title: Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites.
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.
authors.affiliation: Institute of Parasitology, Faculty of Veterinary Medicine, Centre for Infectious Diseases, University Leipzig, Leipzig, Germany.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Thabet
authors.forename: Alaa Aldin
authors.initials: AA
authors.lastname: Alnassan
authors.forename: Arwid
authors.initials: A
authors.lastname: Daugschies
authors.forename: Berit
authors.initials: B
authors.lastname: Bangoura
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003049:Coccidiostats / Q000494:pharmacology*; D016786:Eimeria tenella / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects; D007668:Kidney / Q000166:cytology; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D034101:Sporozoites / Q000187:drug effects*
chemical_list: D003049:Coccidiostats