Putative ß-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative ß-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted ß-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as ß-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly ß-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal ß-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

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.

Short communication: Prevalence of digital dermatitis in Canadian dairy cattle classified as high, average, or low antibody- and cell-mediated immune responders.

Lameness is a major animal welfare issue affecting Canadian dairy producers, and it can lead to production, reproduction, and health problems in dairy cattle herds. Although several different lesions affect dairy cattle hooves, studies show that digital dermatitis is the most common lesion identified in Canadian dairy herds. It has also been shown that dairy cattle classified as having high immune response (IR) have lower incidence of disease compared with those animals with average and low IR; therefore, it has been hypothesized that IR plays a role in preventing infectious hoof lesions. The objective of this study was to compare the prevalence of digital dermatitis in Canadian dairy cattle that were classified for antibody-mediated (AMIR) and cell-mediated (CMIR) immune response. Cattle (n = 329) from 5 commercial dairy farms in Ontario were evaluated for IR using a patented test protocol that captures both AMIR and CMIR. Individuals were classified as high, average, or low responders based on standardized residuals for AMIR and CMIR. Residuals were calculated using a general linear model that included the effects of herd, parity, stage of lactation, and stage of pregnancy. Hoof health data were collected from 2011 to 2013 by the farm's hoof trimmer using Hoof Supervisor software (KS Dairy Consulting Inc., Dresser, WI). All trim events were included for each animal, and lesions were assessed as a binary trait at each trim event. Hoof health data were analyzed using a mixed model that included the effects of herd, stage of lactation (at trim date), parity (at trim date), IR category (high, average, and low), and the random effect of animal. All data were presented as prevalence within IR category. Results showed that cows with high AMIR had significantly lower prevalence of digital dermatitis than cattle with average and low AMIR. No significant difference in prevalence of digital dermatitis was observed between high, average, and low CMIR cows. These results indicate that having more robust AMIR is associated with lower prevalence of digital dermatitis hoof lesions.

Short communication: Efficacy of copper sulfate hoof baths against digital dermatitis--Where is the evidence?

Digital dermatitis is a major problem in modern dairy production because of decreased animal welfare and financial losses. Individual cow treatments are often seen as too time consuming by farmers, and walk-through hoof baths have therefore been used extensively to control digital dermatitis. For decades, copper sulfate hoof baths have been used to treat and prevent digital dermatitis. Copper sulfate has been referred to as the industry gold standard when it comes to hoof-bath chemicals. In several scientific studies testing the efficacy of other hoof-care products, copper sulfate has been used as a positive control, thereby indicating that copper sulfate has a known positive effect. However, this may not be the case. A dilemma may exist between (1) copper sulfate generally being perceived as being effective against digital dermatitis and (2) a possible lack of well-documented scientific evidence of this effect. The objective of this study was to evaluate the existing scientific literature to determine whether the efficacy of copper sulfate used in hoof baths against digital dermatitis has in fact been demonstrated scientifically. A systematic literature search identified 7 peer-reviewed journal articles describing the efficacy of copper sulfate in hoof baths as treatment or prevention of bovine digital dermatitis. Only 2 of the 7 studies compared copper sulfate to a negative control; most studies were relatively small, and often no clear positive effect of copper sulfate was demonstrated. In conclusion, the frequent claim that copper sulfate is widely reported to be effective is supported by little scientific evidence. Well-designed clinical trials evaluating the effect of copper sulfate against digital dermatitis compared with a negative control are needed. Until such studies have been made, the efficacy of copper sulfate in hoof baths against digital dermatitis remains largely unproven.

Immune response against Treponema spp. and ELISA detection of digital dermatitis.

The objective of this longitudinal study was to evaluate the immune response against Treponema spp. infection in dairy heifers affected with digital dermatitis (DD). In addition, the accuracy of an indirect ELISA detecting anti-Treponema IgG antibodies in identifying clinical DD status has been assessed. A cohort of 688 pregnant Holstein heifers was evaluated at least 3 times before calving during a period of 6 mo. Complete clinical assessment of DD presence on the back feet of each heifer and blood extraction were performed in a stand-up chute. Digital dermatitis cases were characterized by the M-stage classification system and size and level of skin proliferation. An ELISA was performed on blood serum samples obtained from a subcohort of 130 heifers. For description purposes, the animals were classified by the number of clinical cases experienced during the study period as type I (no clinical cases were observed), type II (only 1 acute clinical case diagnosed), and type III (at least 2 acute clinical cases diagnosed). Multivariable repeated-measures models were used to evaluate the immune response against Treponema spp. infection. A binormal Bayesian model for the ELISA data without cut-point values was used to assess the accuracy of the ELISA as a diagnostic tool. Animals that never experienced a DD event throughout the study kept a constant low level of antibody titer. A 56% increase in mean ELISA titer was observed in heifers upon a first clinical DD case diagnosis. After topical treatment of an acute DD case with oxytetracycline, the antibody titer decreased progressively in type II heifers, achieving mean levels of those observed in healthy cows after a mean of 223 d. Surprisingly, antibody titer was not increased in the presence of M1 (DD lesion <20mm in diameter surrounded by healthy skin) and M4.1 (DD lesion <20mm in diameter embedded in a circumscribed dyskeratotic or proliferative skin alteration) DD stages. Type III cows showed a slight increase in antibody levels. The presence of skin proliferation at first DD diagnosis was found to be associated with an odds ratio of 2.04 of becoming a type III heifer in relation to heifers presenting first lesions without skin proliferation. The ELISA validity was estimated by an area under the curve of 0.88. Predicted probabilities of infection are provided for a range of ELISA values and prevalence of infection. Early detection and treatment is essential to control DD and the ELISA can be used in understanding the immunopathology of DD and shows great promise for prescreening purposes during DD management programs in combination with traditional clinical inspection.