m bovis bcg strain danish  (ATCC)

Journal: Journal of Advanced Research

Article Title: Enhanced tuberculosis control via leveraging dendritic cell-mediated Th1 responses in preventive and immunotherapeutic vaccine strategies

doi: 10.1016/j.jare.2025.07.056

Figure Lengend Snippet: Comparison of protective efficacy of various DC subsets against Mtb infection. ( A ) After 10 weeks from BCG vaccination, FL-DC1, FL-DC2, and GM-DC were transferred to BCG-immunized mice via an intravenous route. Three days later, the mice were infected with Mtb H37Rv (∼130 CFUs/mouse). Mice were then sacrificed at 1 and 6 weeks post-infection. ( B ) Lung histopathology was analyzed using H&E at 6 weeks post-infection and ( C ) bacterial burdens in the lung at 1 week (upper panel) and in the lung and spleen at 6 weeks post-infection (lower panel) were assessed. n = 3–5. Blue asterisk without designation indicates comparison to infection group. ( D ) One week after the Mtb H37Rv challenge, PPD-specific lung CD4 + T cells producing the indicated cytokines were analyzed. n = 4. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. * p < 0.05, ** p < 0.01, and *** p < 0.001. Asterisk without designation indicates comparison to BCG group. The mean frequencies of cells co-producing IFN-γ, TNF-α, and/or IL-2 are represented in the pie charts. The arcs surrounding the pie charts denote the number of cytokines produced by the T cells. Data are mean ± SD. FL-DC1: FLT3 ligand-derived CD11b + BMDCs matured with CFA, FL-DC2: FLT3 ligand-derived CD11b - BMDCs matured with CFA: GM-DC: GM-CSF derived BMDCs matured with CFA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: M. bovis BCG (Pasteur 1173P2) was generously provided by Dr. Brosch at the Pasteur Institute (Paris, France).

Techniques: Comparison, Infection, Histopathology, Produced, Derivative Assay

Journal: Journal of Advanced Research

Article Title: Enhanced tuberculosis control via leveraging dendritic cell-mediated Th1 responses in preventive and immunotherapeutic vaccine strategies

doi: 10.1016/j.jare.2025.07.056

Figure Lengend Snippet: Protective efficacy of GM-DC transfer after BCG vaccination against various Mtb clinical strains in mice. ( A ) After 6 weeks from BCG vaccination, GM-DC were transferred to non-vaccinated or BCG-immunized mice via an intravenous route. Mice were challenged with HN878, Erdman, or K strain (∼200 CFUs/mouse, each strain). ( B ) After 10 weeks from challenge, mice from each group were sacrificed and bacterial growth of lung and spleen were evaluated. n = 5–6. ( C ) After 10 weeks from challenge with Mtb K, the superior lobes of the right lung were analyzed using H&E staining, and the inflamed areas were presented as dot graph. n = 5. ( D ) One week and ten weeks after the Mtb K challenge, PPD-specific lung CD4 + T cells producing the indicated cytokines were analyzed. n = 3. The mean frequencies of cells co-producing IFN-γ, TNF-α, and/or IL-2 are represented in the pie charts. The arcs surrounding the pie charts denote the number of cytokines produced by the T cells. Data are mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. * p < 0.05, ** p < 0.01, and *** p < 0.001. Asterisk without designation indicates comparison to BCG group. GM-DC, CFA-matured DC differentiated with GM-CSF.

Article Snippet: M. bovis BCG (Pasteur 1173P2) was generously provided by Dr. Brosch at the Pasteur Institute (Paris, France).

Techniques: Staining, Produced, Comparison

Journal: Journal of Advanced Research

Article Title: Enhanced tuberculosis control via leveraging dendritic cell-mediated Th1 responses in preventive and immunotherapeutic vaccine strategies

doi: 10.1016/j.jare.2025.07.056

Figure Lengend Snippet: Analysis of CD4 + T cell response in the lungs of BCG-immunized mice after GM-DC transfer. GM-DC was transferred after 10 weeks from BCG vaccination. After 3 days, mice were sacrificed for analysis. ( A ) The localization of lung immune cells in vascular or parenchymal regions were analyzed. n = 4. ( B ) The localization of lung CD4 + T cells was analyzed according to the expression of CD44, CD69, KLRG1, CCR6, and CXCR3. n = 4. ( C ) PPD-specific CD4 + T cells producing multiple cytokines in the lung parenchymal and vascular areas were examined. The mean frequencies of cells co-producing IFN-γ, TNF-α, and/or IL-2 are represented in the pie charts. The arcs surrounding the pie charts indicate the CD4 + T cells that produce multiple cytokines. n = 4. ( D ) The effect of GM-DC transfer on the formation of memory type of CD4 + T cells (T CM , T EM , and T RM ) in the lung were analyzed. To further analyze the lung CD4 + T RM population, ( E ) the expression of CD103 and ( F ) IFN-γ production of T RM in response to PPD stimulation were analyzed. n = 4. Data are mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. n.s. = not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. Comparison between IV-CD45.2(−) value: red asterisk, comparison between total value: black asterisk. DC, dendritic cell: MΦ, macrophage: Neu, neutrophil: Resident memory T cell, T RM : effector memory T cell, T EM: Central memory T cell, T CM . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: M. bovis BCG (Pasteur 1173P2) was generously provided by Dr. Brosch at the Pasteur Institute (Paris, France).

Techniques: Expressing, Comparison

Journal: Journal of Advanced Research

Article Title: Enhanced tuberculosis control via leveraging dendritic cell-mediated Th1 responses in preventive and immunotherapeutic vaccine strategies

doi: 10.1016/j.jare.2025.07.056

Figure Lengend Snippet: Analysis of CD4 + T cell response in the lung of GM-DC-transferred mice after BCG vaccination at 1 week post Mtb infection. After 10 weeks from BCG vaccination, GM-DC was transferred to BCG-immunized mice. Three days later, mice were infected with the Mtb strain K (∼250 CFUs/mouse). ( A ) One week post-infection, the localization of lung immune cells in vascular and parenchymal regions were analyzed. n = 4. ( B ) The localization of CD4 + T cells in the lungs was analyzed based on the expression of CD44, CD69, KLRG1, CCR6, and CXCR3. n = 4. ( C ) PPD-specific CD4 + T cells producing multiple cytokines in the lung parenchymal and vascular areas were examined. The mean frequencies of cells co-producing IFN-γ, TNF-α, and/or IL-2 are represented in the pie charts. The arcs surrounding the pie charts indicate the CD4 + T cells that produce multiple cytokines. n = 4. ( D ) The expression of Ki67 of memory type of CD4 + T cells in the lung were analyzed. The dot plots represent Ki67 + T RM in the lung of each group. n = 4. ( E ) PPD-specific CD4 + T RM in lung producing IFN-γ were measured. n = 4. Data are mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. n.s. = not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. Comparison between IV-CD45.2(−) value: red asterisk, comparison between total value: black asterisk. DC, dendritic cell: MΦ, macrophage: Neu, neutrophil: Resident memory T cell, T RM : effector memory T cell, T EM: Central memory T cell, T CM . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: M. bovis BCG (Pasteur 1173P2) was generously provided by Dr. Brosch at the Pasteur Institute (Paris, France).

Techniques: Infection, Expressing, Comparison

Journal: Journal of Advanced Research

Article Title: Enhanced tuberculosis control via leveraging dendritic cell-mediated Th1 responses in preventive and immunotherapeutic vaccine strategies

doi: 10.1016/j.jare.2025.07.056

Figure Lengend Snippet: Analysis of long-term protection in GM-DC-transferred mice after BCG vaccination against Mtb infection. ( A ) Six weeks after BCG vaccination, GM-DCs were administered to BCG-vaccinated mice. Twenty-six weeks later, the mice were infected with the Mtb K strain (120 CFUs/mouse). ( B ) Before Mtb K challenge, PPD-specific lung CD4 + T cells producing the indicated cytokines were analyzed. The mean frequencies of cells co-producing IFN-γ, TNF-α, and/or IL-2 are represented in the pie charts. The arcs surrounding the pie charts denote the number of cytokines produced by the T cells. n = 4. ( C ) Ten weeks after Mtb K infection, the superior lobes of the right lung were analyzed using H&E staining, and bacterial growth in the lung and spleen ( D ) were depicted with bar graphs. n = 4. Data are mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. n.s. = not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: M. bovis BCG (Pasteur 1173P2) was generously provided by Dr. Brosch at the Pasteur Institute (Paris, France).

Techniques: Infection, Produced, Staining

Journal: Journal of Advanced Research

Article Title: Enhanced tuberculosis control via leveraging dendritic cell-mediated Th1 responses in preventive and immunotherapeutic vaccine strategies

doi: 10.1016/j.jare.2025.07.056

Figure Lengend Snippet: Protective efficacy of GM-DC transfer in BCG-vaccinated mice against low dose and lethal dose Mtb infection. ( A ) BCG-immunized mice were transferred with GM-DC or saline. Three days after DC transfer, mice were challenged with a low dose (∼10 CFUs/mouse) of Mtb K strain. ( B ) The lung bacterial growth was monitored at 2, 4, 8, and 16 weeks post-infection, and ( C ) lung histopathology at each sacrifice point was analyzed using H&E staining. n = 4. Data are mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. n.s. = not significant, ** p < 0.01, *** p < 0.001. Asterisk without designation indicates comparison to BCG group. ( D ) For the lethal dose infection model, mice were challenged with a high dose (∼1 × 10 5 CFUs/mouse) of Mtb K strain. ( E ) The survival of mice infected with Mtb K was tracked over a period of 14 weeks following infection. n = 6. Statistical analysis was performed by the Log-rank (Mantel-Cox) test. ( F ) Lung histopathology was evaluated at designated time points using H&E staining. The lung sections of the infection, BCG, and BCG/imGM-DC groups were stained after the mice succumbed to Mtb infection. imGM-DC, immature GM-DC.

Article Snippet: M. bovis BCG (Pasteur 1173P2) was generously provided by Dr. Brosch at the Pasteur Institute (Paris, France).

Techniques: Infection, Saline, Histopathology, Staining, Comparison

Journal: Frontiers in Veterinary Science

Article Title: In silico performance of a targeted enriched metagenomics approach to infer Mycoplasma bovis strains in milk

doi: 10.3389/fvets.2026.1770245

Figure Lengend Snippet: Phylogenetic tree (neighbor-joining method) of 162 Mycoplasma bovis whole genome sequences from Bos taurus derived from milk. The tips of the tree are color coded according to the MLST classification. The outer ring is color coded by the genomically clustered sequencing variant (GSV) classification according to the average length of the clade with clustering threshold 0.0001.

Article Snippet: All M. bovis whole genome sequences (WGS) from Bos taurus , along with their associated metadata, were retrieved from the National Center for Biotechnology Information (NCBI) database ( ) on October 23, 2023 ( n = 620, ).

Techniques: Derivative Assay, Sequencing, Variant Assay

Journal: Vaccines

Article Title: Long-Term Protective Immune Responses Induced by rBCG-RBD/rRBD Heterologous Prime/Boost Immunization Strategy: Fusion of RBD-Wuhan with LTB Adjuvant Induces Cross-Reactivity with SARS-CoV-2 Variant Omicron

doi: 10.3390/vaccines14020120

Figure Lengend Snippet: Expression of RBD and LTB-RBD in BCG. ( A ) Schematic representation of the expression cassettes of the vectors pLA71-RBD and pLA73-LTB-RBD. Both vectors contain origins of replication to E. coli and Mycobacterium, a kanamycin resistance gene (KanR), the pBlaF* promoter, and its ATG initiation codon. pLA71-RBD presents the RBD sequence in fusion with the β-lactamase signal sequence (ssBlam), generating ssRBD, while pLA73-LTB-RBD displays the LTB-RBD sequence in fusion with the whole β-lactamase encoding gene (ssBlam + Blam), generating Blam-LTB-RBD. ( B ) Expression of ssRBD and Blam-LTB-RBD in rBCG. Total cell extracts of wtBCG or rBCG (20 µg) were analyzed by Western blotting using an anti-RBD polyclonal antibody. Purified recombinant RBD in fusion with the FH8 tag for solubility and expressed in E. coli was used as positive control (C+), with approximately 45 kDa. Total protein extract of wtBCG was used as negative control (C−). Molecular weights are indicated on the left, and the expected molecular weight of ssRBD (25 kDa) and Blam-LTB-RBD (~67 kDa) are indicated by arrows. A non-specific band at 35 kDa is observed in all samples, including the negative control.

Article Snippet: M. bovis BCG strain Danish (ATCC #35733, Manassas, VA, USA) was used as control and to generate the recombinant BCG strains expressing the RBD and LTB-RBD sequences. wtBCG, rBCG-RBD, and rBCG-LTB-RBD were grown in Middlebrook 7H9 (MB7H9; Difco, Detroit, MI, USA) supplemented with 10% OADC (oleic acid–albumin–dextrose–catalase; BBL, Cockeysville, MD, USA), 0.5% glycerol (Sigma-Aldrich ® ), and 0.05% Tween 80 (Sigma-Aldrich ® ), containing 20 μg/mL kanamycin (Sigma-Aldrich ® ) or not.

Techniques: Expressing, Sequencing, Western Blot, Purification, Recombinant, Solubility, Positive Control, Negative Control, Molecular Weight

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate restores autophagic activity and autophagic flux in bMECs infected with M. bovis . (A) bMECs were divided into 6 experimental groups: control, M. bovis PG45 reference strain infection (MOI = 30, 9 hpi), M. bovis WT21 wild-type strain infection (MOI = 30, 9 hpi), fenofibrate treatment alone, fenofibrate combined with PG45 infection, and fenofibrate combined with WT21 infection. Expression levels of LAMP2, RAB7A, SQSTM1, and LC3, along with the loading control GAPDH, were evaluated by Western blot (using specific antibodies). (B) Densitometric analysis was performed to quantify relative protein levels of LAMP2, RAB7A, SQSTM1, and LC3-II, normalized to GAPDH. (C) Representative confocal images depict autophagic flux in mCherry-GFP-LC3-transfected bMECs across the 6 treatment groups: control, PG45-infected, WT21-infected, fenofibrate-treated, fenofibrate+PG45, and fenofibrate+WT21. Yellow puncta indicate autophagosomes, whereas red puncta represent autolysosomes. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar = 10 μm. (D) Quantification of autophagosomes in bMECs. Twenty cells for each sample and at least 60 cells in each group were used for statistical analyses, Superscript ‘a’: Yellow puncta compared to the control group, Superscript ‘b’: Red puncta compared to the control group. For (A, B) , 2-way ANOVA Dunnett’s multiple comparisons tests were used; for (D) , 1-way ANOVA Dunnett’s multiple comparisons tests and 2-tailed unpaired t -tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Activity Assay, Infection, Control, Expressing, Western Blot, Transfection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate induces nuclear translocation of TFE3 in bMECs infected with M. bovis . (A) bMECs were divided into 6 groups: control, M. bovis PG45 strain infection, M. bovis WT21 wild-type strain infection, fenofibrate-treated, fenofibrate + PG45 infection, and fenofibrate + WT21 infection. Western blot analysis was used to evaluate TFE3 expression in cytoplasmic and nuclear fractions. Relative protein levels were quantified by densitometry and normalized to GAPDH (cytoplasm) or Histone H3 (nucleus). (B) Immunofluorescence staining was conducted to examine subcellular localization of endogenous TFE3 (red) in bMECs across the 6 groups. Nuclei were stained with Hoechst 33258 (blue). Representative confocal images are shown. Scale bar = 10 μm. For (A) 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. # P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Translocation Assay, Infection, Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate induces nuclear translocation of TFEB in bMECs infected with M. bovis . (A) bMECs were divided into 6 groups: control, M. bovis PG45 strain infection, M. bovis WT21 wild-type strain infection, fenofibrate-treated, fenofibrate + PG45 infection, and fenofibrate + WT21 infection. Western blot analysis was used to evaluate TFEB expression in cytoplasmic and nuclear fractions. Relative protein levels were quantified by densitometry and normalized to GAPDH (cytoplasm) or Histone H3 (nucleus). (B) Immunofluorescence staining was conducted to examine subcellular localization of endogenous TFEB (red) in bMECs across the 6 groups. Nuclei were stained with Hoechst 33258 (blue). Representative confocal images are shown. Scale bar = 10 μm. For (A) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. # P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Translocation Assay, Infection, Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects free and total cholesterol concentrations, cholesterol-related gene transcription, and intracellular M. bovis load. (A) Total cholesterol concentrations in bMECs were quantified using the Amplex™ Cholesterol Assay Kit across 6 groups: control, M. bovis PG45 strain infection, M. bovis WT21 wild-type strain infection, fenofibrate treatment, fenofibrate + PG45 infection, and fenofibrate + WT21 infection. (B) Free cholesterol content in bMECs from 6 groups was measured using the Amplex™ Cholesterol Assay Kit. (C–E) The mRNA expression levels of cholesterol esterification gene ACAT1 and key cholesterol biosynthesis regulators HMGCR and SREBF2 were determined by quantitative real-time PCR and normalized to β-actin. (F) Bacterial burden was assessed by plating cell lysates from each group onto PPLOA agar, and colony-forming units (CFU) enumerated. For (A–F) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Cholesterol Assay, Control, Infection, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects co-localization of M. bovis , cholesterol, and LC3. (A) bMECs were divided into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Representative confocal images illustrate triple staining with DiI to label M. bovis (red), GFP-LC3 to visualize the autophagy marker LC3 (green), and Filipin to detect cholesterol (blue). Scale bar = 10 μm.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Control, Infection, Staining, Marker

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects expression of autophagy markers in mammary tissue of mice infected with M. bovis . (A) Mice were allocated into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Immunohistochemical staining was used to detect microtubule-associated protein 1 light chain 3 beta (LC3B) expression in murine mammary tissue. LC3B-positive granule-like cells were observed under a light microscope, with brown staining in nuclei indicating positive signals. (B) Quantification of LC3B-positive staining intensity in mammary tissues. (C) Using the same 6 experimental groups, immunohistochemical staining was performed to assess SQSTM1 expression in mammary tissue. SQSTM1-positive granule-like cells were observed under a light microscope, with brown nuclear staining indicating positive expression. (D) Quantification of SQSTM1-positive staining intensity in mouse mammary tissues. Scale bar = 50 μm. For (B, D) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. NS (Not Significant) P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Expressing, Infection, Control, Immunohistochemical staining, Staining, Light Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects expression of lysosome markers in mammary tissue of mice infected with M. bovis . (A) Mice were allocated into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Immunohistochemical staining was used to detect LAMP1 expression in murine mammary tissue. LAMP1-positive granule-like cells were observed under a light microscope, with brown staining in nuclei indicating positive signals. (B) Quantification of LAMP1-positive staining intensity in mammary tissues. (C) Using the same 6 experimental groups, immunohistochemical staining was performed to assess LAMP2 expression in mammary tissue. LAMP2-positive granule-like cells were observed under a light microscope, with brown nuclear staining indicating positive expression. (D) Quantification of LAMP2-positive staining intensity in mammary tissues. Scale bar = 10 μm. For (B, D) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Expressing, Infection, Control, Immunohistochemical staining, Staining, Light Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects cholesterol concentrations, M. bovis load, tissue morphology, and cytokine concentrations in murine mammary glands. (A) Total cholesterol concentrations in murine mammary glands were quantified using the Amplex™ Cholesterol Assay Kit across 6 experimental groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. (B) Free cholesterol content in mammary tissues from the same 6 groups was determined using the Amplex™ Cholesterol Assay Kit. (C) Hematoxylin and eosin (H&E) staining was performed on mammary gland sections from all 6 groups to assess tissue architecture and histopathological changes. Representative images under light microscopy highlight neutrophils (yellow arrows), lymphocytes (green arrows), and plasma cells (red arrows). (D) Bacterial load was evaluated by plating serial dilutions of tissue lysates from each group onto PPLOA agar, followed by enumeration of colony-forming units (CFU). Scale bar = 50 μm. (E) content of TNF-α in mammary gland tissue. (F) content of IL-1β in mammary gland tissue. (G) content of IL-6 in mammary gland tissue. (H) content of IL-10 in mammary gland tissue. For (A, B, D-H) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Abbreviations: ACAT1, Acetyl-coenzyme a acetyltransferase 1; AMPK, AMP-activated Protein Kinase; ATG5, Autophagy-related protein 5; bMECs, Bovine mammary epithelial cells; CFU, Colony forming unit; DMEM, Dulbecco’s modified eagle medium; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal Bovine Serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; HBSS, Hanks’ balanced salt solution; HMGCR, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; LC3/LC3B – Microtubule-associated protein 1 light chain 3 beta; LAMP1/2, Lysosome-associated membrane protein 1/2; MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PG45, M. bovis reference strain (ATCC 25523); RAB7A, Ras-related protein rab-7a; SBE-β-CD, Sulfobutylether-β-cyclodextrin; SREBF2, Sterol regulatory element-binding transcription factor 2; TFEB, Transcription factor EB; TFE3, Transcription factor E3; WT21, Wild-type M. bovis isolate 21.

Techniques: Cholesterol Assay, Control, Infection, Staining, Light Microscopy, Clinical Proteomics

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate restores autophagic activity and autophagic flux in bMECs infected with M. bovis . (A) bMECs were divided into 6 experimental groups: control, M. bovis PG45 reference strain infection (MOI = 30, 9 hpi), M. bovis WT21 wild-type strain infection (MOI = 30, 9 hpi), fenofibrate treatment alone, fenofibrate combined with PG45 infection, and fenofibrate combined with WT21 infection. Expression levels of LAMP2, RAB7A, SQSTM1, and LC3, along with the loading control GAPDH, were evaluated by Western blot (using specific antibodies). (B) Densitometric analysis was performed to quantify relative protein levels of LAMP2, RAB7A, SQSTM1, and LC3-II, normalized to GAPDH. (C) Representative confocal images depict autophagic flux in mCherry-GFP-LC3-transfected bMECs across the 6 treatment groups: control, PG45-infected, WT21-infected, fenofibrate-treated, fenofibrate+PG45, and fenofibrate+WT21. Yellow puncta indicate autophagosomes, whereas red puncta represent autolysosomes. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar = 10 μm. (D) Quantification of autophagosomes in bMECs. Twenty cells for each sample and at least 60 cells in each group were used for statistical analyses, Superscript ‘a’: Yellow puncta compared to the control group, Superscript ‘b’: Red puncta compared to the control group. For (A, B) , 2-way ANOVA Dunnett’s multiple comparisons tests were used; for (D) , 1-way ANOVA Dunnett’s multiple comparisons tests and 2-tailed unpaired t -tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Activity Assay, Infection, Control, Expressing, Western Blot, Transfection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate induces nuclear translocation of TFE3 in bMECs infected with M. bovis . (A) bMECs were divided into 6 groups: control, M. bovis PG45 strain infection, M. bovis WT21 wild-type strain infection, fenofibrate-treated, fenofibrate + PG45 infection, and fenofibrate + WT21 infection. Western blot analysis was used to evaluate TFE3 expression in cytoplasmic and nuclear fractions. Relative protein levels were quantified by densitometry and normalized to GAPDH (cytoplasm) or Histone H3 (nucleus). (B) Immunofluorescence staining was conducted to examine subcellular localization of endogenous TFE3 (red) in bMECs across the 6 groups. Nuclei were stained with Hoechst 33258 (blue). Representative confocal images are shown. Scale bar = 10 μm. For (A) 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. # P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Translocation Assay, Infection, Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate induces nuclear translocation of TFEB in bMECs infected with M. bovis . (A) bMECs were divided into 6 groups: control, M. bovis PG45 strain infection, M. bovis WT21 wild-type strain infection, fenofibrate-treated, fenofibrate + PG45 infection, and fenofibrate + WT21 infection. Western blot analysis was used to evaluate TFEB expression in cytoplasmic and nuclear fractions. Relative protein levels were quantified by densitometry and normalized to GAPDH (cytoplasm) or Histone H3 (nucleus). (B) Immunofluorescence staining was conducted to examine subcellular localization of endogenous TFEB (red) in bMECs across the 6 groups. Nuclei were stained with Hoechst 33258 (blue). Representative confocal images are shown. Scale bar = 10 μm. For (A) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. # P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Translocation Assay, Infection, Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects free and total cholesterol concentrations, cholesterol-related gene transcription, and intracellular M. bovis load. (A) Total cholesterol concentrations in bMECs were quantified using the Amplex™ Cholesterol Assay Kit across 6 groups: control, M. bovis PG45 strain infection, M. bovis WT21 wild-type strain infection, fenofibrate treatment, fenofibrate + PG45 infection, and fenofibrate + WT21 infection. (B) Free cholesterol content in bMECs from 6 groups was measured using the Amplex™ Cholesterol Assay Kit. (C–E) The mRNA expression levels of cholesterol esterification gene ACAT1 and key cholesterol biosynthesis regulators HMGCR and SREBF2 were determined by quantitative real-time PCR and normalized to β-actin. (F) Bacterial burden was assessed by plating cell lysates from each group onto PPLOA agar, and colony-forming units (CFU) enumerated. For (A–F) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Cholesterol Assay, Control, Infection, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects co-localization of M. bovis , cholesterol, and LC3. (A) bMECs were divided into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Representative confocal images illustrate triple staining with DiI to label M. bovis (red), GFP-LC3 to visualize the autophagy marker LC3 (green), and Filipin to detect cholesterol (blue). Scale bar = 10 μm.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Control, Infection, Staining, Marker

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects expression of autophagy markers in mammary tissue of mice infected with M. bovis . (A) Mice were allocated into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Immunohistochemical staining was used to detect microtubule-associated protein 1 light chain 3 beta (LC3B) expression in murine mammary tissue. LC3B-positive granule-like cells were observed under a light microscope, with brown staining in nuclei indicating positive signals. (B) Quantification of LC3B-positive staining intensity in mammary tissues. (C) Using the same 6 experimental groups, immunohistochemical staining was performed to assess SQSTM1 expression in mammary tissue. SQSTM1-positive granule-like cells were observed under a light microscope, with brown nuclear staining indicating positive expression. (D) Quantification of SQSTM1-positive staining intensity in mouse mammary tissues. Scale bar = 50 μm. For (B, D) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. NS (Not Significant) P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Expressing, Infection, Control, Immunohistochemical staining, Staining, Light Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects expression of lysosome markers in mammary tissue of mice infected with M. bovis . (A) Mice were allocated into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Immunohistochemical staining was used to detect LAMP1 expression in murine mammary tissue. LAMP1-positive granule-like cells were observed under a light microscope, with brown staining in nuclei indicating positive signals. (B) Quantification of LAMP1-positive staining intensity in mammary tissues. (C) Using the same 6 experimental groups, immunohistochemical staining was performed to assess LAMP2 expression in mammary tissue. LAMP2-positive granule-like cells were observed under a light microscope, with brown nuclear staining indicating positive expression. (D) Quantification of LAMP2-positive staining intensity in mammary tissues. Scale bar = 10 μm. For (B, D) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Expressing, Infection, Control, Immunohistochemical staining, Staining, Light Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects cholesterol concentrations, M. bovis load, tissue morphology, and cytokine concentrations in murine mammary glands. (A) Total cholesterol concentrations in murine mammary glands were quantified using the Amplex™ Cholesterol Assay Kit across 6 experimental groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. (B) Free cholesterol content in mammary tissues from the same 6 groups was determined using the Amplex™ Cholesterol Assay Kit. (C) Hematoxylin and eosin (H&E) staining was performed on mammary gland sections from all 6 groups to assess tissue architecture and histopathological changes. Representative images under light microscopy highlight neutrophils (yellow arrows), lymphocytes (green arrows), and plasma cells (red arrows). (D) Bacterial load was evaluated by plating serial dilutions of tissue lysates from each group onto PPLOA agar, followed by enumeration of colony-forming units (CFU). Scale bar = 50 μm. (E) content of TNF-α in mammary gland tissue. (F) content of IL-1β in mammary gland tissue. (G) content of IL-6 in mammary gland tissue. (H) content of IL-10 in mammary gland tissue. For (A, B, D-H) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Two strains of M. bovis , PG45 (ATCC 25523) and WT21 (Wild type isolate) were cultured in PPLO medium (BD Biosciences) and yeast extract (BD Biosciences) broth with 20% horse serum (Solarbio, Beijing, China) and 100 IU/L penicillin (Coolaber) in 5% CO 2 at 37°C for 72 h. The culture was centrifuged (6000 × g for 30 min) and washed with phosphate-buffered saline (PBS).

Techniques: Cholesterol Assay, Control, Infection, Staining, Light Microscopy, Clinical Proteomics