Increasing the Yield of Eimeria tenella Oocysts in Primary Chicken Kidney Cells

Zhang, Jianfei; Wilson, Eric; Yang, Shiguang; Healey, Mark C.

doi:10.2307/1592372

Cited by 6 publications

(4 citation statements)

References 6 publications

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“…Cell culture assays are extensively studied as alternative tests to assess the infectivity and multiplication of Eimeria spp. under controlled and standardized conditions (Schmatz et al 1986;Zhang et al 1996). In our study, there were no fundamental differences between the laboratory strain (Houghton) and field strain (T-376) in their ability to infect and develop in MDBK cells in the absence of any anticoccidial agent (Fig.…”

Section: Discussionmentioning

confidence: 59%

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites

Thabet¹,

Alnassan²,

Daugschies³

et al. 2015

Parasitol Res

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Three in vitro studies were designed to develop an assay for anticoccidial efficacy by use of laboratory (Houghton) and field (T-376) Eimeria tenella strains. In study (1), minimum inhibitory concentrations (MICs) of monensin (Mon), maduramicin (Mad), salinomycin (Sal), and lasalocid (Las) were determined that are able to inhibit more than 50% of sporozoites in host cell (Madin-Darby bovine kidney (MDBK)) penetration and more than 95% of Houghton sporozoites development to mature merozoites (treatment time 24 h) using quantitative real-time PCR (qPCR). MICs were 0.5, 2.5, 1, and 0.5 μg/ml for Mon, Mad, Sal, and Las, respectively. Applying the previous MIC on T-376 strain revealed a different sensitivity profile. Mad reduced T-376 gene copies by only 89.3% after 96 h of infection. In study (2), Houghton strain sporozoites were incubated with MIC of the different tested ionophores for 2 and 4 h, respectively; afterwards, their ability to invade MDBK cells was determined using phase-contrast microscopy and qPCR. Treatment of sporozoites with ionophores for 4 h resulted in significant inhibition of invasion compared with non-treated parasites as assessed both by microscopy as well as qPCR. Inhibition rates for Mon, Mad, Sal, and Las were 90.2, 75.0, 88.3, and 82.6% using phase-contrast microscopy and 83.9, 81.4, 85.8, and 75.4% using qPCR, respectively. T-376 sporozoite invasion into MDBK cells was reduced to 48.9% by Mad. Study (3) was conducted to determine inhibition exerted by toltrazuril (Tol). Tol at 5 μg/ml reduced reproduction of Houghton strain by 95%, whereas T-376 was only reduced by 86.5%. The presented experiments indicate that infectivity inhibition of sporozoites incubated for 4 h with anticoccidials and development inhibition after 96 h of infection by qPCR are suitable means to assess sensitivity of E. tenella strains to anticoccidials.

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Section: Discussionmentioning

confidence: 59%

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites

Thabet¹,

Alnassan²,

Daugschies³

et al. 2015

Parasitol Res

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“…However, the parasite’s life-cycle has been observed to proceed to completion, in vitro only in chick embryonic kidney cells or primary chicken kidney cells (PCKC), where sexual stages (i.e. macrogametes and microgametes) as well as oocysts have been observed after infection with sporozoites [ 7 , 12 , 22 – 25 ]. The complete life-cycle has also been achieved by in ovo infection of chorioallantoic membranes of chick embryos [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…In vitro cell cultures permit limited parasite development, often with cessation of development at the first generation of merozoites (merozoites I) [ 7 – 11 ]. Macrogametes, microgametes and oocysts have only been observed in primary cultures of chick embryonic kidney cells or chicken kidney cells (PCKC), but oocyst yields remain consistently low [ 7 , 12 ]. However, given that Eimeria spp.…”

Section: Introductionmentioning

confidence: 99%

Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development

et al. 2018

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BackgroundEimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide. The life-cycle of E. tenella is monoxenous with the chicken as the exclusive host; infection occurs in caecal epithelial cells. However, in vitro, the complete life-cycle of the parasite has only been propagated successfully in primary chicken kidney cells, which comprise undefined mixed cell populations; no cell line model has been able to consistently support the development of the sexual stages of the parasite. We therefore sought to develop a new model to study E. tenella gametogony in vitro using a recently characterised chicken cell line (CLEC-213) exhibiting an epithelial cell phenotype.MethodsCLEC-213 were infected with sporozoites from a precocious strain or with second generation merozoites (merozoites II) from wild type strains. Sexual stages of the parasite were determined both at the gene and protein levels.ResultsTo our knowledge, we show for the first time in CLEC-213, that sporozoites from a precocious strain of E. tenella were able to develop to gametes, as verified by measuring gene expression and by using antibodies to a microgamete-specific protein (EtFOA1: flagellar outer arm protein 1) and a macrogamete-specific protein (EtGAM-56), but oocysts were not observed. However, both gametes and oocysts were observed when cells were infected with merozoites II from wild type strains, demonstrating that completion of the final steps of the parasite cycle is possible in CLEC-213 cells.ConclusionThe epithelial cell line CLEC-213 constitutes a useful avian tool for studying Eimeria epithelial cell interactions and the effect of drugs on E. tenella invasion, merogony and gametogony.

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“…They reported a purification efficiency of 94% of the total excysted for both E. tenella and E. acervulina. Also, the Percoll ® gradients technique has been investigated by Dulski and Turner [37]: according to their study, it is possible to purify an extensive amount of sporocysts and sporozoites in 50 and 55-60% Percoll ® , respectively. However, both DE52 and Percoll ® rely on the use of reagents that might harm sporozoites.…”

Section: Sample Collection and Purification Of Eimeria Stagesmentioning

confidence: 99%

In Vitro Assessment of Anticoccidials: Methods and Molecules

Felici

Tugnoli²,

Piva

et al. 2021

Animals

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Avian coccidiosis is a disease causing considerable economic losses in the poultry industry. It is caused by Eimeria spp., protozoan parasites characterized by an exogenous–endogenous lifecycle. In vitro research on these pathogens is very complicated and lacks standardization. This review provides a description of the main in vitro protocols so far assessed focusing on the exogenous phase, with oocyst viability and sporulation assays, and on the endogenous phase, with invasion and developmental assays in cell cultures and in ovo. An overview of these in vitro applications to screen both old and new remedies and to understand the relative mode of action is also discussed.

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Increasing the Yield of Eimeria tenella Oocysts in Primary Chicken Kidney Cells

Cited by 6 publications

References 6 publications

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites

Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development

In Vitro Assessment of Anticoccidials: Methods and Molecules

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