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Oxytetracycline reduces inflammation and treponeme burden whereas vitamin D3 promotes β-defensin expression in bovine infectious digital dermatitis

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Abstract

Digital dermatitis (DD), a common ulcerative disease of the bovine foot causing lameness and reducing productivity and animal welfare, is associated with infection by spirochete Treponema bacteria. Topical tetracycline, the most common treatment, has inconsistent cure rates; therefore, new therapeutic options are needed. We compared effects of topical oxytetracycline and vitamin D3 on innate immunity in DD-affected skin. Cows with active DD lesions were treated topically with oxytetracycline or vitamin D3 and skin biopsies were collected from lesions. Tissue samples were examined histologically, transcriptional expression of pro-inflammatory cytokines, Toll-like receptors (TLRs), and host defense peptides assessed, and the presence of specific treponeme species determined. Effects of treatments at a mechanistic level were studied in a human keratinocyte model of treponeme infection. Oxytetracycline promoted hyperplastic scab formation in ulcerated DD lesions and decreased transcriptional expression of Cxcl-8 (neutrophil chemoattractant). Oxytetracycline also reduced numbers of Treponema phagedenis and T. pedis and enhanced Tlr2 mRNA expression. Vitamin D3 did not modify expression of cytokines or Tlrs, or bacterial loads, but enhanced transcription of tracheal antimicrobial peptide (Tap), a key bovine β-defensin. Combing oxytetracycline and vitamin D3 provides complementary clinical benefits in controlling DD through a combination of antimicrobial, immunomodulatory, and pro-healing activities.

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Acknowledgments

We thank certified hoof trimmers Rob Geier (Casper Trimming, Calgary, AB, Canada) and Elbert Koster (No-tilt Hoof Trimming, Calgary, AB, Canada) for facilitating collection of biopsies. We also thank Dr. Brielle Rosa for her critical review and editing the manuscript.

Funding

This work was supported by the Natural Sciences and Engineering Research Council (NSERC) Discovery Grant (RGPAS-2017- 507827) and the Alberta Agriculture and Forestry (2019F040R) to ERC. KW was supported by Queen Elizabeth II Graduate Scholarship.

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Authors and Affiliations

  1. Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, HSC 2519, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada

    Kaitlyn M. Watts, Priyoshi Lahiri, Rakel Arrazuria, Jeroen De Buck, Karin Orsel, Herman W. Barkema & Eduardo R. Cobo

  2. Department of Veterinary Clinical and Diagnostic Science, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N 1N4, Canada

    Cameron G. Knight

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  1. Kaitlyn M. Watts
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  2. Priyoshi Lahiri
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  3. Rakel Arrazuria
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  4. Jeroen De Buck
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  6. Karin Orsel
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Contributions

KW and EC conceived and designed the experiments. KW and KO conducted animal sampling. KW and PL conducted in vitro experiments and analyzed data. RA performed Treponema spp. identification by culture and qPCR. RA and PL conducted immunohistochemistry and CK conducted histopathological examination. KO, CK, RA, JDB, HB, and EC contributed to data analysis and interpretation as well as manuscript editing. KW, PL, CK, and EC drafted and wrote the manuscript.

Corresponding author

Correspondence to Eduardo R. Cobo.

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The authors declare no conflicts of interest. Studies involving cattle were reviewed and approved by the University of Calgary Animal Care Guidelines (AC16-0070) for the care and use of animals. The owner’s written informed consent was obtained prior to the use of any cattle.

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Supplementary Figure S1

Transcriptional gene expression of cytokines, Toll-like receptors (Tlr), host defence peptides, and vitamin D enzymes in foot lesions of cows with active (M2) DD treated with vitamin D3 (VitD3) or oxytetracycline (OTC) (5-d intervals). Relative mRNA expression of Tlr4 (a), Ifn-γ (b), cathelicidin bovine myeloid antimicrobial peptide (Bmap)-28 (c), β-defensin lingual antimicrobial peptide (Lap) (d), and vitamin D3 metabolite Cyp24A1(e) was determined by RT-qPCR. Means + the standard error of the mean (SEM) are presented. Only significant comparisons (P < 0.05) are noted (one-way ANOVA using Tukey’s post hoc test). (PNG 257 kb).

High resolution image (TIFF 9236 kb).

Supplementary Figure S2

Transcriptional gene expressions of CXCL-8 (a, b), CYP24A1 (c, d) and CYP27B1 (e, f) in human keratinocytes (HaCaT cells) treated with tetracycline for 6 h and then challenged by T. phagedenis isolated from active (M2) DD lesions for 2 h (top row) and 24 h (bottom row) under aerobic conditions. Relative mRNA expression was determined by RT-qPCR. Means + the standard error of the mean (SEM) are presented. P < 0.05 was considered significant (Student’s T test). (PNG 303 kb).

High resolution image (TIFF 9148 kb).

Supplementary Figure S3

Transcriptional gene expressions of IL-10 (a, b), CAMP (c, d) and IFN-γ (e, f) in human keratinocytes (HaCaT cells) treated with tetracycline for 6 h and then challenged by T. phagedenis isolated from active (M2) DD lesions for 2 h (top row) and 24 h (bottom row) under aerobic conditions. Relative mRNA expression was determined by RT-qPCR. Means + the standard error of the mean (SEM) are presented. P < 0.05 was considered significant (Student’s T test). (PNG 279 kb).

High resolution image (TIFF 9164 kb).

Supplementary Figure S4

Transcriptional gene expressions of TLR-2 (a, b) and TLR-4 (c, d) in human keratinocytes (HaCaT cells) treated with tetracycline for 6 h and then challenged by T. phagedenis isolated from active (M2) DD lesions for 2 h (top row) and 24 h (bottom row) under aerobic conditions. Relative mRNA expression was determined by RT-qPCR. Means + the standard error of the mean (SEM) are presented. P < 0.05 was considered significant (T-test). (PNG 205 kb).

High resolution image (TIFF 9533 kb).

Supplementary Figure S5

Transcriptional gene expressions of IL-10 (a, b), CAMP(c, d) and IFN-γ(e, f) in human keratinocytes (HaCaT cells) treated with vitamin D3 (VitD3) for 6 h and then challenged by T. phagedenis isolated from active (M2) DD lesions for 2 h (top row) and 24 h (bottom row) under aerobic conditions. Relative mRNA expression was determined by RT-qPCR. Means + the standard error of the mean (SEM) are presented. P < 0.05 was considered significant (Student’s T test). (PNG 260 kb).

High resolution image (TIFF 9316 kb).

Supplementary Figure S6

Transcriptional gene expressions of TLR-2 (a, b) and TLR-4 (c, d) in human keratinocytes (HaCaT cells) treated with vitamin D3 (VitD3) for 6 h and then challenged by T. phagedenis isolated from active (M2) DD lesions for 2 h (top row) and 24 h (bottom row) under aerobic conditions. Relative mRNA expression was determined by RT-qPCR. Means + the standard error of the mean (SEM) are presented. P < 0.05 was considered significant (Student’s T test). (PNG 195 kb).

High resolution image (TIFF 9180 kb).

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Watts, K.M., Lahiri, P., Arrazuria, R. et al. Oxytetracycline reduces inflammation and treponeme burden whereas vitamin D3 promotes β-defensin expression in bovine infectious digital dermatitis. Cell Tissue Res 379, 337–348 (2020). https://doi.org/10.1007/s00441-019-03082-y

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