br Acknowledgements This study was supported by the Science
Acknowledgements This study was supported by the Science Fund for Creative Research Groups of Gansu Province (Grant No.1210RJIA006) and the National Key Project of Scientific and Technical Supporting Program (No.2007BAD40B04) and was partially supported by the National Beef Cattle and Yak Industrial Technology System, NBCITS, MOA (No.CARS-38).
Introduction Avian coccidiosis, an important parasitic disease caused by Eimeria spp., an apicomplexan protozoan parasite, is responsible for a substantial economic burden globally in the avian industry (Györke et al., 2013, Williams, 1999). As the most virulent Eimeria spp. species, has a complex lifestyle, including exogenous (sporogony) and endogenous developmental stages (schizogamy and gametogony, respectively). Although coccidian vaccines have been extensively studied as a novel strategy to provide protection against coccidiosis (Yin et al., 2014, Mansoori and Modirsanei, 2012, Song et al., 2013), these vaccines are not widely used in the poultry industry because of their poor stability. Thus, the control of coccidiosis still mainly depends on anti-coccidiosis drugs. Unfortunately, the large-scale and long-time use of anticoccidials has led to the emergence of anticoccidial resistance problems (Stephan et al., 1997, Kawazoe and Fabio, 1994, Arabkhazaeli et al., 2013). Therefore, studies have been conducted recently to search for novel therapeutic drugs and strategies against coccidiosis. Recently, a novel anti-coccidiosis triazine compound, acetamizuril, was synthesized by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; its structure is similar to those of diclazuril and toltrazuril. Previous studies have shown that this novel Aloperine mg has a high anticoccidial activity with an anticoccidial index above 180 and an acceptable safety profile (unpublished data). Enolase, a key glycolytic enzyme, is an essential cytoplasmic enzyme that catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate in the final steps of the glycolytic pathway (Yang et al., 2014). Enolases have highly conserved amino acid sequences and possess a wide range of additional functions beyond their classical role in glycolysis (Raghunathan et al., 2014, Karina et al., 2012). In E. tenella, enolase also plays a vital role in the interaction of the parasites with host cells (Labbé et al., 2006).
Materials and methods
Result and discussion qRT-PCR was utilized to explore the effect of acetamizuril on the mRNA level of enolase. The results were analyzed according to the Livak 2−ΔΔCT method (Schmittgen and Livak, 2008): the mean CT of the enolase in treated and untreated samples was 29.8 and 28.5, respectively. The mean CT of the 18S rRNA internal control in the treated and untreated samples was 8.0 and 8.3 respectively. The result of 2−ΔΔCT was about 0.3. The fold change in expression of the enolase gene reduced by 3.3-fold due to treatment. This result was graphed as Fig. 1. As shown in Fig. 1, after acetamizuril treatment, the transcription level of enolase was downregulated significantly in the acetamizuril treatment group compared with the untreatment group. The Western blot results showed that no positive band was observed (Fig. 2). At the same time, the subcellular location and spatial expression changes of enolase in mz-Ens isolated from the infected chickens treated with acetamizuril were investigated by immunofluorescence analysis using polyclonal enolase antibodies. Detection by immunofluorescence microscopy showed that the enolase immunostaining was almost undetectable in the mz-Ens of treatment group. In contrast, the considerable enolase immunostaining (green) appeared to be at the top of the second-generation merozoites of the untreatment group (Fig. 3). The results of immunofluorescence assay were somewhat consistent with the outcome of the Western blot analysis.