In vitro susceptibility of contagious ovine digital dermatitis associated Treponema spp. isolates to antimicrobial agents in the UK

Background Contagious ovine digital dermatitis (CODD) is an important cause of infectious lameness in sheep in the UK and Ireland and has a severe impact on the welfare of affected individuals. The three treponemal phylogroups Treponema medium/Treponema vincentii‐like, Treponema phagedenis‐like and Treponema pedis spirochaetes have been associated with clinical CODD lesions and are considered to be a necessary cause of disease. There are scant data on the antimicrobial susceptibility of the treponemes cultured from CODD lesions. Objective The aim of this study was to determine in vitro the miniumum inhibitory concentration/ minimum bactericidal concentration (MIC/MBC) of antimicrobials used in the sheep industry for isolates of the three CODD associated treponeme phylogroups T. medium/T. vincentii‐like, T. phagedenis‐like and T. pedis. Animals Twenty treponeme isolates; from 19 sheep with clinical CODD lesions. Methods A microdilution method was used to determine in vitro the MIC/MBC of 10 antimicrobial agents for 20 treponeme isolates (five T. medium/T. vincentii‐like, 10 T. phagedenis‐like and five T. pedis). The antimicrobials tested were penicillin G, amoxicillin, oxytetracycline, tilmicosin, lincomycin, spectinomycin, tylosin, tildipirosin, tulathromycin and gamithromycin. Results The treponeme isolates tested showed low MICs and MBCs to all 10 antimicrobials tested. They were most susceptible to gamithromycin and tildipirosin (MIC90: 0.0469 mg/L), and were least susceptible to lincomycin, spectinomycin and oxytetracycline (MIC90: 48 mg/L, 24 mg/L and 3 mg/L, respectively). Conclusions These data are comparable to in vitro antimicrobial susceptibility data for treponemes cultured from bovine digital dermatitis lesions. Dependent on local licensing, penicillin and tilmicosin appear to be the best candidates for future in vivo studies.


Introduction
Contagious ovine digital dermatitis (CODD) is a cause of infectious lameness in sheep in the UK and Ireland and has been shown to have a severe impact on the welfare of affected individuals. 1 Recent surveys have shown that CODD may affect approximately 35% of flocks in the UK; while on-farm prevalence is typically low, it may affect up to 50% of the flock at any one time. 1 Information about the microbial flora of CODD lesions is limited, although the bovine digital dermatitis (BDD) associated treponemes Treponema medium/T. vincentiilike, Treponema phagedenis-like and Treponema pedis are currently considered to be a necessary cause of disease. 1 The recent characterization of treponemes associated with CODD demonstrated the presence of at least one BDD phylotype present in all 58 lesions studied, whereas these were totally absent from all healthy sheep foot tissues. 2 There has been a wide range of empirically chosen treatments employed in clinical cases such as parenteral oxytetracycline and topical tylosin, 3 with only one randomized controlled trial conducted comparing parenteral amoxicillin and simultaneous topical chlortetracycline with topical chlortetracycline alone. 4 As with CODD, the successful treatment of BDD has remained problematic with many farms adopting management and control strategies as opposed to affecting a cure. 5 In order to inform the development of effective therapeutic strategies for clinical cases of CODD, a Accepted 25 August 2015 1 The first two authors contributed equally to this manuscript. greater understanding is required of the susceptibility of the treponemes found in CODD lesions to antimicrobials currently available for use in sheep.
The aim of this study was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of a panel of antimicrobials for representatives from each of the three treponeme phylogroups cultured as pure isolates from clinical CODD lesions.

Bacterial isolates
Twenty treponeme isolates from CODD lesions from 19 sheep from six farms in England, Wales and Northern Ireland were used (Table 1). Included were five isolates from the T. medium/T. vincentii-like group, 10 isolates from the T. phagedenis-like group and five isolates from the T. pedis group.

In vitro antimicrobial susceptibility testing
The MIC/MBC for each antimicrobial was determined using a broth microdilution method as previously described. 6 One small adjustment was made to the method such that prior to inoculation, bacterial counts were assessed by determining the optical density (OD) of the cultures using spectrometry with wavelength set at 540 nm. The T. medium/T. vincentii-like cultures had an OD of 0.25; the T. phagedenis-like cultures had an OD of 0.43 and the T. pedis cultures had an OD of 0.37. This corresponds to 8.75 9 10 7 , 1.14 9 10 8 and 2.69 9 10 8 treponemal organisms/mL, respectively. 6 At this time point, cultures were assessed by phase contrast microscopy to determine that the cultures were alive, of the correct morphology and lacking contaminants. The antimicrobials and their test ranges are listed in Table 2.

Determination of MICs
The MIC for each antimicrobial was taken as the lowest concentration of antimicrobial that prevented growth in the wells observed at the same time points. 6 Cell growth was determined by comparison of the absorbance measurement immediately after inoculation with the absorbance measured at the late exponential/early stationary phase. All of the absorbance measurements were at 540 nm using a Multiskan microtitre plate reader (Thermo Scientific; Hampshire, UK).
The MIC values were taken as the median of three repeat experiments, performed on different days.

Determination of MBCs
The MBC for each antimicrobial was determined as previously described. 6 Determination of MIC 90 and MBC 90 The cumulative inhibitory/bactericidal concentration for each antimicrobial tested across all the treponeme isolates was expressed as the concentration at which 90% of CODD-associated treponemes were inhibited from growing (MIC90) or killed (MBC90).

Statistical analysis
Differences in MICs between the three different treponeme phylogroups were assessed using the Kruskal-Wallis test. The Kruskal-Wallis test and the nonparametric equality-of-medians test were used to compare the MICs for penicillin, oxytetracycline, lincomycin and spectinomycin with previous data, 6 and for amoxicillin and gamithromycin. 7 All statistical analyses were conducted using Stata IC 13 (Stata Corp; College Station, TX, USA) and statistical significance was set at P < 0.05.

Study validation
The MIC microdilution method described in this study was validated by comparing the results produced from four antimicrobials (penicillin, oxytetracycline, lincomycin and erythromycin) incubated with two control microorganisms T. phagedenis biotype Reiter and T. phagedenis-like T320A against results previously obtained using a macrodilution method 8 and also results obtained using a similar microdilution method. 6,7 The data were also compared statistically using linear regression.

Results
Antimicrobial susceptibilities of CODD-associated treponemes The individual MIC/MBCs of the antimicrobial agents to each treponeme isolate are summarized in Tables 2 and  3; in this study all isolates showed low MIC/MBCs to all   of the antimicrobials tested. Using Table 2, all treponeme groups were most susceptible to gamithromycin and tildipirosin, and least susceptible to lincomycin, spectinomycin and oxytetracycline. The MIC90 for the other five antimicrobials were all relatively low, being <1.0 lg/mL. No bimodal distributions were identified.

Variation in MIC across the different treponeme phylogroups
There was no significant difference in MIC values between the three different phylogroups for five of the seven macrolides (P = 0.2), with phylogroup differences for lincomycin and spectinomycin approaching significance (P = 0.05). Whilst there was no significant difference between phylogroups in the case of penicillin MIC (P = 0.1), in the case of amoxicillin and oxytetracycline, T. phagedenis-like bacteria were more susceptible compared to T. medium/T. vincentii-like and T. pedis (P = 0.002 and P = 0.001, respectively).

Comparisons with data from previous studies
The MICs for penicillin, oxytetracycline, lincomycin, spectinomycin, amoxicillin and gamithromycin for the 20 isolates investigated here, were not significantly different to those previously reported. 6,7 Study validation The comparison described matched the previous results in all cases except for one antimicrobial (oxytetracycline), which was different by one serial dilution when compared with the macrodilution method. 8 Linear regression for these comparisons showed strong correlations (R = 0.99 P < 0.004) indicating the efficacy and reproducibility of this microdilution method.

Discussion
Study validation and comparisons with data from previous studies The methodological validations described reinforce the comparable nature of these current data with previous studies. Comparisons of these current data for penicillin, oxytetracycline, lincomycin and spectinomycin with previous studies do not reveal any statistically significant differences. 6,7 Therefore, these current data make a valuable contribution to the available data on the in vitro antimicrobial susceptibility of these treponemes.
Antimicrobial use in sheep with CODD All of the isolates in this study were susceptible (in vitro) to all the antimicrobials tested, with gamithromycin, tildipirosin, penicillin, tylosin and tilmicosin demonstrating the lowest MICs and MBCs. This susceptibility, however, may not necessarily be reflected in vivo. To date, there have been very few robust in vivo studies examining effective treatment. In two clinical studies 4,9 systemic amoxicillin together with topical chlortetracycline was found to have a clinical cure rate in clinical cases of CODD of approximately 80%. Anecdotally, systemic tilmicosin was also proposed to be an effective treatment for sheep with CODD 10 and systemic oxytetracycline together with a tylosin footbath were considered to be an effective preventative method. 3 Currently, no antimicrobial product has a license for CODD in the UK. The antimicrobials studied here were selected to include antimicrobials that already have a license for sheep (penicillin, amoxicillin, oxytetracycline and tilmicosin) together with those that in the authors' experience are already used (off label) in the sheep industry. Therefore, given this context and these data as a whole, penicillin and tilmicosin would appear to be the most likely candidates for future in vivo studies.
This study provides the first detailed examination of the in vitro antimicrobial susceptibilities of all three associated phylogroups of treponemes cultured from CODD lesions to antimicrobials. As such, these data provide important in vitro information on antimicrobials currently used to treat this disease and should help inform researchers planning further in vivo studies when considering which products to include.

Resumen
Introducci on -la dermatitis contagiosa digital ovina (CODD) es una causa importante de de cojera infecciosa en ovejas en el Reino Unido e Irlanda y tiene un impacto severo en la salud de los individuos afectados. Los tres filogrupos de espiroquetas de Treponema Treponema medium/Treponema similar a T. vincentii, Treponema similar a T. phagenedis y Treponema pedis han sido asociados con lesiones cl ınicas de CODD y est an considerados como una causa necesaria de enfermedad. Hay pocos datos acerca de la susceptibilidad antimicrobiana de los cultivos de Treponema obtenidos de lesiones de CODD.