Development of Eimeria ninakohlyakimovae in vitro in primary and permanent cell lines
Introduction
Eimeria ninakohlyakimovae is an ubiquitously spread intracellular apicomplexan enteropathogen of goats causing severe haemorrhagic typhlocolitis with up to 30% mortality rates in goat kids (Koudela and Bokova, 1998). In some areas, more than 96% of goat kids, aged 4–10 weeks, may be affected (Ruiz et al., 2006). Clinical E. ninakohlyakimovae coccidiosis is influenced by different factors such as the intensification of production, immune status of animals, age, and even climate conditions (Ruiz et al., 2006). Especially in rural semi-arid areas, depending economically on goat rearing, such as the Canary Islands (Spain) or other zones in Africa or the Middle East exhibiting analogous climate conditions, caprine coccidiosis severely affects animal health and profitability of goat industry (Ruiz et al., 2006). For such, there is a need of basic research allowing for the development of vaccines in the future.
The endogenous development of the obligate intracellular protozoan E. ninakohlyakimovae comprises two merogonies, the first of which results in the formation of large macromeronts (up to 166 μm × 124 μm, Vieira et al., 1997). Macromeronts specifically develop in endothelial cells of the central lymph capillaries in the villi of the distal ileum within 10–12 days p.i. and contain >100,000 merozoites I (Vieira et al., 1997). Much smaller second meronts (17 μm × 12 μm) as well as gamonts of E. ninakohlyakimovae develop in epithelial cells of the crypts of the caecum and colon (Vieira et al., 1997). Occasionally, meronts and oocysts of E. ninakohlyakimovae have also been observed in bile duct epithelial cells of the liver and were described as a cause for hepatic coccidiosis in goats (Dai et al., 1991, Mahmoud et al., 1994).
Given that Eimeria spp. generally are highly host-specific and that reactions induced by different Eimeria spp. within the same host animal are species-specific on both the immunologic (Rose, 1987) and the host cell level (Dalloul et al., 2007), it appears essential that basic research is performed on the precise Eimeria species in question and the respective host cell type. However, suitable in vitro systems, representing one important prerequisite for parasite-specific investigations, are lacking for caprine Eimeria spp. whilst they have already been established for avian Eimeria spp. (Hofmann and Raether, 1990, Augustine, 1994, Bumstead et al., 1998, Heriveau et al., 2000), murine Eimeria spp. (Danforth et al., 1984) and the bovine species Eimeria bovis (Fayer and Hammond, 1967, Hammond et al., 1969, Speer and Hammond, 1973, Speer et al., 1985, Reduker and Speer, 1986, Speer and Whitmire, 1989, Hermosilla et al., 2002). Some of these in vitro systems, however, use cell types which differ from the specific host cell of sporozoites in vivo, and may therefore only partially mirror the unique spectrum of responses. In fact, most of the pathogenic Eimeria spp. in ruminants (e.g. Eimeria bovis, Eimeria zuernii, Eimeria bakuensis, Eimeria arloingi, Eimeria christenseni, Eimeria ninakohlyakimovae) differ from those infecting mice or chicken with respect to sporozoite specificity for host endothelial cells in vivo, macromeront formation and prolonged replication time.
The goal of this investigation was to establish an in vitro system for the caprine pathogen E. ninakohlyakimovae allowing for the production of parasite material. Therefore we tested primary caprine (CUVEC), bovine (BUVEC) and human (HUVEC) umbilical vein endothelial cells as host cells for sporozoites and compared these results with data obtained in permanent cell lines of bovine foetal gastrointestinal cells (BFGC), bovine colonic epithelial cells (BCEC) and African green monkey kidney cells (VERO) with respect to first generation macromeront formation, duration of development and the production of viable merozoites I in vitro. Cells of ruminant origin were identified as the most suitable cell types for studies on immunology and molecular biology of E. ninakohlyakimovae, whilst immortalized BCEC guaranteed the highest and easiest production of merozoites I.
Section snippets
Animals
Goat kids were purchased from a local farmer at the age of 1–7 days, treated with Baycox® (Bayer) and Halocur® (Intervet), assessed for parasitic infections and, when deemed parasite free, maintained under parasite-free conditions in autoclaved stainless steel cages until experimental E. ninakohlyakimovae infection. Goat kids were fed with milk substitute (Bacilactol, Capisa) and commercial concentrates (Starting Concentrate, Capisa). Water and sterilized hay were given ad libitum.
Parasite maintenance
The E.
Gliding motility of Eimeria ninakohlyakimovae sporozoites and host cell invasion
Excystation of sporozoites from sporulated oocysts was successful and resulted in viable sporozoites. After exposure to potential host cells, sporozoites showed typical movements of gliding motility on the surface of all cell types used. Sporozoites of E. ninakohlyakimovae invaded all cell types used, i.e., irrespective of their origin. Highest initial infection rates were observed in BFGC (23.2%), followed by BCEC (18.3%), HUVEC (18.2%), CUVEC (11.2%), BUVEC (7.9%) and VERO (6.3%) (Fig. 1).
Discussion
In this investigation we successfully established in vitro culture systems for E. ninakohlyakimovae resulting the generation of merozoites I. In vivo, the sporozoite stage of this protozoan selectively develops in endothelial cells, mainly of the central lacteals of the ileum. Concerning the selection of suitable host cells and the invasion thereof in vitro, however, neither host- nor cell type-specificity was observed since all cell types exposed to sporozoites were infected. In principle,
Acknowledgements
We greatly acknowledge B. Hofmann and B. Reinhardt for their excellent technical assistance in cell culture. This work was supported by the Ministry of Science and Technology of Spain (MEC, project no. AGL2007-63415) and the FEDER Founds.
References (38)
- et al.
Hepatic coccidiosis in the goat
Int. J. Parasitol.
(1991) - et al.
Unique responses of the avian macrophage to different species of Eimeria
Mol. Immunol.
(2007) - et al.
Toxoplasma gondii: calcium ionophore A23187-mediated exit of trophozoites from infected murine macrophages
Exp. Parasitol.
(1982) - et al.
Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes
Toxicol. In Vitro
(2000) - et al.
Inhibition of Eimeria tenella replication after recombinant IFN-gamma activation in chicken macrophages, fibroblasts and epithelial cells
Vet. Parasitol.
(2000) - et al.
Eimeria bovis modulates adhesion molecule gene transcription in and PMN adhesion to infected bovine endothelial cells
Int. J. Parasitol.
(2006) - et al.
Coccidiosis in goats in the Czech Republic
Vet. Parasitol.
(1998) - et al.
Inhibition of host cell apoptosis by Eimeria bovis sporozoites
Vet. Parasitol.
(2009) - et al.
Hepato-biliary coccidiosis in a dairy goat
Vet. Parasitol.
(1994) - et al.
Migration through host cells by apicomplexan parasites
Microbes. Infect.
(2001)
Immunity to Eimeria infections
Vet. Immunol. Immunopathol.
Lymphocyte subpopulations in the caecum mucosa of rats after infections with Eimeria separata: early responses in naive and immune animals to primary and challenge infections
Int. J. Parasitol.
Toxoplasma gondii: dithiol-induced Ca2+ flux causes egress of parasites from the parasitophorous vacuole
Exp. Parasitol.
Antigen-induced cytokine production in lymphocytes of Eimeria bovis primary and challenge infected calves
Vet. Immunol. Immunopathol.
Establishment of a turkey cecal cell line and development of Turkey coccidia within the cells
Proc. Soc. Exp. Biol. Med.
Alternative mechanism of Eimeria bovis sporozoites to invade cells in vitro by breaching the plasma membrane
J. Parasitol.
Studies on synchronous egress of coccidian parasites (Neospora caninum, Toxoplasma gondii, Eimeria bovis) from bovine endothelial host cells mediated by calcium ionophore A23187
Vet. Res. Commun.
Inhibition of the development of Eimeria tenella in cultured bovine kidney cells by a soluble factor produced by peripheral blood lymphocytes from immune chickens
Parasitology
Ultrastructural observations of host-cell invasion by sporozoites of Eimeria papillata in vivo
Parasitol. Res.
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