First description of an in vitro culture system for Eimeria ovinoidalis macromeront formation in primary host endothelial cells
Graphical abstract
Introduction
Eimeria ovinoidalis is a monoxenous apicomplexan parasite of the genus Eimeria considered one of the most pathogenic species within the sheep industry. Sheep coccidiosis due to E. ovinoidalis infections have been reported worldwide, causing severe typhlocolitis mainly in young lambs [1]. In vivo, the first-generation macromeronts of E. ovinoidalis can reach sizes of up to 300 μm within 9 days post-infection (p.i.) in host epithelial cells of the small intestine and thereby releasing > 120.000 merozoites I. Hereinafter, a rather fast second merogony occur (3 days) in host epithelial cells in the crypts of the large intestine, and resulting merozoites II then have to undergo the sexual gamogony again in the same host cells type [2].
E. ovinoidalis coccidiosis become a substantial livestock farming problem, especially with increased stocking densities and reduced availability of pasture for sheep [2]. The principal symptom observed is profuse diarrhoea related to the loss of gut absorptive capacity combined with watery and mucous faeces, followed by dehydration and consequent weight loss [2].
Currently, the global sheep population is around one billion animals, in which Europe and Central Asia share 12% of the world's ovine population. Moreover, Mediterranean basin countries have been the leaders of small ruminant production in Europe. As such, Spain rears in total 16.5 million head of sheep [3].
Unfortunately, there is no currently available in vitro system for the development of any sheep Eimeria spp. (e.g. E. ovinoidalis, E. bakuensis and E. crandalis), even though suitable in vitro systems have been successfully described for other Eimeria species [4], [5], [6], [7].
We therefore aimed to establish an in vitro culture system for E. ovinoidalis macromeront formation, using primary host endothelial- and permanent host epithelial-cells for consequent production of viable merozoites I, which could be used in future studies concerning basic research on molecular parasite–host cell interactions [4], [8].
Section snippets
Material and methods
E. ovinoidalis oocysts used for the in vitro experiments were isolated from naturally infected sheep. Briefly, faecal samples collected directly from the rectum of naturally infected lambs and ewes, from Murcia Region, Spain, were assessed with a modified McMaster technique for determination of the number of Eimeria oocysts per gram of faeces (OPG). Eimeria species identification was based on morphometric characteristics [9] after sporulation in 2% (w/v) potassium dichromate for 12 days, at room
Results
Field collected E. ovinoidalis sporulated oocysts presented an ovoid shape and were slightly flattened at the micropyle end. The oocysts consisted of two well-formed oocyst wall layers. The outer oocyst wall was smooth and colourless and the inner wall layer was yellowish. Measurements of oocysts revealed a medium length of 19.7 to 31.3 μm (24.1 ± 2.2) and a medium width of 15.4 to 23.7 μm (19.2 ± 1.6), according to the morphology of this species [9]. The achieved E. ovinoidalis sporogony varied from
Discussion
E. ovinoidalis is one of the most pathogenic species in ovine coccidiosis due to its massive replication capacity within the first merogony in host epithelial cells [15]. In this work, we accomplished to isolate an almost pure (90%) E. ovinoidalis field strain from Spain.
Until now, an appropriate ovine Eimeria in vitro culture system is still missed in the literature, despite the fact that more data are available on suitable in vitro systems for other related ruminant species with macromeront
Acknowledgments
The authors would like to thank the Murcian farmers for their collaboration in sample collection and to Brigitte Hofmann and Dr. Christin Ritter (Institute of Parasitology, JLU Giessen) for their help in cell culture maintenance and parasite excystation protocols.
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