The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales

Footrot, caused by the strictly anaerobic bacterium Dichelobacter nodosus, is the most common cause of lameness in sheep in Great Britain but problems exist in association with its diagnosis and control. The fastidious nature of D. nodosus means that complex media and several weeks are required for characterisation. An alternative method to simplify and enhance the detection of D. nodosus in clinical samples is therefore highly desirable. In terms of control, anecdotal evidence from the farming community suggests that the commercially available vaccine, based on Australian isolates of D. nodosus, is not widely employed in this country due to its perceived inefficacy. Seven hundred and six isolates, collected from outbreaks in England and Wales, were therefore used to investigate these issues. A 16S rRNA PCR was adapted to detect D. nodosus in clinical material within 1 day of sampling; a 15% increase in detection compared with culture and less than 1% false negatives were achieved. This represents a major advance in the rapid diagnosis of footrot and will be of great value to practitioners and diagnostic laboratories. Bacterial virulence was tested using protease thermostability and zymogram assays, whilst serogrouping was performed by slide agglutination. All isolates demonstrated virulence patterns previously recorded in Australia and all nine serogroups of D. nodosus (A-I) were represented. Serogroup H was predominant. There was, therefore, no evidence for the presence of novel strains of D. nodosus compared with Australia suggesting the need for further investigation into farmers' views on the use of the commercial vaccine in Great Britain.     

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.     

Genetic characterisation of protective vaccine responses in sheep using multi-valent Dichelobacter nodosus vaccines

Protective vaccine responses to nine distinct serogroups of Dichelobacter nodosus (serogroups A-I) can be readily measured by serogroup-specific K-agglutinating antibody titres. On the basis of a large quantitative genetic experiment (1200 progeny from 129 sire groups), it was shown that variation in antibody responses following vaccination with a multi-valent pilus antigen D. nodosus vaccine (serogroups A-I) is, in part, under genetic control and thus heritable. Based on the genetic relationships between antibody responses to all nine antigens, results suggested that both genes for a broad-based and genes for serogroup-specific response contributed to genetic variation in vaccine response. Furthermore, preliminary data in 389 progeny showed that polymorphism within the ovine major histocompatibility (MHC) based on serological classification accounted for a significant proportion of the variation in vaccine responses. In subsequent experimentation, we examined the importance of genetic polymorphism within the ovine MHC, and the possibility of genes outside the MHC for their involvement in antigen-specific and broad-based vaccine response. Within two large half sib families(131, and 143 progeny), four MHC haplotypes were investigated and found to be associated with differential antibody responses to six out of eight distinct vaccine-antigens presented to the host in a multi-valent vaccine. The model used here shows how well characterised immunogens, quantitative genetic experimentation, and molecular gene mapping tools can be used to unravel genetic differences in host responses to commercial vaccines.     

Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.     

Farmer reported prevalence and factors associated with contagious ovine digital dermatitis in Wales: A questionnaire of 511 sheep farmers

In 2012, 2000 questionnaires were sent to a random sample of Welsh sheep farmers. The questionnaire investigated farmers’ knowledge and views on contagious ovine digital dermatitis (CODD) – an emerging disease of sheep responsible for causing severe lameness, welfare and production problems. The overall response rate was 28.3% with a usable response rate of 25.6%. The between farm prevalence of CODD was 35.0% and the median farmer estimated prevalence of CODD was 2.0%. The disease now appears endemic and widespread in Wales. Furthermore, there has been a rapid increase in reports of CODD arriving on farms since the year 2000. Risk factors for CODD identified in this study include the presence of bovine digital dermatitis (BDD) in cattle on the farm and larger flocks. Farmers also consider concurrent footrot/interdigital dermatitis, buying in sheep, adult sheep, time of year and housing to be associated with CODD. Further experimental research is necessary to establish whether these observations are true associations.     

Effectiveness of different adjuvants in stimulating Dichelobacter nodosus antibody in sheep vaccinated against ovine footrot

This research consists of an evaluation of the effectiveness of different substances administered as adjuvants in the stimulation of humoral immune response induced by the vaccine composed of strains A1, A2 and C of Dichelobacter nodosus. To do this, a total of 120 Merino sheep were vaccinated and revaccinated. These sheep were selected from a farm located in the region of Extremadura (Spain), and they were divided into 12 groups of 10 animals each. An additional group with 10 sheep was used as control. The immune response (titre of antibodies) was determined by agglutination tests and ELISA. The most pronounced immune response was obtained by the use of Freund's incomplete adjuvant and aluminium hydroxide as adjuvants.     

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.     

Effects of parenteral amoxicillin on recovery rates and new infection rates for contagious ovine digital dermatitis in sheep

The present study is a randomised split-flock treatment trial, which compared the effect of foot bathing in a 1 per cent solution of chlortetracycline alone with a treatment protocol that added a single injection of a long-acting amoxicillin. Overall, the prevalence of contagious ovine digital dermatitis in the examined flock was 22 per cent, while 45.7 per cent of affected sheep had infections in two or more feet. Parenteral antibiotic treatment increased the odds of a recovery by 3.8 times (95 per cent confidence interval 1.05 to 14.0) (P=0.008). Moreover, the amoxicillin injection may also have had a preventative effect, reducing the rate of establishment of new infections from 2.5 per cent for foot bathing alone compared with 1.0 per cent with the addition of parenteral amoxicillin.     

中西医结合治疗羊传染性脓疱

2004年10月27日,我县王甫乡一养羊户的羊只发生以唇部皮肤呈菜花样增生、出血、溃烂为特征的疫病,畜主称之为"烂嘴病",该病传播快,发病率高,给养羊户造成严重的经济损失.笔者根据流行病学调查、临床症状诊断为羊传染性脓疱,采用中西医结合治疗,收到了较好的疗效.     

The laboratory culture of Dichelobacter nodosus in a footrot eradication program

SUMMARY 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.     

Host and environmental reservoirs of infection for bovine digital dermatitis treponemes

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.     

Fusobacterium necrophorum,and not Dichelobacter nodosus,is associated with equine hoof thrush

The aim of this study was to determine which of the two species, Fusobacterium necrophorum or Dichelobacter nodosus, are associated with hoof thrush in horses. Fourteen hoof samples, collected from eight horses with thrush and 14 samples collected from eight horses with healthy hooves, were examined for the presence of F. necrophorum, Fusobacterium equinum and D. nodosus. Only isolates with phenotypic characteristics representing Fusobacterium could be cultured. Total DNA extracted from the 28 hoof samples was amplified by using DNA primers designed from gene lktA, present in F. necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. equinum, and gene fimA, present in D. nodosus. The lktA gene was amplified from five of the 14 infected hoof samples and from one hoof sample without thrush. The DNA sequence of the amplified ltkA gene was identical to the lktA gene of the type strain of F. necrophorum (GenBank accession number AF312861). The isolates were phenotypically differentiated from F. equinum. No DNA was amplified using the fimA primer set, suggesting that F. necrophorum, and not D. nodosus, is associated with equine hoof thrush. Hoof thrush in horses is thus caused by F. necrophorum in the absence D. nodosus. This is different from footrot in sheep, goats, cattle and pigs, which is caused by the synergistic action of F. necrophorum and D. nodosus.     

Update on ovine footrot in New Zealand: isolation, identification, and characterization of Dichelobacter nodosus strains [corrected

Dichelobacter nodosus, a Gram-negative strict anaerobe, is the essential causative agent of ovine footrot. Despite its worldwide presence, the disease has significant economic impact in those sheep-farming countries with a temperate climate and moderate to high rainfall, such as New Zealand (NZ) and Australia. In this study, we aimed to isolate, identify, and characterize as many D. nodosus strains as possible from NZ farms by using polymerase chain reaction (PCR)-based technology. Understanding the virulence of this bacterium and showing extensive genomic variation in the fimbrial subunit gene (fimA) in different D. nodosus strains was very important to produce serogroup specific and effective vaccine for NZ. More than 100 footrot samples were collected from four different farming regions in NZ. Thousands of primary plates were cultured anaerobically and examined with Gram-staining in order to detect single colonies of D. nodosus. Approximately 500 plates that had potential D. nodosus colonies were subcultured several times to eliminate contaminating colonies until single colonies were obtained. Variable and a part of the conserved regions of the fimbrial subunit gene (fimA) were amplified directly from bacterial DNA extracted from footrot lesions and also from cultured NZ D. nodosus isolates, using the polymerase chain reaction. Different fimA amplimers were analyzed by DNA sequencing. On the basis of DNA sequence analysis, 16 new D. nodosus isolates belonging to eight different serogroups were identified from NZ. These new D. nodosus fimA sequences from NZ were different to previously reported strains and strains used in a commercial vaccine.