ORIGINAL PAPER Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites Ahmed Thabet & Alaa Aldin Alnassan & Arwid Daugschies & Berit Bangoura Received: 15 January 2015 /Accepted: 27 February 2015 # Springer-Verlag Berlin Heidelberg 2015 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 spo- rozoites 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, respec- tively. 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 mi- croscopy 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 in- vasion 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 re- production 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 sensi- tivity of E. tenella strains to anticoccidials. Keywords Eimeria tenella . Ionophores . Toltrazuril . PCR . In vitro assay Introduction Eimeria tenella is an intracellular protozoon belonging to the family Apicomplexa. It is one of the most important chicken Eimeria species, causing cecal coccidiosis with recognizable lesions and spectacular losses in poultry industry (McDougald and Fitz-Coy 2013). Even though there are dif- ferent types of anticoccidial vaccines available, application of pharmaceuticals by feed or drinking water remains the most widely distributed method to control coccidiosis worldwide (McDougald 1982). Between the different classes of anticoccidial agents, the modes of action vary as well as the developmental stages of Eimeria that are targeted. Polyether ionophores and triazines are considered the most widely used anticoccidial agents to control chicken coccidiosis. Polyether ionophores arrest spo- rozoites by increasing intrasporozoite Na + /K + concentrations and Na + -K + -ATPase activity (Wang et al. 2006). They also act against merozoites by bursting the cell border, endoplasmic reticulum, and internal organelles (Mehlhorn et al. 1983). On the other hand, toltrazuril, a triazine member, acts upon all intracellular stages in the schizogony and gamogony cycles (Haberkorn and Stoltefuss 1987). Different application strategies are used for anticoccidial agents in poultry flocks in order to reduce the emergence of drug resistant isolates, such as shuttle or rotation programs. Nonetheless, the emergence of resistant isolates has been re- ported against almost all compounds introduced on the market (McDougald et al. 1997; Stephan et al. 1997; Mattiello et al. A. Thabet : A. A. Alnassan : A. Daugschies : B. Bangoura (*) Institute of Parasitology, Faculty of Veterinary Medicine, Centre for Infectious Diseases, University Leipzig, Leipzig, Germany e-mail: bangoura@vetmed.uni-leipzig.de Parasitol Res DOI 10.1007/s00436-015-4404-4