Inclusion Complexes of Ethanamizuril with ß- and Hydroxypropyl-ß-Cyclodextrin in Aqueous Solution and in Solid State: A Comparison Study.

Ethanamizuril (EZL) is a new anticoccidial drug developed by our Shanghai Veterinary Research Institute. Since EZL is almost insoluble in water, we conducted a study to improve the solubility of EZL by forming inclusion complexes with ß-cyclodextrin (ß-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD). In this study, we performed molecular docking and then systematically compared the interactions of EZL with ß-CD and HP-ß-CD in both aqueous solution and the solid state, aiming to elucidate the solubilization effect and mechanism of cyclodextrins (CDs). The interactions were also examined in the solid state using DSC, PXRD, and FT-IR. The interactions of EZL with CDs in an aqueous solution were investigated using PSA, UV-vis spectroscopy, MS, 1H NMR, and 2D ROESY. The results of phase solubility experiments revealed that both ß-CD and HP-ß-CD formed inclusion complexes with EZL in a 1:1 molar ratio. Among them, HP-ß-CD exhibited higher Kf (stability constant) and CE (complexation efficiency) values as well as a stronger solubilization effect. Furthermore, the two cyclodextrins were found to interact with EZL in a similar manner. The results of our FT-IR and 2D ROESY experiments are in agreement with the theoretical results derived from molecular simulations. These results indicated that intermolecular hydrogen bonds existing between the C=O group on the triazine ring of EZL and the O-H group of CDs, as well as the hydrophobic interactions between the hydrogen on the benzene ring of EZL and the hydrogen of CDs, played crucial roles in the formation of EZL/CD inclusion complexes. The results of this study can lay the foundation for the future development of high-concentration drinking water delivery formulations for EZL.

Recapitulating familial hypercholesterolemia in a mouse model by knock-in patient-specific LDLR mutation.

Familial hypercholesterolemia (FH) is one of the most prevalent monogenetic disorders leading to cardiovascular disease (CVD) worldwide. Mutations in Ldlr, encoding a membrane-spanning protein, account for the majority of FH cases. No effective and safe clinical treatments are available for FH. Adenine base editor (ABE)-mediated molecular therapy is a promising therapeutic strategy to treat genetic diseases caused by point mutations, with evidence of successful treatment in mouse disease models. However, due to the differences in the genomes between mice and humans, ABE with specific sgRNA, a key gene correction component, cannot be directly used to treat FH patients. Thus, we generated a knock-in mouse model harboring the partial patient-specific fragment and including the Ldlr W490X mutation. LdlrW490X/W490X mice recapitulated cholesterol metabolic disorder and clinical manifestations of atherosclerosis associated with FH patients, including high plasma low-density lipoprotein cholesterol levels and lipid deposition in aortic vessels. Additionally, we showed that the mutant Ldlr gene could be repaired using ABE with the cellular model. Taken together, these results pave the way for ABE-mediated molecular therapy for FH.

Engineered Peptides Harboring Cation Motifs Against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) pathogens pose a serious threat to the health and life of humans, necessitating the development of new antimicrobial agents. Herein, we develop and characterize a panel of nine amino acid peptides with a cation end motif. Bioactivity analysis revealed that the short peptide containing "RWWWR" as a central motif harboring mirror structure "KXR" unit displayed not only high activity against MDR planktonic bacteria but also a clearance rate of 92.33% ± 0.58% against mature biofilm. Mechanically, the target peptide (KLR) killed pathogens by excessively accumulating reactive oxygen species and physically disrupting membranes, thereby enhancing its robustness for controlling drug resistance. In the animal model of sepsis infection by MDR bacteria, the peptide KLR exhibited strong therapeutic effects. Collectively, this study provided the dominant structure of short antimicrobial peptides (AMPs) to replenish our arsenals for combating bacterial infections and illustrated what could be harnessed as a new agent for fighting MDR bacteria.

Engineered Staphylococcus auricularis Cas9 with high-fidelity.

CRISPR-Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off-target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5'-NNGG-3' protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced-fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5'-NNGG-3' than SaCas9 of 5'-NNGRRT-3' PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9-HF1, harboring the N269D mutation; SauriCas9-HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE-seq. At certain sites, reduced off-target effects (approximately 61.6- and 111.9-fold improvements) of SauriCas9-HF2 compared with wild-type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9-HF1 and SauriCas9-HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications.

Peptide Supramolecular Hydrogels with Sustained Release Ability for Combating Multidrug-Resistant Bacteria.

Chronic wound infection caused by multidrug-resistant bacteria is a major threat globally, leading to high mortality rates and a considerable economic burden. To address it, an innovative supramolecular nanofiber hydrogel (Hydrogel-RL) harboring antimicrobial peptides was developed based on the novel arginine end-tagging peptide (Pep 6) from our recent study, triggering cross-linking. In vitro results demonstrated that Hydrogel-RL can sustain the release of Pep 6 up to 120 h profiles, which is biocompatible and exhibits superior activity for methicillin-resistant Staphylococcus aureus (MRSA) biofilm inhibition and elimination. A single treatment of supramolecular Hydrogel-RL on an MRSA skin infection model revealed formidable antimicrobial activity and therapeutic effects in vivo. In the chronic wound infection model, Hydrogel-RL promoted mouse skin cell proliferation, reduced inflammation, accelerated re-epithelialization, and regulated muscle and collagen fiber formation, rapidly healing full-thickness skin wounds. To show its vehicle property for wound infection combined therapy, etamsylate, an antihemorrhagic drug, was loaded into the porous network of Hydrogel-RL, which demonstrated improved hemostatic activity. Collectively, Hydrogel-RL is a promising clinical candidate agent for functional supramolecular biomaterials designed for combating multidrug-resistant bacteria and rescuing stalled healing in chronic wound infections.

Praziquantel promotes protection against Schistosoma japonicum infection in mice.

Praziquantel (PZQ) is the first line drug for the treatment of schistosomiasis. Several studies have confirmed that PZQ regulates host immunity, and we have recently found that pretreatment with PZQ enhances resistance against Schistosoma japonicum infection in buffaloes. We speculate that PZQ induces physiological changes in mice that prevent S. japonicum infection. To test this hypothesis and provide a practical measure to prevent S. japonicum infection, we determined the effective dose (the minimum dose), protection period and onset time of protection by comparing the worm burden, female worm burden and egg burden in PZQ-pretreated mice and blank control mice. Morphological differences between parasites were observed by measuring the total worm length, oral sucker, ventral sucker and ovary. The levels of cytokines, nitrogen monoxide (NO), 5-hydroxytryptamine (5-HT) and specific antibodies were measured using kits or soluble worm antigens. Hematological indicators on day 0 were analyzed in mice that received PZQ on days -15, -18, -19, -20, -21 and -22. The PZQ concentrations in plasma and blood cells were monitored using high performance liquid chromatography (HPLC). The effective dose was found to be two oral administrations (interval of 24 h) at 300 mg/kg body weight (BW) or one injection at 200 mg/kg BW, and the protection period of PZQ injection was 18 days. The optimal preventive effect was observed at two days post-administration, with a >92% worm reduction rate and significant worm reduction until 21 days after administration. Adult worms from PZQ-pretreated mice were runtish showing a shorter length, smaller organs and fewer eggs in the uteri of females. Detection of cytokines, NO, 5-HT and hematological indicators showed that PZQ induced immune-physiological changes, including higher levels of NO, IFN-γ and IL-2, and a lower level of TGF-ß. No significant difference in the anti-S. japonicum specific antibody levels was observed. The PZQ concentrations in plasma and blood cells 8 and 15 days post-administration were lower than the detection limit. Our results confirmed that pretreatment with PZQ promotes the protection of mice against S. japonicum infection within 18 days. Although we observed some immune-physiological changes in the PZQ-pretreated mice, the exact mechanisms involved in the preventive effect require further study.

RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples.

Chicken coccidiosis is one of the most common and economically important diseases in the global poultry industry, and it is caused by at least one of the seven Eimeria species. A simple and reliable way to distinguish Eimeria species in infected chicken is critical for the surveillance, control, and eradication of chicken coccidiosis. In this study, a recombinase polymerase amplification (RPA) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system (RPA-CRISPR/Cas12a) was developed for the detection of Eimeria species in chicken fecal samples. This assay is highly specific to the seven Eimeria species and it does not cross react between species. Assessment of analytical sensitivity revealed that a single copy of plasmid DNA could be detected. Comparative analysis revealed strong agreement between RPA-CRISPR/Cas12a assays and real-time qPCR to reliably detect all seven Eimeria species in fecal chicken samples. Importantly, the cleavage products could be visualized under a blue light instrument, making it possible for the rapid detection of Eimeria species for on-site testing. Collectively, our study demonstrated that RPA-CRISPR/Cas12a assays offer a simple and reliable diagnostic method for Eimeria species.

Effects of ethanamizuril, sulfachlorpyridazine or their combination on cecum microbial community and metabolomics in chickens infected with Eimeria tenella.

Coccidiostat and antibiotics are widely used in poultry industry, but their effects on cecum microbial community and metabolomics in chickens infected with coccidia have been rarely studied. In this study, we analyzed the changes of microbiota and metabolomic which associated with Eimeria tenella infection in 8 days of age chickens in the presence or absence of ethanamizuril, sulfachlorpyridazine or their combinations treatment for 3 consecutive days. 16S rRNA gene sequencing and LC-MS/MS analyses were used to profile the cecal microbiome and metabolome in each group of chickens on 7 days post-infection. The results showed that coccidial infection induced significant perturbations in the distribution of microbial taxonomy and the metabolism of physiological functional molecules in cecal contents. Ethanamizuril treatment seemed to transform microbiota into a steady state conducive to animal health, and sulfachlorpyridazine treatment alleviated the growth of potentially harmful bacteria such as Escherichia-Shigella. The change trends of metabolites such as n-carbamoylglutamic acid were consistent with the anticoccidial effect of ethanamizuril. The combinations of ethanamizuril and sulfachlorpyridazine at low-dose had little effect on gut microbiota, metabolism and anticoccidial effect. These data indicate that the cecal microbiota and metabolic status of chickens infected with E. tenella following ethanamizuril treatment could be used to monitor the response to drug efficacy. This study provides a new system approach to elucidate the microbiota, metabolic and therapeutic effects of the combination of coccidiostat and antibiotics in the context of avian coccidiosis.

Label-free quantitative proteomic analysis of ethanamizuril-resistant versus -sensitive strains of Eimeria tenella.

BACKGROUND: Avian coccidiosis is an important parasitic disease that has serious adverse effects on the global poultry industry. The extensive use of anticoccidial drugs has resulted in an increase in drug resistance. Ethanamizuril (EZL) is a novel triazine with high anticoccidial activity. METHODS: We compared oocyst production and sporulation between EZL-sensitive (S) and EZL-resistant Eimeria tenella strains (R10 and R200) and used label-free quantitative proteomics to identify differentially expressed proteins (DEPs) between these strains. RESULTS: We generated two EZL-resistant E. tenella strains: strain R10, which was induced using a constant dose of 10 mg EZL/kg poultry feed, and strain R200, which was generated by gradually increasing the EZL dosage to 200 mg EZL/kg poultry feed. With an increase in resistance, the total oocyst output decreased, but the percentage of sporulation did not change significantly. We identified a total of 7511 peptides and 1282 proteins, and found 152 DEPs in the R10 strain versus the S strain, 426 DEPs in the R200 strain versus the S strain and 494 DEPs in the R200 strain versus the R10 strain. When compared with the S strain, 86 DEPs were found to have consistent trends in both resistant strains. The DEPs were primarily involved in ATP and GTP binding, invasion, and membrane components. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the DEPs suggested that they are involved in transcription and translation processes. Protein-protein interaction network analysis of the 86 DEPs showed that 10 proteins were hubs in the functional interaction network (≥ 8 edges) and five of them were ribosomal proteins. CONCLUSIONS: The results of the present study indicate that the resistance mechanisms of E. tenella against EZL might be related to the transcriptional and translational processes, especially in the factors that inhibit the growth of parasites. The DEPs found in this study provide new insights into the resistance mechanisms of E. tenella against EZL. Further research on these potential targets holds promise for new chemotherapeutic approaches for controlling E. tenella infections.

Niclosamide as a repurposing drug against Gram-positive bacterial infections.

OBJECTIVES: Niclosamide is commonly used as an antiparasitic drug in veterinary clinics. The objectives of this study were to evaluate the efficacy of niclosamide against resistant Gram-positive bacteria in vitro and in an in vivo experimental model of topical bacterial infection. Moreover, to study the antibacterial mechanism of niclosamide to Staphylococcus aureus. METHODS: A mouse topical infection model was established to detect the antibacterial activity of niclosamide in vivo. The antimicrobial mechanism was probed by visualizing the bacterial morphologies using scanning electron microscopy and transmission electron microscopy. Moreover, the haemolytic assay and western blotting analysis were performed to evaluate whether niclosamide could inhibit the secretion of alpha-haemolysin (α-HL) from S. aureus. RESULTS: The MICs of niclosamide were below 0.5 mg/L for Gram-positive bacteria, showing excellent antibacterial activity in vitro. The in vivo antibacterial activity results indicated that niclosamide treatment at 10 mg/kg of body weight caused a significant reduction in the abscess area and the number of S. aureus cells. Moreover, the antibacterial mechanism of niclosamide showed that the surface morphology of S. aureus displayed noticeable shrinkage, with an increasing number of small vacuole-like structures observed as the drug concentration increased. Intracellular ATP levels were found to decrease in a niclosamide dose-dependent manner. Haemolysis and western blotting analyses revealed that niclosamide inhibited the haemolytic activity of S. aureus by inhibiting α-HL expression under subinhibitory concentration conditions. CONCLUSIONS: Niclosamide has significant potential for development into drugs that prevent and treat diseases caused by Gram-positive bacteria such as Staphylococcus and Streptococcus.

Effects of salinomycin and ethanamizuril on the three microbial communities in vivo and in vitro.

The fate of a drug is not only the process of drug metabolism in vivo and in vitro but also the homeostasis of drug-exposed microbial communities may be disturbed. Anticoccidial drugs are widely used to combat the detrimental effects of protozoan parasites in the poultry industry. Salinomycin and ethanamizuril belong to two different classes of anticoccidial drugs. The effect of salinomycin and ethanamizuril on the microbiota of cecal content, manure compost, and soil remains unknown. Our results showed that although both salinomycin and ethanamizuril treatments suppressed some opportunistic pathogens, they failed to repair the great changes in chicken cecal microbial compositions caused by coccidia infection. Subsequently, the metabolite5 profiling of cecal content by LC-MS/MS analyses confirmed the great impact of coccidia infection on chicken cecum and showed that histidine metabolism may be the main action pathway of salinomycin, and aminoacyl tRNA biosynthesis may be the major regulatory mechanism of ethanamizuril. The microbial community of manure compost showed a mild response to ethanamizuril treatment, but ethanamizuril in soil could promote Actinobacteria reproduction, which may inhibit other taxonomic bacteria. When the soil and manure were exposed to salinomycin, the Proteobacteria abundance of microbial communities showed a significant increase, which suggested that salinomycin may improve the ability of the microbiota to utilize carbon sources. This hypothesis was confirmed by a BIOLOG ECO microplate analysis. In the animal model of coccidia infection, the treatment of salinomycin and ethanamizuril may reconstruct a new equilibrium of the intestinal microbiota. In an in vitro environment, the effect of ethanamizuril on composting and soil microbiota seems to be slight. However, salinomycin has a great impact on the microbial communities of manure composting and soil. In particular, the promoting effect of salinomycin on Proteobacteria phylum should be further concerned. In general, salinomycin and ethanamizuril have diverse effects on various microbial communities.

Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips.

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella.

Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as "FnCpf1") protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase-thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

Pharmacokinetics and anticoccidial activity of ethanamizuril in broiler chickens.

The treatment effect of ethanamizuril (EZL) to broiler chickens experimentally infected with 8 × 104Eimeria tenella was evaluated. On the third day after infection, the broiler chickens were treated with EZL by gavage at doses of 2, 4, and 8 mg/kg body weight (bw) for once. For double administration, the challenged broiler chickens were administered EZL at doses of 1, 2, 4, and 8 mg/kg bw by gavage continually on the third day and fourth day and once a day. Throughout the experimental period, performance parameters including body weight gain, mortality, cecal lesion score, bloody diarrhoea and oocyst output were recorded. The anticoccidial efficacy was evaluated using the anticoccidial index (ACI). Meanwhile, the concentrations of EZL in chicken cecal contents were measured, and the data were analyzed with a non-compartmental model. The results indicated that EZL showed good anticoccidial activity at single dose of 4 mg/kgbw, with the corresponding ACI of 175.73. When the challenged chickens were treated with EZL under double administration, the EZL showed a medium level of anticoccidial activity at a dose of 2 mg/kg bw, with the corresponding ACI of 162.48. The maximum concentrations (Cmax) of EZL in content were 2.43 ±â€¯1.16, 4.28 ±â€¯1.56, and 8.57 ±â€¯1.33 mg/kg after the chickens were administrated at doses of 2, 4, and 8 mg/kg bw, respectively. The respective areas under the curve were 36.93 ±â€¯8.91, 96 ±â€¯16.31, and 262.76 ±â€¯51.52 mg/kg h. The respective half-lives (T1/2) were 10.82 ±â€¯2.02, 10.53 ±â€¯2.23, and 10.60 ±â€¯1.50 h. The results show that when the concentrations of EZL in chicken cecal contents reached 4.28 ±â€¯1.56 mg/kg, there is a significant therapeutic effect on chicken coccidiosis.

Beagle dog 90-day oral toxicity study of a novel coccidiostat - ethanamizuril.

BACKGROUND: Triazine coccidiostats are widely used in chickens and turkeys for coccidiosis control. Ethanamizuril is a novel triazine compound that exhibits anticoccidial activity in poultry. This study was designed to evaluate the subchronic toxicity of ethanamizuril in beagle dogs at doses of 12, 60 or 300 mg/kg/day in diet for 90 days. RESULTS: Ethanamizuril was well tolerated at low and middle dosages in beagle dogs, and no drug-related toxical effects were observaed in terms of survival, clinical observations, organs weight and damage in these dose groups. However, in high dose administration group, food consumption and histologic changes in kidneys were noticed in both sexes of beagle dog, although the renal lesions were finally resolved at the end of 4 weeks exposure of ethanamizuril. CONCLUSIONS: No-observed-adverse-effect level (NOAEL) was considered for ethanamizuril at dose of 60 mg/kg/day in Beagle dog. This result added toxicity effects of ethanamizuril to the safety database, which might guide safely using of ethanamizuril as a novel coccidiostat.

Population pharmacokinetics of ethanamizuril in normal and coccidiosis-infected chickens through ultra-performance liquid chromatography-Tandem Mass Spectrometry.

Ethanamizuril is a new triazine compound that shows potential for application in novel anticoccidial treatment. In this study, a pharmacokinetic model of ethanamizuril was established on the basis of the blood concentration of 81 experimental animals. The final model showed that ethanamizuril was distributed as a two-compartment model with first-order absorption after oral administration in chickens. Its clearance rate and volumn of central compartment distribution (Vc ) were affected by age and body weight, and volumn of central compartment distribution (Vc ) and volume of peripheral compartment distribution(Vp ) were influenced by weight and infection. External verification revealed that the model had good prediction accuracy and stability.

Effects of polysaccharide from Pueraria lobata on gut microbiota in mice.

Polysaccharide was derived from Pueraria lobata (PPL) which was considered as one of the traditional Chinese medicinal and edible herbs. In the present study, PPL was administered in equal doses (12.5 mg/kg) to both normal mice and antibiotic-associated diarrhea (AAD) mice for two weeks, and was evaluated in terms of body weight, organ indices, gut structure, gut microbiota and short chain fatty acids. The results showed that normal mice treated with PPL not only reduced the isovaleric acid concentration (P < 0.05), but also significantly increased the abundance of beneficial bacteria, involving Oscillospira and Anaerotruncus (P < 0.05). In addition, PPL could relieve colonic pathological changes and gut microbiota dysbiosis caused by AAD. It indicated that PPL was a potential functional food ingredient by modulating gut microbiota.

Two-generation reproduction and limited teratology studies of ethanamizuril fed to rats.

Ethanamizuril, a new anticoccidial agent that belongs to triazine derivatives, has a broad and good anticoccidial activity. To evaluate the reproductive toxicity and teratogenic potential of ethanamizuril, different concentrations of ethanamizuril were administered to Sprague-Dawley rats by feeding diets containing 0, 2, 8, and 30 ppm, respectively. Each group consisting of 30 males and 30 females (F0) was treated with different concentrations of ethanamizuril through a 13-week period before mating and during mating, gestation, parturition and lactation. In the 30 ppm dose group, pup body weight on days 7 and 21 in F1 offspring and day 21 in the F2a offspring were significantly decreased. A limited teratogenicity study was performed in combination with the F1 generation of a two-generation reproduction study. F1 offspring of the reproduction study were mated after weaning of the F2a offspring. Pregnant female rats were subjected to cesarean section on gestational day 20 for teratogenic examination. No obvious body weights, fetal body lengths, tail lengths, litter weights, number of viable fetuses, external, skeletal, or visceral malformations in fetuses were noted in any groups in the teratogenic test, but ethanamizuril could be passed on to offspring through lactation.

Preliminary study of the mechanism of action of ethanamizuril against Eimeria tenella.

Ethanamizuril (EZL) is a novel triazine compound with excellent anticoccidial activity. We carried out a preliminary investigation of the effects of EZL on the different life cycle stages of Eimeria tenella. EZL mainly acted on the schizogony stage, with peak activity during the second-generation merozoite stage. We also studied the possible target of EZL by identifying the majorly differentially expressed gene affected by EZL in second-generation merozoites using real-time polymerase chain reaction, and screening for surface antigen proteins (SAGs). The relative expression levels of SAGs were compared by Western blot analysis showing that expression levels of surface antigen family member (SAGfm) and SAG19 were significantly downregulated by EZL. Immunofluorescence analysis indicated that SAGfm and SAG19 were localized on the surface of second-generation merozoites. In addition, fluorescence signals were significantly stronger in second-generation merozoites of infected non-medicated control (INC) group compared with that of the EZL group. Therefore, it was speculated that SAGs might be a potential target of EZL action. The inhibitory effects of anticoccidial drugs on SAG levels in coccidia thus warrant further research.