Previously described Taqman qPCR primer and probes targeting a 61-bp sequence within the RNA polymerase sigma-70 factor gene (rpoD) of Dichelobacter nodosus (29) and an 86-bp sequence within the β-subunit of RNA polymerase gene (rpoB) of Fusobacterium necrophorum (17) were employed. In addition, three novel Taqman qPCR assays were designed to individually target 254, 234, and 247 bp sequences within the Recombinase A (RecA) genes of T. medium (accession number CP027017), T. phagedenis (accession number CP027018), and T. pedis (accession number CP045760), respectively. The RecA gene was selected because of its singular occurrence in the genomes of Treponema spp. and its relatively low within-species diversity across a global panel of digital dermatitis-associated treponeme isolates (30). RecA gene sequences were extracted from the sequenced genomes, aligned with all available recA alleles from the previous multilocus sequence typing study and subjected to primer design using Mega X (31) and Primer 3 (32). The quality of the primer and probe sequences were analyzed using OligoCalc (33). The primer and probe sequences for the T. medium, T. phagedenis, and T. pedis qPCR assays are shown in Table 1. All probes were labeled with the fluorophore, 6-carboxyl-fluorescein (FAM), at the 5′-end and the non-fluorescent Black Berry Quencher (BBQ) at the 3′-end. Primers and probes were synthesized and purified commercially (TIB MOLBIOL, GmbH, Berlin, Germany).

A 61-bp rpoD gene fragment from D. nodosus strain VCS1703A was commercially synthesized and inserted into a pGM plasmid (GeneMill, University of Liverpool, UK). The plasmid was propagated in Top10 Escherichia coli (Life Technologies, Paisley, UK) according to the manufacturer's instructions and purified using the Plasmid Mini Kit (Qiagen, Manchester, UK). Fusobacterium necrophorum subs. funduliforme, previously isolated from a bovine digital dermatitis lesion by our laboratory, was grown in oral treponeme enrichment broth (OTEB, Anaerobe Systems, Morgan Hill, CA, USA). T. medium T19 was cultured in OTEB supplemented with 10% (v/v) rabbit serum, while T. phagedenis T320A and T. pedis T3552B^T were cultured in OTEB supplemented with 10 % (v/v) fetal calf serum as previously described (34). Chromosomal DNA was extracted using the Wizard kit (Promega, Southampton, UK) according to manufacturer's instructions and quantified using the Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Loughborough, UK). For each bacterial DNA preparation, genome copy numbers were calculated using the following equation: number of copies of DNA template per μl = (DNA concentration (ng/μl) × Avogadro's number)/[length of template (bp) × conversion factor to ng × average weight of a base pair (Da)]. Serial dilutions of the purified bacterial DNA were subsequently prepared in 10 mM Tris-Cl, pH 8.5, to yield estimated genome copy number of 3.25 × 10⁶ to 3.25 × 10⁻¹, 1.03 × 10⁶ to 1.03 × 10⁻¹, 2.66 × 10⁶ to 2.66 × 10⁻¹, 6.31 × 10⁸ to 6.31 × 10⁻¹, and 4.37 × 10⁶ to 4.37 × 10⁻¹ for T. medium, T. phagedenis, T. pedis, D. nodosus, and F. necrophorum, respectively.

All qPCR assays were performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and each 25 μl reaction comprised of the following: 5 μl 5 × HOT FIREPol® Probe qPCR Mix Plus (ROX) (Solis Biodyne, Tartu, Estonia), 1 μl (0.4 μM) forward primer, 1 μl (0.4 μM) reverse primer, 0.625 μl (0.25 μM) probe, 16.375 μl PCR-grade water, and 1 μl of DNA template. All reactions were performed in triplicate. The F. necrophorum (17) and D. nodosus (29) qPCR assays were performed using the cycling conditions previously described. Optimal qPCR cycling conditions for the three Treponema qPCR assays were empirically determined and comprised of a single activation step of 95°C for 12 min followed by 40 cycles of 95°C for 15 s and 61°C for 15 s and 72°C for 30 s. Analytical specificity and sensitivity were empirically defined for each assay. The baseline fluorescence signal was automatically calculated from the first 3–15 qPCR cycles. Where no increase in fluorescence signal was detected after 40 cycles, the bacterial load was classified as “undetectable,” i.e., below the limit of detection.

Analytical specificity of the D. nodosus and F. necrophorum qPCR assays have been previously described (17, 29). The specificity of the F. necrophorum rpoB assay was verified by analyzing the genomic DNA of Fusobacterium varium, a species closely related to F. necrophorum, while the genomic DNA of F. necrophorum subsp. necrophorum (DSM21784) (DSMZ, Germany) was used as a positive control. The specificity of the D. nodosus rpoD assay was verified by analyzing the genomic DNA of the two closest relatives of D. nodosus, namely, Suttonella indologenes (DSM8309) and Cardiobacterium hominis (DSM8339), while using the genomic DNA of D. nodosus (DSM23057) as a positive control. The specificities of the three novel pathogenic Treponema qPCR assays were individually established by analyzing the genomic DNA of T. medium, T. phagedenis, and T. pedis, to confirm the absence of cross-reactivity between the three assays, and by analyzing the genomic DNA of two commensal treponemes, namely, T. ruminis (DSM 103462) and T. rectale (DSM 103679). The lower limits of detection for the T. medium, T. phagedenis, T. pedis, D. nodosus, and F. necrophorum assays were 32.5, 10.3, 26.6, 63.1, and 43.7 genome copies/μl template, respectively; below these concentrations, DNA detection failed. The calibration standards for the T. medium, T. phagedenis, T. pedis, D. nodosus, and F. necrophorum assays generated R²-values of >0.99 and mean slopes of −3.5 (SEM ± 0.04), −3.5 (SEM ± 0.01), −3.5 (SEM ± 0.07), −3.4 (SEM ± 0.07), and −3.7 (SEM ± 0.06), respectively, indicating that the amplification efficiency of these assays was >85%.

Each sample assay was performed in triplicate, and the mean genome copy number (MGCN) was calculated from the three assay results.

At the start of the study, foot swab samples were collected from all the 10 source sheep (with a veterinary clinical diagnosis of CODD) and the 30 experimental study sheep (with a veterinary clinical diagnosis of healthy). All five target bacterial pathogens were present in the source sheep, confirming the introduction of these pathogens into the experimental flock as anticipated. F. necrophorum, D. nodosus, T. medium, and T. phagedenis were also detected in the healthy study sheep despite these sheep showing no clinical signs of disease in their feet (Figure 5). However, the foot level mean log₁₀ MGCN for D. nodosus, T. medium, T. pedis, and T. phagedenis were all significantly lower in the study sheep compared with the source sheep, and the proportion of colonized feet was also significantly lower in the study compared to the source sheep (Table 2). The degree of colonization of the feet by F. necrophorum in the source and study sheep was the same (Table 2).

A total of 2,320 samples were collected throughout the study period from the 30 sheep, with 877 samples (37.8%) yielding a positive qPCR result for at least one of the five target pathogens. There was considerable heterogenity in qPCR results, with positive results for all bacteria, namely, F. necrophorum, D. nodosus, T. medium, T. pedis, and T. phagedenis being recorded in both healthy and diseased feet, albeit with differing frequencies and pathogen loads (Figure 6). There was a tendency for both FR and CODD lesions to have higher treponeme mean log₁₀ MGCN compared to ID and other lesions (Figure 6).

While all microorganisms were detected on all sampling occasions during the study period, there were apparent broad temporal trends in detection frequency with all organisms being identified with greater frequency as the study progressed, up until commencement of treatment (Figure 7).

Survival analysis was performed to investigate time to first identification of an organism in a foot and Kaplan–Meier survival curves plotted (Figure 8). These suggest an apparent trend for early colonization by T. phagedenis (median survival time, 39 days; 95% CI, 32–36 days) followed by F. necrophorum and D. nodosus (median survival time, 81 days; 95% CI, 74–88 days), T. medium (median survival time, 88 days; 95% CI, 81–95 days), and T. pedis (median survival time, 116 days; 95% CI, 95–123 days).

As can be seen from Figure 6, there was considerable heterogeneity in bacterial mean log₁₀ MGCN by recorded lesion type (ID, FR, and CODD). To further examine associations between each of the target bacterial species and lesion type (healthy, ID, FR, and CODD), two sets of mixed linear regression models were fitted with outcome variable being the specific bacterial species log₁₀ MGCN and explanatory variables being lesion type or CODD grade. Model outputs are in Appendix 1 (Supplementary Material), and predicted marginal means estimated from the models are displayed graphically in Figures 9, 11. While all bacterial species were present in ID, FR, and CODD, there were some trends apparent. At lesion level (Figure 9), D. nodosus was present at a significantly higher predicted mean log₁₀ MGCN in FR lesions compared to both ID and CODD, while Treponema species were significantly higher in CODD and FR lesions compared to ID lesions (p < 0.001), although there was no statistically significant difference between predicted mean log₁₀ MGCN in FR and CODD lesions (p = 0.47).

A total of 139 samples were collected from lesions described as CODD, from 31 affected feet. As described earlier, in the case of five feet, lesions of grade 1 CODD arose de novo, with no prior foot disease recorded. In 26 feet, G#1 lesions were recorded, following prior lesions of ID and/or FR (progressive CODD lesions). Bacterial colonization (as a binary event) and mean bacterial log₁₀ counts were compared within grade 1 lesions by origin, i.e., arising de novo or progressive. In the case of all bacterial species, a high proportion (>86%) of progressive G#1 lesions were colonized compared to the CODD lesions, which arose de novo. Furthermore, in the de novo CODD lesions, the qPCR signal for all bacterial species was totally absent, or insufficient as to indicate colonization, suggesting an absence of all bacterial species tested for in lesions that arose de novo (Table 3).

Within CODD lesions, there was considerable heterogeneity in both proportion and mean log₁₀ genome copy number by CODD grade (Figure 10).

While all bacterial species were present in all CODD lesions irrespective of grade (Figure 11), all predicted log₁₀ mean genome copy numbers were significantly lower in grade 5 lesions compared to other grades. Both predicted mean log₁₀ MGCN for F. necrophorum and D. nodosus were significantly higher (p < 0.001) in G#1 lesions compared to all other grades. No other clear apparent trends in colonization could be observed within CODD grade.

All 12 CODD lesions (G#1–G#4), present in the flock at the time of the treatment intervention, were colonized by the five target pathogens (Figure 12). One week following completion of treatment, when all of the CODD foot lesions had resolved to G#5 or healthy status, bacterial colonization and the number of colonized feet had reduced substantially in the treated animals; however, their feet remained colonized albeit at much lower levels. By 3 weeks post-treatment, when one CODD foot lesion (8.33%) had clinically recurred, the level of colonization remained at a low level and was broadly similar for F. necrophorum, D. nodosus, and T. phagedenis, while T. medium and T. pedis were now absent from the treated feet (Table 4).

In order to understand, as closely as possible, the etiopathogenesis of CODD as it would occur in natural field cases of the disease, the experimental study was designed to transmit and induce CODD, by mixing healthy and CODD-infected sheep in a typical, UK, indoor, sheep husbandry environment. Therefore, in this study, CODD-infected sheep were introduced to a naive flock (2); animals were housed with exposure to wet underfoot conditions (18), and there was regular close gathering of sheep for sampling (19). Transmission of CODD is hypothesized to occur between sheep indirectly through the underfoot environment or via fomites such as gloves and hoof trimming equipment (35, 36). However, to avoid cross-contamination of foot lesions for microbiological sampling, no hoof trimming was carried out in the group, and samplers' gloves were changed between handling each sheep.

The conditions described were sufficient to induce CODD lesions in 18 of the 30 sheep (60%) and 31 of the 120 feet (25.7%). The median survival time taken for the lesions to develop was 116 days (95% CI, 103–123 days). There are no other CODD experimental transmission studies for direct comparison; however, anecdotal evidence on CODD transmission in the field and experimental studies of induction of FR and BDD suggest that the time to disease induction in our study was surprisingly long. For example, in a study where BDD lesion material was directly inoculated onto abraded and wet bandaged sheep's feet (37), lesions consistent with BDD were observed after 28 days in the sheep. Experimental induction of FR in other studies report a lag of 7 days to the onset of FR when scarification of feet was combined with continued exposure to wet bedding and accumulation of fecal matter (38). Following continued exposure to a wet underfoot environment, wet bandaging of feet and direct inoculation of lesions with D. nodosus, FR lesions developed after 10 days (27). Similarly, recent pasture transmission studies of induction of footrot by direct inoculation of foot skin and pasture contamination with D. nodosus induced FR lesions in the feet typically by 8 days post-exposure (39). This compares with a median survival time (in the present study) for ID of 88 days (95% CI, 74–109 days) and for FR of 108 days (95% CI, 95–116 days). Therefore, it is apparent that foot disease induction in our experimental model was much slower than previously reported. This may be because we did not directly damage the integrity of the foot through scarification or bandaging or directly inoculate pathogens or infected material directly onto the feet. It may also reflect the time taken for the relevant pathogens in the environment to reach infective levels. It is also highly likely that the environmental conditions of the experimental model may have affected the rate of CODD disease expression in the sheep. Environmental conditions of moisture and temperature are known to affect the expression of footrot in sheep (39), and seasonal trends in field case occurrence of footrot and CODD have also been observed (18, 40). In our experimental study, the straw under the feed troughs was kept wet and the bedding deep littered; however, the sheep were not continuously exposed to wet underfoot conditions, as this would not be consistent with the management of typical UK housed sheep, which the study aimed to replicate. Environmental temperature and moisture parameters were not recorded in this study and are an area of important further work. A final reason for the slow expression of CODD in our study could be that there was no opportunity for transmission of bacterial pathogens between sheep's feet via fomites from manual handling of the feet or foot trimming equipment, which would be expected to occur in a typical farm environment.

All progressive CODD lesions (i.e., not the de novo ones) did develop from ID and/or FR lesions. Therefore, consistent with field evidence (2, 18, 19) and the experimental FR studies (27, 38), pre-existing damage to the foot was a prerequisite for CODD lesions to occur in this study, and CODD lesions represented the end stage of an infectious foot disease process, which began with ID/FR.

Two distinct patterns of CODD lesion development were observed: progressive and de novo CODD lesions. The majority (83%) of CODD lesions in the study were progressive CODD lesions, which developed from pre-existing ID and/or FR lesions; they progressed through stages of CODD lesions 1–5 (although there was a degree of waxing and waning of lesion grades over time), and were all heavily colonized by all five pathogens: T. medium, T. phagedenis, T. pedis, D. nodosus, and F. necrophorum. A smaller number of de novo CODD G#1 lesions were observed to develop from clinically healthy sheep's feet; these did not progress beyond G#1. Colonization of these de novo CODD lesions by the five-foot pathogens was substantially less than that observed with the progressive CODD lesions. Therefore, it is likely that in the de novo foot lesions, there was insufficient initial damage to the foot (by ID or FR) to allow effective colonization of the skin by the pathogenic bacteria in order for CODD lesions to fully develop.

In this experiment, CODD was a disease of polymicrobial etiology. All five bacterial species, namely, T. medium, T. phagedenis, T. pedis, D. nodosus, and F. necrophorum were identified in all CODD lesion stages; however, the proportion of colonized samples and mean log₁₀ MGCN of all five bacteria species were substantially lower in healthy feet, healed CODD grade 5, and treated CODD foot lesions, compared with feet affected by an active CODD lesion (grades 1–4). Thus, a clear association between bacterial colonization and foot pathology is observed, and further evidence for disease causality for the five pathogens consortium is demonstrated. This polymicrobial etiology is consistent with previous cross-sectional studies of field cases of CODD etiology where the same three DD treponeme bacteria were found in all CODD lesions; D. nodosus and F. necrophorum were also identified, but to a lesser extent (12). Metagenomic analysis of the foot microbiome during the development of CODD (from the present study) confirmed the polymicrobial etiology of CODD and clear associations between the same bacterial consortium and ID, FR, and CODD (28).

While it is clear that CODD is polymicrobial in nature, there were apparent trends in the data regarding the temporal development of CODD. As foot pathology progressed from ID to FR to CODD (grades 1–4), linear regression models exploring bacterial colonization suggested that D. nodosus was present at significantly higher mean log₁₀ concentrations in FR lesions compared to both ID and CODD (Figure 9); the treponeme bacteria colonized FR and CODD lesions at a higher mean log₁₀ concentrations compared to the ID lesions (Figure 9); and finally, D. nodosus and F. necrophorum counts were significantly higher (p < 0.001) in the earlier G#1 CODD lesions compared to the later ones (G#2–G#4) (Figures 9, 11). The survival analysis is not entirely consistent with the modeling data, as early colonization by T. phagedenis is then followed by F. necrophorum and D. nodosus, and then colonization by T. medium and T. pedis is observed.

In the pathogenesis of FR, the strain of D. nodosus present affects the ability of the bacteria to invade the epidermis and cause clinical disease (due to the expression of the AprV₂ gene) (41). In the current study, the strain of D. nodosus was not examined, but now that we have shown a clear link in the pathogenesis of FR and CODD, this is an important area of future work.

Biosecurity measures and practices to prevent introduction of disease-causing agents into a population are an essential component of almost all infectious disease control strategies. The findings of the current study have important implications for flock biosecurity. The current advice for the prevention of the introduction of CODD into naive flocks is that all feet of all brought sheep should be examined upon arrival at the farm for CODD lesions (42). However, this may not be sufficient alone to prevent the introduction of CODD. At the start of the study, there was evidence of low-level colonization of the healthy experimental study sheep's feet with T. medium (0.83% of feet), T. phagedenis (13.3% of feet), D. nodosus (0.83% of feet), and F. necrophorum (4.17% of feet) (but not T. pedis). The flock, from which the sheep were sourced, did have a history of infection with ID and FR but had never observed CODD. It would be expected to find low-level colonization with F. necrophorum in healthy sheep's feet and D. nodosus, as reported in studies of FR in sheep (43). However, it was surprising to find low levels of CODD- and DD-associated treponemal species in the healthy feet from a farm with no known history of CODD in sheep or DD in the beef cattle on the farm. This contrasts with previous reports, employing routine gel-based PCR techniques, which demonstrated an absence of CODD/BDD treponemes in healthy vs. infected CODD foot biopsy tissue (12). The present study offers no evidence that the bacterial load detected in healthy feet is sufficient to initiate infection or was viable or may even be an artifact associated with the chosen qPCR cutoff values. Alternatively, given that T. phagedenis has been previously reported as a commensal of the GI tract in humans, chimpanzees (44), and healthy cattle (45), its presence on healthy feet could in fact represent commensal contamination from the GI tract and not be associated with foot disease per se. Indeed, recent work comparing bovine DD and human non-pathogenic T. phagedenis demonstrates the presence of unique gene clusters encoding key survival factors and a putative secretion system within the disease-associated bovine T. phagedenis strains (46). Using qPCR assays to target such virulence factors may enable better pathogen load determination to better describe disease progression in the future. However, based on the current data we have presented here, while the current advice regarding the examination of sheep feet at entry should serve to reduce the risk of introducing CODD—given that diseased sheep have a much greater bacterial load—it may not totally eliminate all risk.

There was 100% clinical cure rate of CODD lesions (CODD G#5), observed 1 week after the treatment intervention of two doses of long-acting amoxicillin and environmental decontamination. However, one lesion (8.3%) was observed to recur 2 weeks later. The microbiology of the feet also clearly demonstrates substantial reduction in the mean log₁₀ MGCN of all five bacterial species 1 and 3 weeks post-treatment. Importantly, bacterial cure was not established in all feet. Of all feet, 16.7% remained colonized by D. nodosus and 8.3% by F. necrophorum and T. phagedenis, although bacteriological cure was achieved for T. pedis and medium. The antibiotic therapy chosen (amoxicillin) was selected based on its previously reported field clinical cure rates for CODD (71%) (19) and in vitro bacterial sensitivity assays for treponeme bacteria (47). Therefore, the results of the current study are consistent with these data and emphasize that treated animals may still be infectious to the flock.

The principal limitation of the study is that is an experimental study, and the results require validation in the field. However, every effort was made to mimic known field conditions, and the data are consistent with previous field studies. Furthermore, a dual sampling strategy of swabs and different biopsy depths in the field together with fluorescent in situ hybridization of key pathogens may help confirm and dissect findings in the future. Here, we used housekeeping genes as targets of qPCR, a well-established methodology; however, in future studies, if these were to be supplemented with qPCR assays targeting recently identified pathogenic determinants, this may enable better associations of pathogen presence with disease progression.