Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis).

BACKGROUND: Treponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock. RESULTS: The bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci. CONCLUSIONS: The bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations.

Phenotypic and proteomic characterization of treponemes associated with bovine digital dermatitis.

Bovine digital dermatitis (BDD) is a multifactorial polymicrobial infectious disease associated with multiple species and phylotypes of treponemes. However, despite the abundance of molecular signatures for treponemes that are identified in bovine lesions, relatively few isolates are cultured, and even fewer have been characterized at the level of protein expression. Here we report the successful isolation and characterization of novel strains of T. brennaborense and T. phagedenis from cases of BDD in Iowa dairy cows, and compare them to a well characterized strain of T. phagedenis, and the type strain of the more recently recognized T. pedis. Propagation of T. brennaborense was only possible at room temperature in Cooked Meat Medium, and not in oral treponeme enrichment medium at 37 °C as used for T. phagedenis and T. pedis. A prominent and rapid motility is observed by T. brennaborense under dark-field microscopy. The highly motile T. brennaborense strain 11-3 has an identical enzymatic profile to that of the only other isolate of T. brennaborense to be cultured from a lesion of BDD. Outer membrane protein profiles of each strain were compared by 2-D gel electrophoresis, and the five most abundant proteins in each strain were identified by mass spectrometry. All identified proteins are predicted to have signal peptides. Results identified outer membrane proteins specific to each strain including predicted membrane lipoproteins, ABC transporters and, as yet, uncharacterized proteins. Collectively, our results provide for the identification and characterization of outer membrane components of multiple phylotypes of treponemes associated with BDD which can facilitate development of vaccines and diagnostics in our efforts to eradicate the disease.

Short communication: Lymphocyte proliferative responses in cattle naturally infected with digital dermatitis consist of CD8+ and γδ-T cells but lack CD4+ T cells.

Digital dermatitis is an infectious disease of cattle and the leading cause of lameness. This disease is complicated by the reoccurrence of the lesions and the observation of lesions on more than one limb at different time points, indicating infection may not result in a protective immune response. The objective of this study was to characterize the peripheral blood cellular response in naturally infected and naïve cattle to bacterial antigens derived from pathogens associated with digital dermatitis lesions. Peripheral blood mononuclear cells were isolated from dairy cattle identified as having active or chronic lesions during routine hoof-trimming. Following bacterial antigen stimulation, cells were analyzed for proliferation and phenotype by flow cytometry, and culture supernatants were analyzed for IFN-γ secretion. Digital-dermatitis-infected animals had greater serum antibody titers to treponemal antigens, higher percentages of proliferating CD8+, γδ-T cells, and B cells, and increased IFN-γ secretion in vitro when compared with responses of naïve animals. No increase in proliferation of CD4+ T cells was detected in infected or naïve cattle. Although CD8+ and γδ-T cell responses may be antigen specific, the memory nature or long-lived response is yet unknown. The lack of responsiveness of CD4+ memory cells to treponemal antigens could explain the high rate of reoccurrence of digital dermatitis in infected animals.

Experimental Transmission of Bovine Digital Dermatitis to Sheep: Development of an Infection Model.

Digital dermatitis is an infectious cause of lameness primarily affecting cattle but also described in sheep, goats, and wild elk. Digital dermatitis is a polymicrobial infection, involving several Treponema species and other anaerobic bacteria. Although the exact etiology has not been demonstrated, a number of bacterial, host, and environmental factors are thought to contribute to disease development. To study host-bacterial interactions, a reproducible laboratory model of infection is required. The objective of this study was to demonstrate key aspects of bovine digital dermatitis lesions in an easy-to-handle sheep model. Crossbred sheep were obtained from a flock free of hoof disease. Skin between the heel bulb and dewclaw was abraded before wrapping to emulate a moist, anaerobic environment. After 3 days, abraded areas were inoculated with macerated lesion material from active bovine digital dermatitis and remained wrapped. By 2 weeks postinoculation, experimentally inoculated feet developed erosive, erythematous lesions. At 4 weeks postinoculation, microscopic changes in the dermis and epidermis were consistent with those described for bovine digital dermatitis, including erosion, ulceration, hyperkeratosis, ballooning degeneration of keratinocytes, and the presence of neutrophilic infiltrates. Silver staining of lesion biopsy sections confirmed that spirochetes had penetrated the host epidermis. The model was then perpetuated by passaging lesion material from experimentally infected sheep into naïve sheep. This model of bovine digital dermatitis will allow for future novel insights into pathogenic mechanisms of infection, as well as the development of improved diagnostic methods and therapeutics for all affected ruminants.

Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion.

BACKGROUND: Bovine papillomatous digital dermatitis (DD) is the leading cause of lameness in dairy cattle and represents a serious welfare and economic burden. Found primarily in high production dairy cattle worldwide, DD is characterized by the development of an often painful red, raw ulcerative or papillomatous lesion frequently located near the interdigital cleft and above the bulbs of the heel. While the exact etiology is unknown, several spirochete species have been isolated from lesion material. Four isolates of Treponema phagedenis-like spirochetes were isolated from dairy cows in Iowa. Given the distinct differences in host, environmental niche, and disease association, a closer analysis of phenotypic characteristics, growth characteristics, and genomic sequences of T. phagedenis, a human genitalia commensal, and the Iowa DD isolates was undertaken. RESULTS: Phenotypically, these isolates range from 8.0 to 9.7 µm in length with 6-8 flagella on each end. These isolates, like T. phagedenis, are strictly anaerobic, require serum and volatile fatty acids for growth, and are capable of fermenting fructose, mannitol, pectin, mannose, ribose, maltose, and glucose. Major glucose fermentation products produced are formate, acetate, and butyrate. Further study was conducted with a single isolate, 4A, showing an optimal growth pH of 7.0 (range of 6-8.5) and an optimal growth temperature of 40 °C (range of 29 °C-43 °C). Comparison of partial genomic contigs of isolate 4A and contigs of T. phagedenis F0421 revealed > 95% amino acid sequence identity with amino acid sequence of 4A. In silico DNA-DNA whole genome hybridization and BLAT analysis indicated a DDH estimate of >80% between isolate 4A and T. phagedenis F0421, and estimates of 52.5% or less when compared to the fully sequenced genomes of other treponeme species. CONCLUSION: Using both physiological, biochemical and genomic analysis, there is a lack of evidence for difference between T. phagedenis and isolate 4A. The description of Treponema phagedenis should be expanded from human genital skin commensal to include being an inhabitant within DD lesions in cattle.

Papillomatous digital dermatitis spirochetes suppress the bovine macrophage innate immune response.

Papillomatous digital dermatitis (PDD) is a polymicrobial infection in soft tissue adjacent to the hoof and is the leading cause of lameness in dairy cattle. Treponema phagedenis-like (TPL) spirochetes are a constant feature of PDD lesions and are localized deep in infected tissue. Host-cell response mechanisms to TPL spirochetes are poorly understood. To assess how bovine macrophages respond to cellular constituents of TPL spirochetes, changes in transcription were analyzed using serial analysis of gene expression (SAGE) and real time RT-PCR. This analysis revealed that some proinflammatory cytokines (e.g. GCP-2 and IL-8) are induced in treated macrophages, while receptors and their accessory proteins for IL-1, IL-6 and IL-11 are either down regulated or unchanged. Two genes encoding proteins having negative effects on NFkappaB, IkappaB and SIVA-1, are significantly induced in stimulated cells. Several genes associated with the cytoskeleton and antigen presentation are down regulated after exposure to sonicated TPL spirochetes, as are genes associated with wound repair. Combined, these data suggest that the innate immune and wound repair functions of bovine macrophages exposed to TPL cellular constituents are impaired thereby enabling bacteria to resist clearance and induce lesion formation. Use of this in vitro bovine macrophage model should be useful in elucidating host-spirochete interactions and facilitate identification of potential virulence traits.

Lesion formation and antibody response induced by papillomatous digital dermatitis-associated spirochetes in a murine abscess model.

Papillomatous digital dermatitis (PDD), also known as hairy heel wart, is a growing cause of lameness of cows in the U.S. dairy industry. Farms with PDD-afflicted cows experience economic loss due to treatment costs, decreased milk production, lower reproductive efficiency, and premature culling. While the exact cause of PDD is unknown, lesion development is associated with the presence of anaerobic spirochetes. This study was undertaken to investigate the virulence and antigenic relatedness of four previously isolated Treponema phagedenis-like spirochetes (1A, 3A, 4A, and 5B) by using a mouse abscess model with subcutaneous inoculation of 10(9), 10(10), and 10(11) spirochetes. Each of the PDD isolates induced abscess formation, with strain 3A causing cutaneous ulceration. Lesion development and antibody responses were dose dependent and differed significantly from those seen with the nonpathogenic human T. phagedenis strain. Strains 3A, 4A, and 5B showed two-way cross-reactivity with each other and a one-way cross-reaction with T. phagedenis. Strain 5B showed one-way cross-reactivity with 1A. None of the isolates showed cross-reactivity with T. denticola. In addition, distinct differences in immunoglobulin G subclass elicitation occurred between the PDD strains and T. phagedenis. From these data, we conclude that spirochetes isolated from PDD lesions have differential virulence and antigenic traits in vivo. Continuing investigation of these properties is important for the elucidation of virulence mechanisms and antigenic targets for vaccine development.

Characterization of Treponema phagedenis-like spirochetes isolated from papillomatous digital dermatitis lesions in dairy cattle.

Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar to Treponema phagedenis and 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity among T. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.