A Differential Innate Immune Response in Active and Chronic Stages of Bovine Infectious Digital Dermatitis

Digital dermatitis (DD) commonly associated with Treponema spp. infection is a prevalent infectious bovine foot disease characterized by ulcerative and necrotic lesions. Lesions associated with DD are often classified using the M-stage scoring system, with M0 indicating healthy heel skin and M4 indicating chronic lesions. Current treatments utilizing antimicrobials or chemical footbaths are often ineffective and rarely cure DD lesions. Understanding the function of the innate immune response in the pathogenesis of DD will help to identify novel therapeutic approaches. In this study, the expression of the local innate host defense peptides cathelicidins and β-defensins was investigated in cows with DD and associated with the presence of treponemes and inflammatory reactions. Samples from active ulcerative DD lesions (M2) had considerable epidermal neutrophilic infiltration and increased gene expression of β-defensin tracheal antimicrobial peptides compared to control skin. Samples from acute lesions also had elevated local Cxcl-8 and TLR4 gene expression and abundant treponemes as identified by direct visualization, immunohistochemistry, and culture. Conversely, the anti-inflammatory peptide IL-10 was elevated in skin from chronic (M4) lesions, whereas bovine cathelicidin myeloid antimicrobial peptide 28 (Bmap-28) was increased in skin from oxytetracycline-treated M2 lesions. Experiments using cultured human keratinocytes challenged with Treponema spp. isolated from clinical cases of bovine DD showed that structural products from treponemes are able to initiate the innate immune response, in part through TLR2 signaling. These findings indicate that neutrophil influx, Cxcl-8, and β-defensin are key markers of active DD. Cathelicidins and IL-10 seem important in response to treatment or during the chronic proliferative stages of the disease

Distribution of Treponema species and antimicrobial resistance genes in digital dermatitis lesions

Digital dermatitis lesions are ulcerative or proliferative masses between the heel bulbs. The most significant clinical outcome of digital dermatitis (DD) is lameness, leading to animal welfare concerns and economic loss for dairy producers worldwide due to premature culling, milk loss, and decreased fertility. While there is insufficient evidence to determine the etiological agent(s) responsible for DD, it is widely accepted that DD is a polymicrobial disease significantly associated with anaerobic bacteria, Treponema. Difficulties in obtaining pure cultures and the nearly exclusive presence of Treponema in diseases as members of a polytreponemal or polymicrobial communities has led to insufficient species identification within lesions and incomparable prevalence estimates based on phylotype. Our primary research objectives were to develop a diagnostic tool to identify Treponema spp. within DD lesions to associate presence and abundance with DD lesion grades. Further, we examined the distribution of antimicrobial resistance genes (AMGs) within sequenced isolates of DD-associated Treponema and designed targeted PCR for Treponema AMGs (AMGsTrep) within lesions. We provide significant evidence that the absolute quantities of Treponema and AMGsTrep are lower in chronic and early, active DD lesions suggesting Treponema spp. within these lesions may be more susceptible to antimicrobials (AMs) compared to species in advanced, active lesions. We have identified potential interactions among Treponema spp. that may facilitate DD progression and enhance pathogenicity and may affect AM susceptibility and lesion chronicity. With the novel diagnostic tool developed here, future research should elucidate interactions among Treponema spp. and in vitro susceptibility profiles and the efficacy of treating early and chronic lesions compared to advanced, active lesions on infection resolution

Associations between digital dermatitis lesion grades in dairy cattle and the quantities of four Treponema species

Abstract Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity

Image_1_A Differential Innate Immune Response in Active and Chronic Stages of Bovine Infectious Digital Dermatitis.TIFF

<p>Digital dermatitis (DD) commonly associated with Treponema spp. infection is a prevalent infectious bovine foot disease characterized by ulcerative and necrotic lesions. Lesions associated with DD are often classified using the M-stage scoring system, with M0 indicating healthy heel skin and M4 indicating chronic lesions. Current treatments utilizing antimicrobials or chemical footbaths are often ineffective and rarely cure DD lesions. Understanding the function of the innate immune response in the pathogenesis of DD will help to identify novel therapeutic approaches. In this study, the expression of the local innate host defense peptides cathelicidins and β-defensins was investigated in cows with DD and associated with the presence of treponemes and inflammatory reactions. Samples from active ulcerative DD lesions (M2) had considerable epidermal neutrophilic infiltration and increased gene expression of β-defensin tracheal antimicrobial peptides compared to control skin. Samples from acute lesions also had elevated local Cxcl-8 and TLR4 gene expression and abundant treponemes as identified by direct visualization, immunohistochemistry, and culture. Conversely, the anti-inflammatory peptide IL-10 was elevated in skin from chronic (M4) lesions, whereas bovine cathelicidin myeloid antimicrobial peptide 28 (Bmap-28) was increased in skin from oxytetracycline-treated M2 lesions. Experiments using cultured human keratinocytes challenged with Treponema spp. isolated from clinical cases of bovine DD showed that structural products from treponemes are able to initiate the innate immune response, in part through TLR2 signaling. These findings indicate that neutrophil influx, Cxcl-8, and β-defensin are key markers of active DD. Cathelicidins and IL-10 seem important in response to treatment or during the chronic proliferative stages of the disease.</p