Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens
Abstract
:Simple Summary
Abstract
1. Introduction
2. Methods
2.1. Primary Cell Isolation and Culture
2.2. Immunofluorescent Analysis of Primary Isolated Cells
2.3. Cell Stimulation
2.4. RNA Isolation, cDNA Synthesis and RT-qPCR
2.5. ELISA
2.6. Statistical Analysis
3. Results
3.1. Characterisation of Ovine Interdigital Skin Keratinocytes and Fibroblasts
3.2. Expression of Pro-Inflammatory Mediators in Primary Ovine Skin Keratinocytes and Fibroblasts in Response to D. nodosus Stimulation
4. Discussion
4.1. Primary Ovine Interdigital Skin Cell Cultures as a Model for Infection
4.2. Response of Ovine Interdigital Skin Fibroblasts and Keratinocytes to Microbial Preparations
4.3. Limitations
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Target | Primer | Size | GenBank ID | Source |
---|---|---|---|---|
Ov_Actin_F | TGTGCGTGACATCAAGGAGAA | 67 bp | AF129289 | [21] |
Ov_Actin_R | CGCAGTGGCCATCTCCTG | |||
Ov_PPIA_F | TGAGCACTGGAGAGAAAGGATTTG | 84 bp | AY251270 | [22] |
Ov_PPIA_R | AGTCACCACCCTGGCACATAA | |||
Ov_IL1 β _F | TTCTGCATGAGCTTCGTACAA | 115 bp | X54796 | [23] |
Ov_IL1 β _R | GGGTCGGTGTATCACCTTTTT | |||
Ov_CXCL8_F | GAGAAGTCCTCTGGGACAGC | 102 bp | NM_001009401.1 | [15] |
Ov_CXCL8_R | CAGCCAGCTTGGAAGTCATA |
Target | Capture Antibody | Protein Standard | Secondary Antibody | Tertiary Antibody | Standard Curve Range |
---|---|---|---|---|---|
IL-1β | Bio-Rad Mouse anti Ovine IL-1β clone 1D4 (MCA1658, 1:200) | Ovine IL-1β RP0656V-005 | rabbit anti sheep IL-1β polyclonal (AHP423, 1:500) | Dako (PO448) Goat anti rabbit-HRP conjugate (1:500) | 375–30,000 pg/mL |
CXCL8 | Bio-Rad Mouse anti Ovine IL-8 clone 8M6 (MCA1660, 1:200) | Kingfisher Biotech Ovine & Caprine IL-8 (CXCL8) RP0488V-005 | Bio-Rad rabit anti-sheep IL-8 polyclonal (AHP425, 1:500) | Dako (PO448) goat anti rabbit-HRP (1:500) | 25.6–1640 pg/mL |
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Blanchard, A.M.; Baumbach, C.-M.; Michler, J.K.; Pickwell, N.D.; Staley, C.E.; Franklin, J.M.; Wattegedera, S.R.; Entrican, G.; Tötemeyer, S. Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens. Animals 2022, 12, 3235. https://doi.org/10.3390/ani12233235
Blanchard AM, Baumbach C-M, Michler JK, Pickwell ND, Staley CE, Franklin JM, Wattegedera SR, Entrican G, Tötemeyer S. Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens. Animals. 2022; 12(23):3235. https://doi.org/10.3390/ani12233235
Chicago/Turabian StyleBlanchard, Adam M., Christina-Marie Baumbach, Jule K. Michler, Natalie D. Pickwell, Ceri E. Staley, Jemma M. Franklin, Sean R. Wattegedera, Gary Entrican, and Sabine Tötemeyer. 2022. "Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens" Animals 12, no. 23: 3235. https://doi.org/10.3390/ani12233235
APA StyleBlanchard, A. M., Baumbach, C.-M., Michler, J. K., Pickwell, N. D., Staley, C. E., Franklin, J. M., Wattegedera, S. R., Entrican, G., & Tötemeyer, S. (2022). Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens. Animals, 12(23), 3235. https://doi.org/10.3390/ani12233235