Abstract
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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Thabet, A., Alnassan, A.A., Daugschies, A. et al. Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites. Parasitol Res 114, 2155–2163 (2015). https://doi.org/10.1007/s00436-015-4404-4
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DOI: https://doi.org/10.1007/s00436-015-4404-4