Journal: Communications Biology

Article Title: Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella

doi: 10.1038/s42003-025-09386-w

Figure Lengend Snippet: a Strategy for generating maduramicin-resistant introgression lines through iterative backcrossing and drug selection. Resistance progeny were backcrossed with the sensitive parental strain over two generations under maduramicin pressure, yielding lines enriched for resistance-associated alleles within a largely sensitive genetic background. This figure was originally created by the authors using Adobe Illustrator. b Schematic overview of the QTL-seq approach on whole-genome resequencing of resistant and sensitive bulks to identify genomic regions under selection. This figure was originally created by the authors using Adobe Illustrator. c ΔSNP-index plots highlight genomic regions associated with maduramicin resistance across the E. tenella genome. The X -axis represents chromosomal positions, and the Y -axis shows the smoothed ΔSNP-index values (resistant minus sensitive) using 100kb sliding windows. Arrows mark significant peaks corresponding to candidate loci potentially involved in drug resistance.

Article Snippet: Subsequently, three 4-week-old AA broilers each received an inoculum of 10,000 F 1 oocysts via the crop, and 5 mg/kg maduramicin (MedChemExpress, Monmouth Junction, NJ, USA) was supplemented in their feed.

Techniques: Selection

Journal: Communications Biology

Article Title: Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella

doi: 10.1038/s42003-025-09386-w

Figure Lengend Snippet: a Construction strategy of overexpression transgenic parasites. The purple regions indicate mutation sites. b Fluorescence observation of F 1 generation transgenic parasites. Scale bar, 50 µm. c mCherry expression across different stages of ETH2_0402100 Mut -OE strain. Unsporulated oocyst, sporulated oocyst, sporozoites, schizont, merozoite, and gametocyte. Scale bar, 5 µm. d Indirect immunofluorescence microscopy showing the localization of ETH2_0402100 Mut -OE-mCherry. Mouse anti-Flag mAb was used to detect the Flag-tagged ETH2_0402100 Mut protein followed by FITC-conjugated goat anti-mouse IgG. Scale bar, 10 µm. e Stable expression of the target protein in ETH2_0402100 Mut -OE strain validated by western blot. H was used as the wild-type control. f Oocyst output curves comparing ETH2_0402100 Mut -OE and wild-type H strains. “−” and “+” indicate treatment without and with maduramicin (5 mg/kg), respectively. g Total oocyst output measured during infection. Strain H served as the wild-type control. “−” and “+” indicate treatment without and with maduramicin (5 mg/kg), respectively. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. P -values: *** p < 0.001; ns not significant. Data are presented as mean ± SD. Each dot represents an individual biological replicate ( n = 3).

Article Snippet: Subsequently, three 4-week-old AA broilers each received an inoculum of 10,000 F 1 oocysts via the crop, and 5 mg/kg maduramicin (MedChemExpress, Monmouth Junction, NJ, USA) was supplemented in their feed.

Techniques: Over Expression, Transgenic Assay, Mutagenesis, Fluorescence, Expressing, Immunofluorescence, Microscopy, Western Blot, Control, Infection, Two Tailed Test

Journal: Communications Biology

Article Title: Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella

doi: 10.1038/s42003-025-09386-w

Figure Lengend Snippet: a Cas9-mediated strategy for introducing a single point mutation. b PCR amplification and c western blot analysis confirming successful generation of homologous recombination (HR) parasites. EtHCYA strain was used as the control. d ETH2_0402100 Mut protein expression across different developmental stages of ETH2_0402100 Mut -HR transgenic parasites: unsporulated oocyst, sporulated oocyst, sporozoites, schizont, merozoite, and gametocyte. Scale bar, 5 µm. e Indirect immunofluorescence microscopy showing localization of the ETH2_0402100 Mut protein. Rabbit anti-mCherry pAb was used to detect the ETH2_0402100 Mut protein, followed by Alexa Fluor ® 647 goat anti-rabbit IgG. Scale bar, 5 µm. f Oocyst output curves comparing ETH2_0402100 Mut -HR and EtHCYA strains. “−” and “+” indicate treatment without and with maduramicin (5 mg/kg), respectively. g Total oocyst outputs. EtHCYA was used as the control. “−” and “+” indicate treatment without and with maduramicin (5 mg/kg), respectively. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. P -values: *** p < 0.001. Data are presented as mean ± SD. Each dot represents an individual biological replicate ( n = 3).

Article Snippet: Subsequently, three 4-week-old AA broilers each received an inoculum of 10,000 F 1 oocysts via the crop, and 5 mg/kg maduramicin (MedChemExpress, Monmouth Junction, NJ, USA) was supplemented in their feed.

Techniques: Mutagenesis, Amplification, Western Blot, Homologous Recombination, Control, Expressing, Transgenic Assay, Immunofluorescence, Microscopy, Two Tailed Test

Journal: Communications Biology

Article Title: Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella

doi: 10.1038/s42003-025-09386-w

Figure Lengend Snippet: a Phylogenetic tree of the ETH2_0402100 protein in eukaryotes. b Transcriptional patterns of the ETH2_0402100 gene across different developmental stages. SO sporulated oocysts, UO unsporulated oocysts, MZ-2 second-generation merozoites, SZ sporozoites. The relative expression levels were normalized to the 18S rRNA internal control and calculated using the 2 −ΔΔCt method. Data are presented as mean ± SEM from three biological replicates. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. P -values: * p < 0.05 ; ** p < 0.01; *** p < 0.001. Data are presented as mean ± SD. Each dot represents an individual biological replicate ( n = 3). c Expression of the ETH2_0402100 protein across different developmental stages. SO sporulated oocysts, UO unsporulated oocysts, MZ-2 second-generation merozoites, SZ sporozoites. d Secondary structure of the ETH2_0402100 protein. Mutated amino acids highlighted in red. e Structural comparison of wild-type and mutant ETH2_0402100 proteins. The mutated amino acid is highlighted in red. The inset on the left shows the side chain of the wild-type residue (leucine, L419), while the inset on the right illustrates the mutant residue (phenylalanine, F419), emphasizing the structural change from an aliphatic side chain to an aromatic benzene ring. f Molecular docking visualization between the ETH2_0402100 Mut protein and maduramicin drug molecule. The right panel shows the predicted binding interaction between the mutant protein and maduramicin, with the drug displayed in stick representation. The left inset is a magnified view, highlighting the spatial relationship between the drug and the mutated amino acid residue located near the putative binding pocket.

Article Snippet: Subsequently, three 4-week-old AA broilers each received an inoculum of 10,000 F 1 oocysts via the crop, and 5 mg/kg maduramicin (MedChemExpress, Monmouth Junction, NJ, USA) was supplemented in their feed.

Techniques: Expressing, Control, Two Tailed Test, Comparison, Mutagenesis, Residue, Binding Assay

Journal: Communications Biology

Article Title: Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella

doi: 10.1038/s42003-025-09386-w

Figure Lengend Snippet: a Principal component analysis (PCA) revealing variability among samples. Circles represent samples from the MZ-2 stage, and triangles represent samples from the SZ stage. Different colors indicate treatment groups: H (no drug treatment), MRR (no drug treatment), MRR (treated with 5 mg/kg maduramicin), and MRR (treated with 7 mg/kg maduramicin). b Pearson correlation analysis uncovers resistance patterns across developmental stages of SZ and MZ-2. c PCA highlights variance in resistance profiles across groups and stages. d Visualization of gene modules through dendrogram clustering and TOM similarity analysis. e Heatmap visualization of gene expression correlations across groups and stages. The asterisk marks the target gene. The red box highlights a co-expression module comprising genes with expression patterns similar to the target gene.

Article Snippet: Subsequently, three 4-week-old AA broilers each received an inoculum of 10,000 F 1 oocysts via the crop, and 5 mg/kg maduramicin (MedChemExpress, Monmouth Junction, NJ, USA) was supplemented in their feed.

Techniques: Gene Expression, Expressing

Journal: Communications Biology

Article Title: Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella

doi: 10.1038/s42003-025-09386-w

Figure Lengend Snippet: a PCR amplification of a 306 bp fragment at the ETH2_0402100 locus using laboratory-prepared mixtures containing defined proportions (5–95%) of maduramicin-resistant E. tenella (MRR). b Amplicon sequencing of these PCR products revealed the C > T mutation at position 1255, with mutation frequencies increasing proportionally from 3 to 89%. c PCR amplification of the 306 bp fragment from 11 DNA samples collected from poultry farms. d Amplicon sequencing of field samples identified the C > T mutation at position 1255 in all eight PCR-positive farms, with mutation frequencies ranging from 1.43 to 100%.

Article Snippet: Subsequently, three 4-week-old AA broilers each received an inoculum of 10,000 F 1 oocysts via the crop, and 5 mg/kg maduramicin (MedChemExpress, Monmouth Junction, NJ, USA) was supplemented in their feed.

Techniques: Amplification, Sequencing, Mutagenesis