Failure to eradicate ovine footrot associated with Dichelobacter nodosus strain A198 by repeated daily footbathing in zinc sulphate with surfactant.

OBJECTIVE: To investigate the effect of repeated daily footbathing in zinc sulphate on virulent ovine footrot associated with S1, U1 and U5 zymogram types of Dichelobacter nodosus, including the highly virulent S1 strain A198. DESIGN: A field trial with experimentally infected sheep. PROCEDURE: At week 0, 50 sheep were infected with D. nodosus strains A198 (S1), C305 (U1), BC3993 (U5) and BC3995 (U5). At weeks 1 and 47, respectively, 169 and 235 uninfected sheep were added. At week 60, sheep were allocated to control and treatment groups each containing 220 sheep. Every 2 or 4 weeks to week 113, feet were inspected, and lesions were scored and sampled. Treatment sheep were footbathed in 15 to 18% (w/v) zinc sulphate with surfactant for 5 consecutive days (10 min per day) during week 61. During week 110, the footbathing protocol was applied again, this time to all surviving treatment and control sheep. RESULTS: BC3993 and A198 were isolated from 57% (162 of 285) and 20% (58 of 285), respectively, of new lesions sampled between weeks 3 and 13, and 21% (57 of 271) and 50% (136 of 271) between weeks 49 and 59. Percentages of new lesions associated with C305 and BC3995 remained constant. During the initial 17 weeks after footbathing at week 61, 90% (75 of 83) and 19% (95 of 490) of lesions in treatment and control sheep, respectively, were score 4 or 5, and 94% (47 of 50) and 38% (33 of 87) of those were associated with A198. CONCLUSIONS: Repeated daily footbathing did not eradicate virulent ovine footrot because strain A198 produced deep, covert lesions that facilitated the survival of D. nodosus.

Eradication of ovine footrot by repeated daily footbathing in a solution of zinc sulphate with surfactant.

OBJECTIVE: To investigate the effect on ovine footrot of repeated daily footbathing in a solution of zinc sulphate with surfactant. DESIGN: Merino sheep were allocated to control and treatment groups of 119 sheep each at week 0. The sheep had a history of S1, U1, T and/or U6 types of Dichelobacter nodosus in interdigital and underrunning footrot lesions. Feet were not pared prior to treatment. PROCEDURE: Treatment sheep were footbathed in a 15 to 18% (w/v) solution of zinc sulphate with surfactant for 10 min on five consecutive days during week 1. At week 2, and fortnightly to week 52, all feet were inspected, lesion scores were recorded and samples were taken for laboratory tests. At week 53, all feet with no lesions at week 52, but with underrunning lesions prior to week 1, were pared and samples were taken. RESULTS: After footbathing, there were no lesions in any treatment sheep at any inspection to week 52. The percentage of feet of control sheep with lesions increased from 9% (391 of 4,284) between weeks 20 and 36, to 14% (593 of 4,284) between weeks 36 and 52. Ninety-five of 96 control sheep with no lesions at week 20 were still asymptomatic at week 52. D nodosus was not isolated from samples taken from 99 and 87 pared feet of treatment and control sheep, respectively. CONCLUSION: Repeated daily footbathing combined with prolonged exposure to a dry environment eradicated footrot in sheep with both interdigital and underrunning lesions in feet that were not pared prior to treatment.

Occurrence of different strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections.

OBJECTIVE: To investigate the occurrence of S1, U1 and T strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections. DESIGN: Seventy-seven donor sheep were grazed with 84 recipients for 33 weeks. The donor sheep were Merinos with a history of clinically virulent footrot associated with protease type S1, U1 and T strains of D nodosus that hybridised with gene sequences pJIR314B, pJIR318 and/or pB645-335. The recipient sheep were Merinos with no history of footrot. PROCEDURE: Each fortnight, all feet were examined, their lesion scores were recorded and samples of lesion material were taken for laboratory tests. RESULTS: Eighty-nine percent (299 of 336) of feet of recipient sheep developed new clinical lesions. S1, U1 and T strains of D nodosus were recovered from 58%, 22% and 18%, respectively, of these lesions at a ratio that remained constant during two apparent peaks in footrot transmission. Gene sequences homologous to pJIR314B and pB645-335 were detected in 56% (93 of 166) and 29% (48 of 166), respectively, of S1 strains of D nodosus at a ratio that was not constant during the experiment. CONCLUSIONS: S1 was the dominant protease type of D nodosus in new clinical lesions. The occurrence of S1 strains did not increase relative to U1 and T strains of D nodosus during the experiment. S1, U1 and T strains of D nodosus remained in equilibrium despite changes in environment, genetic types in the population of S1 strains, and host resistance to footrot.

Severity and persistence of footrot in Merino sheep experimentally infected with a protease thermostable strain of Dichelobacter nodosus at five sites.

OBJECTIVE: To test the hypothesis that ovine footrot associated with a thermostable protease strain of Dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment. DESIGN AND PROCEDURE: Forty Merino sheep from a single blood line were infected with a protease thermostable strain of D nodosus at each of five sites in Western Australia. Footrot lesions and microscopic evidence of D nodosus were recorded every fortnight for 2.5 years, supplemented by laboratory culture. Rainfall, soil and air temperature, pasture quantity and composition and soil types were also recorded. Flocks that apparently self cured were relocated to a more favourable site for footrot in the final spring season. RESULTS: The maximum prevalence of feet with clinical footrot lesions was 80.6, 1.3, 14.4, 3.8 and 88.1% at the five sites. Severe footrot occurred for three consecutive spring seasons at one site that had clay loam soil and at least 3500 kg/ha total pasture dry matter annually. However, the infection was asymptomatic for up to 10 weeks between outbreaks. D nodosus was isolated from flocks for 2.5 years at only two sites, although there was microscopic evidence of the organism at other sites in the final year. A thermolabile variant (strain U6) of D nodosus was isolated from the two sites where footrot persisted. CONCLUSION: Depending on time and location, ovine footrot induced by a protease thermostable strain of D nodosus either self cured or persisted as annual outbreaks interspersed with periods of asymptomatic infection.

The laboratory culture of Dichelobacter nodosus in a footrot eradication program.

As part of a program to eradicate virulent footrot from Western Australia, 2745 isolates of Dichelobacter nodosus were isolated from 5263 specimens from 1883 submissions. The virulence of each isolate was assessed using protease thermostability and isoenzyme zymogram. We describe changes to the materials and methods required to handle these specimens and to reduce the interval between submission and report to 8 days.

Identification of three gene regions associated with virulence in Dichelobacter nodosus, the causative agent of ovine footrot.

Dichelobacter nodosus (formerly Bacteroides nodosus) is a Gram-negative strict anaerobe and is the primary pathogen involved in ovine footrot. A comparative hybridization strategy was used to isolate recombinant clones which hybridized to DNA from a virulent strain of D. nodosus but not with a benign isolate. Three virulence-associated gene regions were identified and one of these regions was shown to be present in multiple copies in the D. nodosus genome. Hybridization studies on 101 clinical isolates of D. nodosus showed that these strains could be divided into three hybridization categories which could be correlated with the virulence of the isolates. The recombinant clones have considerable potential for the development of a gene-probe-based method for the differential diagnosis of ovine footrot.

Characterisation of virulent and benign strains of Bacteroides nodosus.

The extracellular proteases of 395 isolates of B. nodosus from ovine, bovine and caprine foot lesions were classified as either thermostable or thermolabile. Stable protease was associated with one and unstable protease with four distinctive isoenzyme patterns, each pattern differentiated by the relative mobility of paired isoenzymes. Pathogenicity tests on 64 isolates showed a correlation between the production of stable protease and the production of virulent ovine footrot lesions. The mean values for total protease activity, twitching motility and colony diameter were significantly higher for virulent compared to benign isolates, but the range of values overlapped. SDS-PAGE whole-cell electrophoretic profiles of virulent isolates were similar to the profiles of some benign isolates.

Measurement of protease thermostability, twitching motility and colony size of Bacteroides nodosus.

Total extracellular protease activity of Bacteroides nodosus in TAS liquid culture varied directly with cell mass and buffer concentration between 20 and 50 mM HEPES, MOPS and TES buffers, but not with Tris which gave anomalous high cell counts, nor with Na2Co3 which showed a decline of protease activity and cell mass. The stability of HEPES-buffered crude protease preparations were estimated on the basis of temperature or Ca2+ activity. Variation of the estimates for cellular twitching was greater than that for colony diameter in benign and virulent strains of B. nodosus. Surface translocation, quantified on the basis of colony diameter, reached a limit after 72 h incubation on modified TAS agar, ranging from 0.04 to 0.14 mm per h for six isolates tested.

Motility in relation to virulence of Bacteroides nodosus.

Fourteen Bacteroides nodosus isolates from footrot lesions of sheep were examined microscopically and all were found to have twitching motility. The mean percentage of cells showing motility was 40% and 9% for virulent and benign strains, respectively. This corresponded with mean agar colony diameters of 17 mm and 7 mm, respectively, for these strains. Two strains of intermediate virulence had values of motility and colony diameter similar to the benign strains. However, the intermediate and the virulent strains produced relatively stable protease compared to the benign strains. All virulent, benign and intermediate strains produced abundant pili. Included for comparison in this study was an avirulent variant strain which was highly motile, formed large colonies and produced stable protease, but showed no pili on electron microscopy. It was concluded that the properties of motility and protease stability may be used to distinguish, in the laboratory, wild-type virulent, benign and intermediate strains of B. nodosus.

The thermostability of proteases from virulent and benign strains of Bacteroides nodosus.

Protease enzymes, produced by Bacteroides nodosus strains isolated from animals with virulent and benign forms of ovine footrot, were partially purified by ultra-filtration, ion exchange chromatography and gel permeation chromatography. Each enzyme had a similar pH optimum, was inhibited by phenylmethylsulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA) and ethyleneglycot-bis-aminoethylether-N,N-tetraacetic acid (EGTA), but was not inhibited by 1,10-phenanthroline. The results suggest that these enzymes are serine proteases that require divalent cations for activity. The enzymes could be distinguished by their differential temperature stability and differing susceptibility to irreversible inactivation by EDTA. Both enzymes were stabilised by incubation in the presence of Ca2+, but the enzyme purified from the virulent isolate required less Ca2+ for maximum stability. These results suggest that the differential thermostability of the protease activity detected in virulence tests is an intrinsic property of the protease enzymes.

Isolation and characterisation of Bacteroides nodosus from foot lesions of cattle in Western Australia.

Thirty one isolates of Bacteroides nodosus were obtained from foot lesions observed on cattle at 3 abattoirs. All isolates were similar to the B. nodosus of ovine benign footrot (BFR) in their response to the degrading proteinase test. At one abattoir, where the interdigital lesions were examined in detail, 9 of 10 isolates were obtained from hyperkeratotic lesions with deep fissures. Traceback to 8 of the farms of origin which carried both sheep and cattle, revealed BFR in sheep on 4 farms. The significance of B. nodosus in interdigital lesions in cattle, and its possible pathogenicity, are discussed.

A degrading proteinase test to distinguish benign and virulent ovine isolates of Bacteroides nodosus.

Bacteroides nodosus was recovered from naturally occurring cases of virulent ovine footrot (VFR) and benign footrot (BFR) using an artificial culture medium which incorporated trypticase, arginine, serine and 5% agar. A degrading proteinase (DP) test was developed which measured the proteinase activity of broth cultures of B. nodosus for a period of time after organism death to assess the stability of the enzyme. The spectrophotometric measurement of the release of dye from a hide powder--azure conjugate by the action of proteinase provided an objective analysis of enzyme activity. The DP test differentiated VFR and BFR isolates and promises to be a useful laboratory method for the diagnosis of benign and virulent footrot in sheep.