In vitro and ex vivo testing of alternative disinfectants to currently used more harmful substances in footbaths against Dichelobacter nodosus.

A footbath-based control program for ovine footrot, a contagious disease caused by Dichelobacter nodosus, will be implemented in Switzerland. The currently used footbath disinfectants formaldehyde, zinc sulfate and copper sulfate are carcinogenic or environmental pollutants. Hence, the aim of this study was to identify alternative disinfectants, which are highly effective, non-carcinogenic, environmentally acceptable, inexpensive, available as concentrate and suitable for licensing. The antimicrobial effect of a series of potential chemicals such as lactic acid, propionic acid, hydrogen peroxide, sodium hypochlorite, octenidine dihydrochloride, chlorocresol, Ampholyt 20 and the registered biocide DESINTEC® Hoof Care Special D (Desintec) were investigated by culture based in vitro testing. The microcidal effect of various Desintec concentrations were then compared against routinely used 4% formaldehyde and 10% zinc sulfate in ex vivo assays on sheep feet from slaughter. For this purpose a newly established PMA (propidium monoazid) real-time PCR using the improved dye PMAxx™ was applied that allows discrimination of viable and dead D. nodosus. In the ex vivo experiments, 4% formaldehyde was significantly more effective than 10% zinc sulfate and was chosen as positive control for assessing the new disinfectant. The disinfectant effect of Desintec in a minimal concentration of 6% was equally effective as 4% formaldehyde, meaning that it offers a comparable antimicrobial effect against virulent D. nodosus. In conclusion, Desintec is a promising disinfectant for replacing formaldehyde, copper sulfate and zinc sulfate in footbaths against footrot.

Antimicrobial Resistance in Clostridium and Brachyspira spp. and Other Anaerobes.

This article describes the antimicrobial resistance to date of the most frequently encountered anaerobic bacterial pathogens of animals. The different sections show that antimicrobial resistance can vary depending on the antimicrobial, the anaerobe, and the resistance mechanism. The variability in antimicrobial resistance patterns is also associated with other factors such as geographic region and local antimicrobial usage. On occasion, the same resistance gene was observed in many anaerobes, whereas some were limited to certain anaerobes. This article focuses on antimicrobial resistance data of veterinary origin.

The prevalence of Dichelobacter nodosus in clinically footrot-free sheep flocks: a comparative field study on elimination strategies.

BACKGROUND: Ovine footrot caused by Dichelobacter nodosus (D nodosus) is an infectious disease affecting sheep worldwide. Switzerland plans a nationwide footrot eradication program, based on PCR-testing of interdigital swab samples. The aim of this study was to test for the presence of D nodosus in clinically footrot-free sheep flocks which had been subjected to different treatment strategies, to assess whether they were feasible for the eradication process, especially focussing on antimicrobial flock treatments. Clinical scoring and PCR-results were compared. Ten farms had used hoof bathing and hoof trimming without causing bleeding, ten had used individual treatments and flock vaccines to gain the free status and ten had become free through whole-flock systemic macrolide treatment. For every farm, three risk-based collected pool samples were analysed for the occurrence of virulent and benign D nodosus by PCR detection of aprV2/aprB2. RESULTS: Six flocks from any treatment group tested positive for aprB2 in all pools. Clinical signs were absent at the time of sampling, but some flocks had experienced non-progressive interdigital inflammation previously. Two flocks tested aprV2-positive in the high-risk pool. One of them underwent a progressive footrot outbreak shortly after sampling. Individual retesting indicated, that virulent D nodosus most likely was reintroduced by a recently purchased ram. In the second flock, a ram was tested positive and treated before clinical signs occurred. CONCLUSIONS: All treatment strategies eliminated the causative agent and were found to be suitable for implementation in the PCR-based eradication process. PCR-testing proved to be more sensitive than visual scoring, as it also detected clinically healthy carriers. It will be of benefit as a diagnostic tool in elimination and surveillance programs.

Relationship between the likelihood of footrot elimination from a flock and the virulence of the strain of Dichelobacter nodosus present.

OBJECTIVE: To assess ability to eliminate different strains of footrot in sheep using inspection and culling of affected sheep. METHODS: A flock of 1417 Polwarth sheep that had deliberately been infected with seven different strains of Dichelobacter nodosus and undergone different control measures prior to eradication, including zinc sulfate footbathing and vaccination, were subjected to visual foot inspection on a number of occasions. Sheep identified as infected or having any foot abnormalities were removed from the flock at each inspection. The experiment had three replicates and a small number of untreated control animals. Sheep were examined following the inspections to assess the effectiveness of elimination and additional measures were implemented in two of the three replicates in an attempt to eliminate all strains of D. nodosus. RESULTS: Three strains of D. nodosus were apparently successfully eliminated from all replicates (strains A, E and H). Strains B and C were detected in one replicate each, despite additional inspections. The three stains that were eliminated were the more virulent strains and the two strains that remained were the least virulent. No assessment could be made on a further two strains. CONCLUSIONS: The application of a rigorous inspection and culling program resulted in the elimination of the more virulent D. nodosus strains, but did not result in the elimination of all D. nodosus strains on all occasions.

Elimination of virulent strains (aprV2) of Dichelobacter nodosus from feet of 28 Swiss sheep flocks: A proof of concept study.

Virulent ovine footrot caused by Dichelobacter nodosus is an endemic disease worldwide. It is associated with severe pain, impaired animal welfare and economic losses. The competitive real-time PCR for the differentiation of virulent aprV2 and benign aprB2 strains of Dichelobacter nodosus provides an objective, rapid and sensitive diagnostic tool for footrot surveillance, especially as it enables early detection of subclinical carriers of virulent strains. The aim of this study was to evaluate the feasibility of complete elimination of Dichelobacter nodosus strains carrying the aprV2 gene from sheep flocks. The treatment protocol was based on careful removal of loose and severely overgrown claw horn, weekly stand-in foot baths of the entire flock for 10 min per sheep, using a 10% zinc sulphate disinfectant solution, clinical and PCR follow-up and isolation or culling of non-responders. Dichelobacter nodosus strains carrying the aprV2 gene were successfully eliminated from the feet of the sheep of all 28 flocks (100%) participating in the study within 6-19 weeks of weekly foot bathing. A strong correlation between the length of time for weekly foot bathing to eliminate the virulent strains and the within-flock prevalence of clinical footrot at the beginning was observed (rho, 0.68; P <0.001). A statistically significant correlation was not detected between flock size and the length of time for weekly foot bathing (rho, 0.28; P = 0.14), or the prevalence of clinical footrot at study commencement (rho, -0.04; P = 0.82), respectively. In conclusion, a complete elimination of Dichelobacter nodosus strains carrying the aprV2 gene in sheep flocks was possible with a protocol based on careful trimming, weekly stand-in foot baths, and identification of infection using a specific PCR-test and isolation or culling of non-responders.

Impact of footrot vaccination and antibiotic therapy on footrot and contagious ovine digital dermatitis.

Footrot and contagious ovine digital dermatitis (CODD) are common causes of foot disease of sheep in the UK. The study reported here is a split flock randomised treatment trial undertaken on a group of 748 fattening lambs on a UK sheep farm affected by CODD and footrot. The sheep were randomly assigned to one of two treatment protocols. In protocol A, all sheep were given two doses of footrot vaccine (Footvax, MSD), plus targeted antibiotic therapy (long-acting amoxicillin, Betamox LA, Norbrook Pharmaceuticals) to sheep with foot lesions likely to be associated with a bacterial infection. In protocol B, the sheep only received targeted antibiotic therapy. Sheep were re-examined and foot lesions recorded five and nine weeks later. New infection rates in the footrot vaccinated group were lower compared with the vaccinated group for both CODD (18.2 per cent compared with 26.4 per cent, P=0.014) and footrot (12.55 per cent compared with 27.5 per cent, P<0.001). Recovery rates were unaffected for CODD (80.46 per cent compared with 70.97 per cent, P=0.14) but higher for footrot (92.09 per cent compared with 81.54 per cent, P=0.005) in sheep which received the vaccine. On this farm, a footrot vaccine efficacy of 62 per cent was identified against footrot and 32 per cent against CODD infection. An association between a sheep having footrot at visit 1 and subsequently acquiring CODD was identified (odds ratio [OR] 3.83, 95 per cent CI 2.61 to 5.62, P<0.001). These results suggest a role for infection with Dichelobacter nodosus in the aetiopathogenesis of CODD on this farm.

Comparative study of biochemical methods for the identification of Dichelobacter nodosus.

The aim of this study was to compare four identification procedures to detect Dichelobacter nodosus and develop a rapid, simple and effective method to identify D. nodosus strains isolated from cases of ovine footrot. The four methods used were: (a) the classic guidelines set down by Holdeman et al. (1977) and Summanen et al. (1993) which are based on gas liquid chromatography (GLC) and different biochemical tests, this method was considered as landmark; (b) Baron and Citron's flowchart for the rapid identification of Gram-negative rod-shaped anaerobes (1997); (c) the API rapid 32 A system (bio Mérieux), and (d) Mast ID Anaerobe ID Ring (MID8) (Mast Diagnostics). None of the four methods used allowed us to correctly identify the D. nodosus strains (neither the strains isolated from cases of ovine footrot nor those originating from type collection). Because of the difficulties encountered in obtaining a correct identification of D. nodosus, we propose a simple, rapid and effective way to achieve this task. Our flowchart will provide the means to identify this microorganism in any laboratory of general microbiology without having to use any specialised equipment.

Minimum inhibitory concentrations for 25 selected antimicrobial agents against Dichelobacter nodosus and Fusobacterium strains isolated from footrot in sheep of Portugal and Spain.

The agar dilution method was used to determine the inhibitory activity of 25 antimicrobial agents against 69 strains of Dichelobacter nodosus and 108 strains of the genus Fusobacterium, all of which were isolated from 90 clinical cases of ovine footrot between October 1998 and November 2000. In the case of the micro-organisms belonging to the genus Fusobacterium, the six beta-lactams studied (benzyl penicillin, ampicillin, cloxacillin, cefadroxil, cefuroxime and cephalexine) proved to be, in general, the most effective antimicrobial agents. Chloramphenicol, clindamycin and doxycycline were also quite active against Fusobacterium spp. With regard to the 69 strains of D. nodosus tested, the levels of resistance remain low.

Comparison of erythromycin and oxytetracycline for the treatment of ovine footrot.

A microbiological study of 25 cases of ovine footrot was performed. Cultures belonging to Dichelobacter nodosus were isolated in 48% of the sampled animals. The sensitivity of the 99 strict anaerobic bacterial isolates to 5 antibiotics (penicillin G, amoxycillin, spiramycin, erythromycin and oxytetracycline) was studied. The percentage of resistant cultures was in all cases higher than 30%. The efficacy of erythromycin and oxytetracycline in the treatment of ovine footrot was studied. To conduct this test, an intramuscular injection was applied, of one antimicrobial or the other, at the beginning of the treatment. The tolerance of animals to the antimicrobials, the success rate of treatment and the severity of lameness were evaluated. The percentage of animals cured within 15 days was around 75%. In contrast, only 44% improvement was achieved in the lameness. No differences were found between the two antimicrobials in the above indices.

Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus.

Studies on the potential virulence genes of the ovine footrot pathogen Dichelobacter nodosus have been hindered by the lack of a genetic system for this organism. In an attempt to accomplish the transformation of D. nodosus cells, we constructed a plasmid that contained part of a native D. nodosus plasmid and carried a tetracycline resistance gene that was located between the D. nodosus rrnA promoter and terminator. This plasmid was used to transform several D. nodosus strains to tetracycline resistance. Analysis of two independent transformants from each parental strain showed that in nearly all of these derivatives, the plasmid was not replicating independently, but that the tetracycline resistance gene had inserted by homologous recombination into one of the three rrn operons located on the chromosome. In most of the transformants, double reciprocal crossover events had occurred. These results are highly significant for genetic studies in D. nodosus and for footrot pathogenesis studies, since by using reverse genetics it will now be possible to examine the role of putative D. nodosus-encoded virulence genes in the disease process.