Efficacy of dietary supplements of Glycyrrhiza glabra (Licorice) and maduramicin alone or in combination with Eimeria tenella infected chicks: A clinical study and molecular docking.

Background: Coccidiosis is one of the most economically significant poultry diseases worldwide, caused by the pathogenic Eimeria species, and is characterized by decreased weight gain (WG) and failure to grow due to malabsorption, low feed conversion rate, bloody diarrhea, and dehydration. Aim: This study investigated the effectiveness of licorice root extract (LRE) in controlling cecal coccidiosis to determine whether its combination with maduramicin could help alleviate the pathological, biochemical, and histopathological effects of cecal coccidiosis in Sasso broiler chicks. Methods: A total of 125 one-day-old Sasso broiler chicks were categorized into five equal groups (n = 25), each consisting of five replicates (n = 5 per replicate). G1-LE received a basal diet supplemented with LRE (3 g/kg); G2-ME received a basal diet containing maduramycin (0.5 g/kg); and G3-LME received a basal diet containing LRE and maduramicin together with the same rates. G4-E (positive control) and G5-N (negative control) received no additives in their feed. Birds in groups (G1-4) were challenged on day 14 of the experiment by orally intercropping a 1 ml suspension of Eimeria tenella sporulated oocysts. Results: Groups of birds fed on LRE and maduramicin separately or together appeared to be in good condition where no deaths or clinical abnormalities were observed, based on the analysis of clinicopathological examination. Compared with the G4-E positive control, the dropping scoring and oocyst shedding of groups G1-LE, G2-ME, and G3-LME along the 10th-day post-challenge (dpc), as well as macroscopic and microscopic lesions scoring at the 7th dpc, was considerably lower. The dual supplementation use of LRE and maduramicin in G3-LME's reduced the harmful effects of coccidian, which appeared only as a mononuclear cellular infiltration and a small number of oocysts invading the intestinal glands. Molecular docking revealed that LRE and maduramicin interacted with E. tenella DNA polymerase, E. tenella apical membrane antigen 1, and microneme protein binding sites resulting in reduced E. tenella replication and invasion. Conclusion: The inclusion of LRE and maduramicin, individually or in combination, in the diet might effectively mitigate the detrimental effects of coccidiosis.

Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.

One-Step Platform for Maduramicin and Salinomycin Detection Based on Bispecific Monoclonal Antibody and Interpretation of Molecular Recognition Mechanism.

Maduramicin (MAD) and salinomycin (SAL) are the widely used poly(ether ionophore) antibiotics to control coccidiosis in animals. Due to their strong cytotoxicity, strict control over their dosage and residue in animal food is necessary. To improve the detection efficiency of the existing single-residue detection methods, a tetraploid tumor hybrid system was constructed using drug mutagenesis, and the bispecific monoclonal antibody (BsMAb) against MAD and SAL was obtained by hybridization-hybridoma technology. By optimizing the optimal working concentration of the tracer and antibody, a multiresidue fluorescence polarization immunoassay method based on BsMAb was successfully established. The whole detection process takes 10 min, and the LOD values of MAD and SAL were 4.71 and 3.49 ng·g-1, respectively. IC50 values were 6.45 and 6.24 ng·mL-1, respectively. There was no cross-reactivity with other polyether ionophore antibiotics. Finally, a breakthrough in detection was achieved: bispecific monoclonal antibody prepared by the hybridization-hybridoma technology was used to detect maduramicin and salinomycin.

V-ATPase V0 subunit activation mediates maduramicin-induced methuosis through blocking endolysosomal trafficking in vitro and in vivo.

Methuosis, a novel cell death phenotype, is characterized by accumulation of cytoplasmic vacuolization upon external stimulus. Methuosis plays a critical role in maduramicin-induced cardiotoxicity despite the underlying mechanism is largely unknown. Herein, we aimed to investigate the origin and intracellular trafficking of cytoplasmic vacuoles, as well as the molecular mechanism of methuosis caused by maduramicin (1 µg/mL) in myocardial cells. H9c2 cells and broiler chicken were used and were exposed to maduramicin at doses of 1 µg/mL in vitro and 5 ppm-30 ppm in vivo. Morphological observation and dextran-Alexa Fluor 488 tracer experiment showed that endosomal compartments swelling and excessive macropinocytosis contributed to madurdamcin-induced methuosis. Cell counting kit-8 assay and morphology indicated pharmacological inhibition of macropinocytosis largely prevent H9c2 cells from maduramicin-triggered methuosis. In addition, late endosomal marker Rab7 and lysosomal associated membrane protein 1 (LAMP1) increased in a time-dependent manner after maduramicin treatment, and the recycling endosome marker Rab11 and ADP-ribosylation factor 6 (Arf6) were decreased by maduramicin. Vacuolar-H+-ATPase (V-ATPase) was activated by maduramicin, and pharmacological inhibition and genetic knockdown V0 subunit of V-ATPase restore endosomal-lysosomal trafficking and prevent H9c2 cells methuosis. Animal experiment showed that severe cardiac injury included the increase of creatine kinase (CK) and creatine kinase-MB (CK-MB), and vacuolar degeneration resembled methuosis in vivo after maduramicin treatment. Taken together, these findings demonstrate that targeting the inhibition of V-ATPase V0 subunit will prevent myocardial cells methuosis by restoring endosomal-lysosomal trafficking.

Molecular characterization and analysis of drug resistance-associated protein enolase 2 of Eimeria tenella.

Eimeria tenella, an intestinal parasite, has brought huge economic losses to the poultry industry. The prevalence and severity of the development of drug resistance has increased the challenge of coccidiosis control. We previously identified the enolase 2 of E. tenella (EtENO2) was differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In this study, the expression of EtENO2 in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains was analyzed by quantitative real-time PCR (qRT-PCR) and western blots. EtENO2 was highly expressed in several drug-resistant strains compared with the DS strain. The qRT-PCR showed that the transcription level of EtENO2 in the field-isolated resistant strains was upregulated compared with the DS strain. The enzyme activity results indicated that the catalytic activity of EtENO2 in the drug-resistant strains was higher than in the DS strain. In addition, qRT-PCR and western blots showed that the expression level of EtENO2 was higher in second generation merozoites (SM) and unsporulated oocysts (UO) than that in sporozoites (SZ) and sporulated oocysts (SO). Immunofluorescence localization revealed that EtENO2 was distributed throughout SZ and SM and on the surface of the parasites. After the SZ invasion DF-1 cells, it was also observed on the parasitophorous vacuole membrane. Our secretion experiments found that EtENO2 could be secreted outside the SZ. This study indicated that EtENO2 might be related to the interaction between E. tenella and host cells and be involved in the development of E. tenella resistance to some anticoccidial drugs.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion.

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.

Optimization of Maduramicin Ammonium-Loaded Nanostructured Lipid Carriers Using Box-Behnken Design for Enhanced Anticoccidial Effect against Eimeria tenella in Broiler Chickens.

Maduramicin ammonium (MAD) is one of the most frequently used anticoccidial agents in broiler chickens. However, the high toxicity and low solubility of MAD limit its clinical application. In this study, MAD-loaded nanostructured lipid carriers (MAD-NLCs) were prepared to overcome the defects of MAD by using highly soluble nanostructured lipid carriers (NLCs). The formulation was optimized via a three-level, three-factor Box-Behnken response surface method. Then, the optimal MAD-NLCs were evaluated according to their hydrodynamic diameter (HD), zeta potential (ZP), crystal structure, encapsulation efficiency (EE), drug loading (DL), in vitro release, and anticoccidial effect. The optimal MAD-NLCs had an HD of 153.6 ± 3.044 nm and a ZP of -41.4 ± 1.10 mV. The X-ray diffraction and Fourier-transform infrared spectroscopy results indicated that the MAD was encapsulated in the NLCs in an amorphous state. The EE and DL were 90.49 ± 1.05% and 2.34 ± 0.04%, respectively, which indicated that the MAD was efficiently encapsulated in the NLCs. In the in vitro study, the MAD-NLCs demonstrated a slow and sustained drug release behavior. Notably, MAD-NLCs had an excellent anticoccidial effect against Eimeria tenella in broiler chickens. In summary, MAD-NLCs have huge potential to form a new preparation administered via drinking water with a powerful anticoccidial effect.

Comparative effect of potentiated zinc oxide and antibiotic growth promoters on intestinal morphometry and nutrient digestibility in broiler chickens.

The comparative effects of potentiated zinc oxide (pZnO) and antibiotic growth promoters (AGP) supplementation on intestinal morphometry and nutrient digestibility in broiler chickens were studied. Four hundred straight-run Cobb 500-day-old broiler chicks were randomly allotted to four dietary treatments replicated 10 times with 10 birds per replicate. Dietary treatments were as follows: T1: basal diets without AGP (negative control; NC), T2: basal diets with 500 g/t maduramicin 10 g and 500 g/t zinc bacitracin 150 (positive control; PC), T3: NC added with 150 g/t pZnO, and T4: PC added with 150 g/t pZnO in a 2 × 2 factorial design in RCBD. At days 18 and 35, 10 birds were randomly selected per treatment for morphometry of the duodenum, jejunum, and ileum. At day 38, eight birds per treatment were used for the nutrient digestibility study. Results showed significant interaction effects (P < 0.05) of AGP and pZnO supplementation on day 35 intestinal morphometry of duodenum's villi height and villi height: crypt depth, and ileum's crypt depth; apparent CODGE, AME, CP, DM, and EE. Significant differences (P < 0.05) with pZnO supplementation were only observed on feed intake and FCR of birds fed with pZnO at days 8-14 and fecal quality at days 0-7. Results of present study suggested that pZnO has the potential to replace AGPs without negatively affecting the intestinal morphometry, digestibility, and growth performance of broiler chickens.

Maduramicin arrests myocardial cells at G0/G1 phase of the cell cycle through inhibiting AKT-Cyclin D1 signaling.

Maduramicin, a polyether ionophore antibiotic used as an anticoccidial feed additive in poultry, is toxic to animals and humans and can cause heart failure. The present study was initiated to explore the underlying mechanism of toxicity in H9c2 myocardial cells. We observed using cell imaging and counting methods that maduramicin inhibited cell growth in a concentration-dependent manner. Furthermore, MTT assays showed that maduramicin inhibited cell proliferation in a concentration- and time-dependent manner, and was also confirmed by the finding that maduramicin time dependently blocked the incorporation of BrdU into DNA in H9c2 myocardial cells. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle and concurrently, there was down regulation of expression of Cyclin D1. In addition, exposure to maduramicin pruned phosphorylation of AKT at both T308 and S473 sites. Finally, we found that pre-treatment of H9c2 myocardial cells with AKT activator SC79, attenuated the inhibitory effects of maduramicin on Cyclin D1 expression and cell proliferation. Collectively, our results suggest that maduramicin-suppressed AKT-Cyclin D1 signaling which results in G0/G1 phase cell cycle arrest, leading to the inhibition of myocardial cell proliferation.

Maduramicin induces cardiotoxicity via Rac1 signaling-independent methuosis in H9c2 cells.

Maduramicin frequently induces severe cardiotoxicity in target and nontarget animals in clinic. Apoptotic and non-apoptotic cell death mediate its cardiotoxicity; however, the underlying non-apoptotic cell death induced by maduramicin remains unclear. In current study, a recently described non-apoptotic cell death "methuosis" caused by maduramicin was defined in mammalian cells. Rat myocardial cell H9c2 was used as an in vitro model, showing excessively cytoplasmic vacuolization upon maduramicin (0.0625-5 µg/mL) exposure for 24 h. Maduramicin-induced reversible cytoplasmic vacuolization of H9c2 cells in a time- and concentration-dependent manner. The vacuoles induced by maduramicin were phase lucent with single membrane and were not derived from the swelling of organelles such as mitochondria, endoplasmic reticulum, lysosome, and Golgi apparatus. Furthermore, maduramicin-induced cytoplasmic vacuoles are generated from micropinocytosis, which was demonstrated by internalization of extracellular fluid-phase marker Dextran-Alexa Fluor 488 into H9c2 cells. Intriguingly, these cytoplasmic vacuoles acquired some characteristics of late endosomes and lysosomes rather than early endosomes and autophagosomes. Vacuolar H+ -ATPase inhibitor bafilomycin A1 efficiently prevented the generation of cytoplasmic vacuoles and decreased the cytotoxicity of H9c2 cells triggered by maduramicin. Mechanism studying indicated that maduramicin activated H-Ras-Rac1 signaling pathway at both mRNA and protein levels. However, the pharmacological inhibition and siRNA knockdown of Rac1 rescued maduramicin-induced cytotoxicity of H9c2 cells but did not alleviate cytoplasmic vacuolization. Based on these findings, maduramicin induces methuosis in H9c2 cells via Rac-1 signaling-independent seriously cytoplasmic vacuolization.

Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method to Determine Maduramicin in Crayfish (Procambarus clarkii) and Evaluate Food Safety.

Maduramicin (MAD) is widely introduced into aquatic environments and results in the contamination of fish products. Worryingly, the consumption of MAD-contaminated crayfish (Procambarus clarkii) may induce symptoms of Haff disease. In this study, to monitor this potential contamination and to understand the residue and elimination characteristics of MAD in edible tissues of crayfish, a sensitive and efficient ultra-performance liquid chromatography-tandem mass spectrometry method was developed, validated, and applied. After extraction with acetonitrile and purification by solid-phase extraction column, multiple-reaction monitoring mass spectrometry with positive ionization mode was used to determine MAD's residues. The limits of detection and of quantification were 6 µg·kg-1 and 20 µg·kg-1, respectively. The fortified recoveries ranged from 74.2% to 110.4%, with relative standard deviation of 1.2% to 10.1%. Furthermore, MAD was completely eliminated after 3 and 5 days from abdominal muscle and hepatopancreas tissues of crayfish, respectively. The maximum residue limits (MRLs) of MAD respectively was 200 µg·kg-1 in muscle and 600 µg·kg-1 in the hepatopancreas, and its withdrawal time in both edible tissues was 25.8 °C·d. Collectively, the results of this study indicate the proposed method is an efficient tool to evaluate the public health risk associated with crayfish consumption.

Effect of maduramicin on crayfish (Procambius clarkii): Hematological parameters, oxidative stress, histopathological changes and stress response.

Maduramicin, an extensively used anticoccidial drug, has been introduced into environment due to poorly absorbed in the intestine of broiler chicken. To understand the potential ecological toxicity of maduramicin on aquatic organisms, acute and subacute toxicity, hemolymph biochemistry, histopathology and the expressions of drug metabolism and stress response genes of crayfish (Procambius clarkii) were investigated in this study. For the first time, the 96 h median lethal concentration (LC50) of maduramicin on crayfish was 67.03 mgL-1 with a 95% confidence interval (54.06-81.32 mgL-1). Then, the crayfish were exposed to 0.7 mgL-1 (1/100 LC50), 3.5 mgL-1 (1/20 LC50) and 7.0 mgL-1 (1/10 LC50) maduramicin for 28 days. Maduramicin significantly altered biochemical parameters including AST, ALT, CK, LDH and ALP of hemolymph in crayfish at several time points. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of crayfish gills, hepatopancreas and abdominal muscle were significantly decreased or elevated by different concentrations of maduramicin treatment at varying time points. Furthermore, histopathological damage of crayfish gills, hepatopancreas and abdominal muscle were observed in a concentration-dependent manner. The expressions of metabolic and stress response genes (CYP450, GST, COX1, COX2, HSP70 and MT) in hepatopancreas of crayfish were significantly up-regulated by maduramicin (7.0 mgL-1) treatment for 8 h to 7 d, and returned to normal levels after the removal of maduramicin for 3-7 days. In conclusion, our findings demonstrated that environmental exposure of maduramicin threaten to the health of crayfish living in the areas nearby livestock farms or pharmaceutical factory. Crayfish exhibited resistance to the stress of maduramicin via activating drug metabolite and detoxification pathways.

Green one-pot synthesis of nitrogen and sulfur co-doped carbon quantum dots as new fluorescent nanosensors for determination of salinomycin and maduramicin in food samples.

A simple green hydrothermal method was proposed for synthesis of highly fluorescent nitrogen and sulfur co-doped carbon quantum dots (N,S-CQDs) using citric acid and thiosemicarbazide. The produced N,S-CQDs were subjected to extensive spectroscopic characterization and applied as fluorescent nanosensors for the sensitive spectrofluorimetric determination of salinomycin and maduramicin directly without prior derivatization for the first time. The obtained N,S-CQDs showed strong emission band at 430 nm after excitation at 360 nm. The native fluorescence of N,S-CQDs was found to be quenched by the addition of increased concentrations of each drug. Method validation revealed a wide linear relationship between the fluorescence quenching of N,S-CQDs and the concentration of each drug in the range of 10.0-300.0 µM with detection limits of 2.07 µM and 1.34 µM for salinomycin and maduramicin, respectively. The developed method has been efficiently applied for estimation of analytes in six raw matrices with high recoveries.

Ionophore Toxin Maduramicin Produces Haff Disease-Like Rhabdomyolysis in a Mouse Model.

Maduramicin is a toxic ionophore antibiotic that is isolated from Streptomyces, frequently occurring in an aquatic environment. To understand the potential role of maduramicin in crayfish consumption related Haff disease, a mouse model was established in this study. Two exposure routes of maduramicin in the abdominal muscle and the hepatopancreas tissue homogenates of crayfish were given intragastrically to mice in different doses for seven days. Action changes, clinical symptoms, feed consumption, body weight, blood biochemistry, and histopathology examination of mice were observed and analyzed. In the natural exposure group, relatively low concentration of maduramicin in crayfish muscle and hepatopancreas had no obvious effects on mental state, body weight, blood biochemical indexes, or histologic appearance. However, in the artificial exposure group, with increasing concentrations, maduramicin in crayfish muscle and hepatopancreas homogenates both induced mental sluggishness and weight loss of mice. Blood biochemical examination showed that 3.5 mg·kg-1 and 7 mg·kg-1 maduramicin in crayfish tissue homogenates significantly increased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and creatine kinase (CK). Additionally, histopathological examination showed that multiple organs were damaged by maduramicin, including degeneration of liver cells, shedding of renal epithelial cells, and disturbance and partial lysis of myocardial and skeletal muscle filaments in the mice. In summary, maduramicin may not cause Haff disease through contamination of the aquatic environment under normal conditions. Maduramicin can be used as a potential toxin tool to establish a rhabdomyolysis disease animal model for drug development.

Efficacy of Cygro® 10G (maduramicin ammonium-α) for turkeys.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the efficacy of Cygro® 10G. The active substance of Cygro® 10G is the polyether ionophore maduramicin ammonium-α, a coccidiostat intended to be used in feed for turkeys for fattening. In a former opinion, the FEEDAP Panel concluded that the efficacy of Cygro® 10G in turkeys for fattening had not been sufficiently demonstrated. In the present submission, new efficacy studies have been provided by the applicant. A positive effect of Cygro® 10G in preventing coccidiosis in turkeys was shown in three anticoccidial sensitivity tests (ASTs). However, owing to the lack of floor pen studies showing a positive effect, the FEEDAP Panel is not in the position to conclude on the efficacy of Cygro® 10G for turkeys for fattening.

Maduramicin triggers methuosis-like cell death in primary chicken myocardial cells.

Maduramicin frequently induces severe cardiotoxicity in broiler chickens as well as in humans who consume maduramicin accidentally. Apoptosis and non-apoptotic cell death occur concurrently in the process of maduramicin-induced cardiotoxicity; however, the underlying mechanism of non-apoptotic cell death is largely unknown. Here, we report the relationship between maduramicin-caused cytoplasmic vacuolization and methuosis-like cell death as well as the underlying mechanism in primary chicken myocardial cells. Maduramicin induced a significant increase of cytoplasmic vacuoles with a degree of cell specificity in primary chicken embryo fibroblasts and chicken hepatoma cells (LMH), along with a decrease of ATP and an increase of LDH. The accumulated vacuoles were partly derived from cellular endocytosis rather than the swelling of endoplasm reticulum, lysosomes, and mitochondria. Moreover, the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) did not prevent maduramicin-induced cytoplasmic vacuolization. DNA ladder and cleavage of PARP were not observed in chicken myocardial cells during maduramicin exposure. Pretreatment with 3-methyladenine (3-MA) and cholorquine (CQ) of chicken myocardial cells did not attenuate cytoplasmic vacuolization and cytotoxicity, although LC3 and p62 were activated. Bafilomycin A1 almost completely prevented the generation of cytoplasmic vacuoles and significantly attenuated cytotoxicity induced by maduramicin, along with downregulation of K-Ras and upregulation of Rac1. Taken together, "methuosis" due to excessive cytoplasmic vacuolization mediates the cardiotoxicity of maduramicin. This provides new insights for understanding a nonclassical form of cell death in the field of drug-induced cytotoxicity.

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella.

Avian coccidiosis is a widespread and economically significant poultry disease caused by several Eimeria species, including Eimeria tenella. Previously, E. tenella serine/threonine protein phosphatase (EtSTP) was found to be differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In the present study, we found that transcription and translation levels of EtSTP were higher in diclazuril-resistant (DZR) strains and maduramicin-resistant (MRR) strains than in DS strains using quantitative real-time PCR (qPCR) and Western blotting. Enzyme activity results indicated that the catalytic activity of EtSTP was higher in the two drug-resistant strains than in DS strains. Western blot and qPCR analysis also showed that expression levels of EtSTP were higher in unsporulated oocysts (UO) and second-generation merozoites (SM). Indirect immunofluorescence localization showed that EtSTP was located in most areas of the parasite with the exception of refractile bodies, and fluorescence intensity was enhanced during development. In vitro inhibition experiments showed that the ability of sporozoites (SZ) to invade cells was significantly decreased after treatment with anti-rEtSTP antibody. These results indicated that EtSTP acted mainly during the developmental and reproductive stages of the parasite and may be related to the resistance of coccidia to external drug pressure.

Validated spectrophotometric determination of maduramicin ammonium using three charge transfer complexation reactions.

Direct spectrophotometric determination of Maduramicin ammonium (MAD) represents an analytical challenge since it is a weak UV-absorbing and lacking a strong chromophore. This work represents the first spectrophotometric determination of MAD as no direct spectrophotometric or colorimetric determination methods for MAD are available in the literature. The present study illustrates the development of three simple, rapid and inexpensive colorimetric methods for the routine quality control analysis of MAD based on the formation of colored charge transfer complexes with three electron acceptors namely p-chloranilic acid (p-CA), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and picric acid (PA). The color products of MAD with p-CA, DDQ and PA were measured at 519, 588 and 405nm respectively. The proposed methods were validated in terms of linearity, ranges, precision, accuracy, robustness and limits of detection and quantification. MAD was effectively determined over concentration ranges of 100-1000, 25-250 and 30-150µg/mL using p-CA, DDQ and PA, respectively with good linearity as shown by the values of correlation coefficients not less than 0.9991. The developed methods were successfully implemented in the assay of MAD powder pharmaceutical formulation for veterinary use.

Comparative Transcriptome Analyses of Drug-sensitive and Drug-resistant Strains of Eimeria tenella by RNA-sequencing.

Avian coccidiosis is a widespread and economically significant disease in poultry. At present, treatment of coccidiosis mainly relies on drugs. Anticoccidial drugs can be divided into two categories: ionophorous compounds and synthetic drugs. However, the emergence of drug-resistant strains has become a challenge for coccidiosis control with anticoccidial drugs. To gain insights into the molecular mechanism governing the drug resistance of Eimeria tenella, two drug-resistant strains of E. tenella, one maduramicin-resistant (MRR) strain and one diclazuril-resistant (DZR) strain, were generated. We carried out comparative transcriptome analyses of a drug-sensitive strain (DS) and two drug-resistant MRR and DZR strains of E. tenella using RNA-sequencing. A total of 1,070 differentially expressed genes (DEGs), 672 upregulated and 398 downregulated, were identified in MRR vs. DS, and 379 DEGs, 330 upregulated and 49 downregulated, were detected in DZR vs. DS. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to better understand the functions of these DEGs. In the comparison of DZR vs. DS, some DEGs were involved in peroxisome, biosynthesis of unsaturated fatty acids, and fatty acid metabolism. In the comparison of MRR vs. DS, some DEGs were involved in glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and DNA replication. In addition, some DEGs coded for surface antigens that were downregulated in two drug-resistant strains involved invasion, pathogenesis, and host-parasite interactions. These results provided suggestions for further research toward unraveling the molecular mechanisms of drug resistance in Eimeria species and contribute to developing rapid molecular methods to detect resistance to these drugs in Eimeria species in poultry.