corroborate and detail the previous descriptions of the ultrastructure of Babesia equi (Simpson et al. 1967, Simpson 1970, Frerichs & Holbrook 1974, Simpson & Neal 1980). The electronmicrographs of this paper show clearly that B. equi trophozoite is surrounded by a single membrane and is not contained in a parasitophorous vacuole. It is apparent that its presence does not provoke evident changes in the cytoplasm of the host cell. All the stages have a prominent nucleus, well-developed rough and smooth endoplasmic reticulum, and many free ribosomes. Mitochondrial organelles were not observed in the intra-erythrocytic stages of B. equi, and typical mitochondria have not been recorded in any previous studies (Simpson et al. 1967, Simpson 1970, Frerichs & Holbrook 1974, Simpson & Neal 1980).

A singular aspect of the morphology of the B. equi trophozoites is the presence of a cytostome and the tubular feeding structure involved in the feeding mechanism (Frerichs & Holbrook 1974). The cytostome was first described in Plasmodium spp (Aikawa et al. 1966), and a cytostome together with a tubular food vacuole has been described in some species of Theileria (Higuchi et al. 1984, Conrad et al. 1986, Fawcett et al. 1987). In this study, the analysis of electron-micrographs allowed discussion of only certain aspects of the feeding mechanism of B. equi. These included the detection of pinocytosis, and the formation of food vacuoles, along with the presence of a cytostome and tubular feeding structure. Food vacuoles were observed in the B. equi trophozoites which had the same density as the erythrocyte cytoplasm, lacked a limiting membrane, and showed continuity between the vacuole contents and the cytoplasm of the host cell. A similar situation has been described in Babesia rodhaini (Rudzinska & Trager 1960, 1962), Babesia canis and Babesia caballi (Simpson et al. 1963), Babesia felis (Denning & Hebel 1969) and Babesia microti (Shortt & Blackie 1965, Mcmillan & Brocklesby 1971). Rudzinska (1976) demonstrated that these food vacuoles are not formed through phagocytosis and called them pseudo-food vacuoles. According to this author, the reason for this phenomenon is unknown, but they probably serve to increase the surface area of the parasite which is in direct contact with the host cytoplasm, and facilitate as such the ingestion of nutrients.

In this study, some trophozoites were found to have a well-developed cytostome and food vacuole. The material in the cytostome cavity was of the same density as that observed for the erythrocyte cytoplasm. Some B. equi trophozoites were eventually observed with food vacuoles surrounded by a single membrane, without continuity between the vacuole contents and cytoplasm of the erythrocyte. This type of vacuole did not, therefore, arise from the surface of the parasite through pinocytosis, but probably through expansion of the base of the cytostome and its subsequent separation by constriction. In this case, it can be considered a true food vacuole formed by cytostomal phagocytosis of the hemoglobin. The fact that a continuity between the cytostome and the food vacuole was not observed in this study may be attributed in part to the transitory nature of the connection between the two structures. In contrast to other Babesia species, it is possible that the trophozoites of B. equi are capable of ingesting hemoglobin through both cytostomes and pinocytosis. Intra-erythrocytic stages of Theileria spp with a cytostome and the formation of food vacuoles were described in the electron microscope studies of Conrad et al. (1986) and Fawcett et al. (1987).

B. equi is able to form a tubular food vacuole from the extensive invagination of the plasma membranes of the erythrocyte and the trophozoite, when the parasite is located very close to the periphery of the host cell (Simpson et al. 1967, Simpson & Neal 1980). A similar structure has been described in species of Theileria (Conrad et al. 1986, Fawcett et al. 1987). The tubular structure extends from the interior of the trophozoite to the periphery of the erythrocyte, where its lumen frequently maintains direct contact with the blood plasma, probably allowing to ingest nutrients during periods of rapid growth and development of the parasite (Frerichs & Holbrook 1974). Higuchi et al. (1984) observed similar tubules in Theileria sergenti along with ''excreta-like'' material outside the erythrocyte in the region where the tubule was in contact with the parasite. Higuchi et al. (1984) argued that the parasite probably excretes actively from the tubular structure. Controversy persists, however, with regard to the role of this structure (ingestion or excretion), and further studies are required to determine its true function.

While taking into account the inherent limitations of the method, based on an analysis of the electronmicrographs, it is possible to conclude that Babesia equi can form true food vacuoles and use the cytostome, the tubular feeding structure and pinocytosis as feeding mechanisms.

Received on June 4, 2002

Accepted for publication on February 14, 2003

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Publication Dates

• Publication in this collection
  05 Dec 2003
• Date of issue
  Sept 2003

History

• Received
  04 June 2002
• Accepted
  14 Feb 2003