First description of an in vitro culture system for Eimeria ovinoidalis macromeront formation in primary host endothelial cells

https://doi.org/10.1016/j.parint.2016.05.003Get rights and content

Highlights

  • Establishment of a suitable E. ovinoidalis excystation protocol.

  • In vitro first E. ovinoidalis merogony BUVEC leading to macromeront I formation.

  • Development and viable merozoites I stages of E. ovinoidalis.

Abstract

The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine coccidiosis. As one of the most pathogenic species in sheep, the principal clinical sign is profuse diarrhoea in young animals, which leads to important economic losses in the ovine industry. We here aimed to establish an in vitro culture system for the development of E. ovinoidalis macromeronts, as no suitable systems are currently available for any ovine Eimeria species. Faecal samples containing more than 90% of E. ovinoidalis oocysts were collected from naturally infected lambs and ewes in Murcia Region (Spain). E. ovinoidalis oocysts were collected, left to sporulate in potassium dichromate and stored at 4 °C until further studies were conducted. Moreover, a suitable excystation protocol was effectively established, resulting in the release of viable sporozoites, which were allowed to infect primary bovine umbilical vein endothelial cells (BUVEC) and permanent bovine colonic epithelial cells (BCEC). In vitro first merogony was successfully accomplished exclusively in BUVEC leading to macromeront formation (up to 100 μm) and the release of fully developed and viable merozoites I stages. Given that we were able to establish a suitable in vitro system for the first merogony of such pathogenic Eimeria species in sheep, advances might be further made not only on studies regarding the control of ovine coccidiosis, such as drug screenings, but also on the better understanding of molecular parasite–host cell interactions as already demonstrated for other ruminant Eimeria species.

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.

References (20)

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