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  • br Materials and methods br Result


    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. An earlier study reported that E. tenella uses Calcium Colorimetric Assay Kit glycolysis during schizogony but transitions to aerobic metabolism before merozoite release (Lal et al., 2009). While enolase participates in the glycolysis pathway, recent investigations have indicated that enolase has several functions in addition to its innate glycolytic function and plays a significant role in various physiological and pathological processes (Pancholi, 2001). Enolase is a plasminogen-binding protein and may be involved in parasite invasion (Hernández et al., 2011, Jiang et al., 2014, de la Torre et al., 2010). In this study, we also observed enolase at the apical end of mz-En, which indicates that enolase may participate in the parasite invasion process. In addtion, enolase may also be involved in the control of gene regulation, as enolase was also partially observed inside the nucleus of sporozoites and schizonts (Labbé et al., 2006). In Toxoplasma gondii, the enolases (ENO1 and ENO2) are expressed in a stage-specific manner. ENO1 and ENO2 are expressed in slowly growing bradyzoites and rapidly growing tachyzoites, respectively (Holmes et al., 2010). In Plasmodium falciparum, the enolase protein is detected at every stage. In the asexual stages, enolase is principally presented in the soluble fraction forms, whereas it is largely correlated with the particulate fraction in the sexual stages (Bhowmick et al., 2009). Nevertheless, in the present work, we could barely detect enolase in the nucleus, indicating that the expression of enolase in different parasitic stages may vary. Meanwhile, some studies have shown that enolase is a potential vaccine candidate against parasite infection, such as Ascariasis and Clonorchis sinensis (Chen et al., 2012Wang et al., 2011). In the immunoproteomic analysis of the mz-En proteins of E. tenella, enolase was detected as having a high immunogenicity (Liu et al., 2009). These findings indicated that enolase may be a potential drug target. Moreover, the anti-malarial drug mefloquine has also been reported to interfere with glycolysis in schistosomula of Schistosoma mansoni by inhibiting enolase (Manneck et al., 2012). Our data demonstrated that enolase may be a potential target of acetamizuril, a novel anti-coccidiosis triazine compound.
    Autoimmune retinopathy (AIR) is a syndrome caused by antiretinal antibodies resulting in retinal impairments., , AIR is classified into cancer-associated retinopathy (CAR), melanoma-associated retinopathy, and nonparaneoplastic AIR. Recently, benign tumor-associated AIR has also been reported. While the initial funduscopy in AIR patients often reveals no abnormal findings, there may be abnormalities on the visual field and/or electroretinography (ERG). On the other hand, the patients may experience retinal degeneration resembling retinitis pigmentosa (RP), often with cystoid macular edema or other atypical features. A diagnosis of AIR is based on the clinical features and symptoms and on the presence of antiretinal antibodies on immunoblotting or enzyme-linked immunosorbent assay, especially if the patients have antibodies against recoverin and/or α-enolase.