Virulent Footrot Research Articles

Preliminary association of microsatellite heterozygosity with footrot in domestic sheep

Genetic heterozygosity in wild, unmanaged animal populations is often associated with protection against infectious disease. However, little is known about the relationship between heterozygosity and disease susceptibility in domesticated livestock, where disease resistance has the potential to improve animal welfare and productivity. We have investigated whether susceptibility to footrot, an important cause of poor welfare and reduced productivity in sheep, is associated with heterozygosity at 14 candidate microsatellite loci. Heterozygosity at locus BMC5221 was associated with resistance to footrot (P=0.0034). This locus was selected based on a gene ontology classification of ‘response to Gram-negative bacteria’. Sheep homozygous at BMC5221 were at increased risk of virulent footrot (OR=4.8, 95% CI=1.5–15.3), with a dose response relationship between homozygosity and disease severity. A highly significant homozygote deficit was observed in sheep without virulent footrot (observed=4, expected=21, χ2=13.76, P=0.0002) but not in sheep that had clinical disease, suggesting homozygotes were disproportionately likely to contract virulent footrot. Our results indicate that genetic heterozygosity might be important for healthy immune function in domesticated livestock. The use of gene ontology codes might prove a useful strategy to target selection of candidate markers in future studies.

Prevalence of footrot in Swedish slaughter lambs

BackgroundFootrot is a world-wide contagious disease in sheep and goats. It is an infection of the epidermis of the interdigital skin, and the germinal layers of the horn tissue of the feet. The first case of footrot in Swedish sheep was diagnosed in 2004. Due to difficulties in distinguishing benign footrot from early cases of virulent footrot and because there is no possibility for virulence testing of strains of Dichelobacter nodosus in Sweden, the diagnosis is based of the presence or absence of clinical signs of footrot in sheep flocks. Ever since the first diagnosed case the Swedish Animal Health Service has worked intensively to stop the spread of infection and control the disease at flock level. However, to continue this work effectively it is important to have knowledge about the distribution of the disease both nationally and regionally. Therefore, the aims of this study were to estimate the prevalence of footrot in Swedish lambs at abattoirs and to assess the geographical distribution of the disease.MethodsA prevalence study on footrot in Swedish lambs was performed by visual examination of 2000 feet from 500 lambs submitted from six slaughter houses. Each foot was scored according to a 0 to 5 scoring system, where feet with score ≥2 were defined as having footrot. Moreover, samples from feet with footrot were examined for Dichelobacter nodosus by culture and PCR.ResultsThe prevalence of footrot at the individual sheep level was 5.8%, and Dichelobacter nodosus was found by culture and PCR in 83% and 97% of the samples from feet with footrot, respectively. Some minor differences in geographical distribution of footrot were found in this study.ConclusionsIn a national context, the findings indicate that footrot is fairly common in Swedish slaughter lambs, and should be regarded seriously.

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet.

Determination of prevalence and economic impact of ovine footrot in central Kashmir India with isolation and molecular characterization of Dichelobacter nodosus

The present study determines the prevalence, economic impact of virulent footrot in central Kashmir, India, along with isolation and molecular characterization of Dichelobacter nodosus ( D. nodosus) where so far no such work has been carried out. Over all 12.54% prevalence of footrot was recorded in central Kashmir with highest (15.84%) in district Srinagar, and least (10.89%) in district Budgam, while it was 13.28% in district Ganderbal. Overall economic impact of footrot was estimated to the tune of Rs 15.82 million annually to the sheep farming in central Kashmir. Out of 370 samples collected from footrot lesions of naturally infected sheep, 200 (54.05%) detected D. nodosus positive by polymerase chain reaction (PCR). Out of these, 132 (66.00%) samples carried serogroup B of D. nodosus, five (2.50%) serogroup E, one (0.50%) serogroup I, while, 53 (26.50%) had mixed infection of serogroups B and E, four (2.00%) of serogroups B and I, two (1.00%) of serogroups B and G and the remaining three (1.50%) samples harboured the mixed infection of serogroups B, E and I. Serogroup G was detected for the first time in India. Over all serogroup B was most frequent (97.0%) followed by E (30.5%), while serogoups I (4.0%) and G (1.0%) were least prevalent. A total of 265 D.nodosus strains were isolated out of which 194 (73.20%) were typed as serogroup B, 61 (23.01%) as serogroup E, eight (3.01%) as serogroup I and remaining two (0.75%) belonged to serogroup G. Out of 265 D. nodosus isolates, 164 (61.88%) possessed intA (integrase) gene, thus were considered as virulent strains. Serogroup wise intA gene was found in 121(62.37%) isolates of serogroup B, 36 (59.01%) of E, two (100%) of G and five (62.50%) of I. Out of 20 randomly selected isolates subjected to gelatin gel test, 16 isolates with intA gene produced thermostable protease while four isolates without intA gene revealed the production of thermolabile protease. This indicated a good co-relation between presence of intA gene and gelatin gel test in determination of the D. nodosus virulence. Thus the present investigation suggests the incorporation of serogroups B and E, based on their predominant prevalence, in the formulation of an effective bivalent vaccine to combat footrot in central Kashmir.

Detection and diversity of a putative novel heterogeneous polymorphic proline-glycine repeat (Pgr) protein in the footrot pathogen Dichelobacter nodosus

Dichelobacter nodosus, a Gram-negative anaerobic bacterium, is the essential causative agent of footrot in sheep. Currently, depending on the clinical presentation in the field, footrot is described as benign or virulent; D. nodosus strains have also been classified as benign or virulent, but this designation is not always consistent with clinical disease. The aim of this study was to determine the diversity of the pgr gene, which encodes a putative proline-glycine repeat protein (Pgr). The pgr gene was present in all 100 isolates of D. nodosus that were examined and, based on sequence analysis had two variants, pgrA and pgrB. In pgrA, there were two coding tandem repeat regions, R1 and R2: different strains had variable numbers of repeats within these regions. The R1 and R2 were absent from pgrB. Both variants were present in strains from Australia, Sweden and the UK, however, only pgrB was detected in isolates from Western Australia. The pgrA gene was detected in D. nodosus from tissue samples from two flocks in the UK with virulent footrot and only pgrB from a flock with no virulent or benign footrot for >10 years. Bioinformatic analysis of the putative PgrA protein indicated that it contained a collagen-like cell surface anchor motif. These results suggest that the pgr gene may be a useful molecular marker for epidemiological studies.

Chronic contagious ovine digital dermatitis-like lesions in a Scottish flock

apparently new, severe condition (Figs. 1-7) first described in 1997. While CODD is reported to present a different clinical picture from virulent footrot (Fig. 8) and white line lesions (Fig. 9) there are few convincing published images. The isolation of spirochaetes resembling those involved in digital dermatitis in cattle from some, but not all, suspected clinical cases in sheep has led to the adoption of the current name. However, the isolation of the same spirochaetes from apparently normal feet, and the occurrence of mixed infections of ovine foot lesions with the causal agents of virulent foot-rot, provide ample opportunity for confusion. This report describes severe lameness of several months’ duration in a group of ten Cheviot ewes in poor body condition presenting with CODD-like lesions.

Crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen Dichelobacter nodosus.

Dichelobacter nodosus is the principal causative agent of ovine footrot, a disease of significant economic importance to the sheep industry. D. nodosus secretes a number of subtilisin-like serine proteases which mediate tissue damage and presumably contribute to the pathogenesis of footrot. Strains causing virulent footrot secrete the proteases AprV2, AprV5 and BprV and strains causing benign footrot secrete the closely related proteases AprB2, AprB5 and BprB. Here, the cloning, purification and crystallization of AprV2, AprB2, BprV and BprB are reported. Crystals of AprV2 and AprB2 diffracted to 2.0 and 1.7 A resolution, respectively. The crystals of both proteases belonged to space group P1, with unit-cell parameters a = 43.1, b = 46.0, c = 47.2 A, alpha = 97.8, beta = 115.2, gamma = 115.2 degrees for AprV2 and a = 42.7, b = 45.8, c = 45.7 A, alpha = 98.4, beta = 114.0, gamma = 114.6 degrees for AprB2. Crystals of BprV and BprB diffracted to 2.0 and 1.8 A resolution, respectively. The crystals of both proteases belonged to space group P2(1), with unit-cell parameters a = 38.5, b = 89.6, c = 47.7 A, beta = 113.6 degrees for BprV and a = 38.5, b = 90.5, c = 44.1 A, beta = 109.9 degrees for BprB. The crystals of all four proteases contained one molecule in the asymmetric unit, with a solvent content ranging from 36 to 40%.

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot ( Dichelobacter nodosus) vaccines in sheep

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.

Pilot trials in Australia on eradication of footrot by flock specific vaccination

Footrot is a contagious disease of ruminants requiring strains of Dichelobacter nodosus that possess virulence factors including proteases and fimbriae. Sheep can be immunised against footrot using vaccine-containing fimbriae, either native or recombinant. The fimbriae are responsible for the serological K-agglutination reaction, which has been used to classify field isolates into nine major serogroups. The range of protection conferred by vaccination is largely restricted to the serogroup involved, but antigenic competition precludes effective vaccination with multivalent vaccines that contain all serogroups. However, vaccination with specific bivalent recombinant fimbrial vaccine led to eradication of virulent footrot from small ruminants in Nepal and the same result was obtained in Bhutan using a specific whole cell vaccine. In the study reported here two pilot trials have been conducted in Australian sheep flocks, one with a virulent form of footrot caused by a single serogroup F, and the other with an intermediate form also caused by a single serogroup C. In trial 1 pre-vaccination prevalence of clinical footrot in a group of randomly selected animals was 44%. This reduced to 2% at 3 months and 0.5% at 4 months, and there were no clinical cases at 5 months or at 16 months post-vaccination in the whole flock. Similarly in trial 2 pre-vaccination whole flock prevalence was 8.5%, while it was 2% at 3 months, 0.3% at 6 months and zero at 18 months post-vaccination. Use of flock specific monovalent whole cell vaccines over whole flocks for only one season and culling of the few non-responders has been a successful approach in eradication of the disease from both these flocks. This is the first study to report the successful use of specific vaccine for the intermediate form of footrot.

Improved diagnosis of virulent ovine footrot using the intA gene

Footrot is a mixed bacterial infection of the hooves of sheep. The Gram-negative anaerobic bacterium Dichelobacter nodosus is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. In Australia, in the state of New South Wales (NSW), only virulent footrot is subject to regulatory action, including quarantine. However, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection, or under adverse climatic conditions. The gelatin gel test, which measures the thermostability of secreted bacterial proteases, is the laboratory test most widely used in Australia to aid in the differential diagnosis of footrot. The proteases of virulent strains are, in general, more thermostable than the proteases of benign strains. However, there are some false positives in the gelatin gel test, which may lead to unnecessary quarantine procedures. We used Southern blot analysis on 595 isolates of D. nodosus from 124 farms on which sheep had benign or virulent footrot to test for the presence of the intA gene. We found that for D. nodosus strains which are stable in the gelatin gel test, there is a high correlation between the presence of the intA gene and the ability of the strain to cause virulent footrot. We also developed a PCR-based assay for the rapid detection of intA, which can be used to test DNA extracted from colonies grown on plates, or DNA extracted from cotton swabs of culture plates.

The use of an autogenous Dichelobacter nodosus vaccine to eliminate clinical signs of virulent footrot in a sheep flock in Bhutan

An outbreak of virulent footrot was investigated in a flock of 605 Merino cross-bred sheep in Bhutan. Conventional control methods in the preceding eight years had reduced its prevalence from 36–79% in different components of the flock to about 15% overall. Only one serogroup (B) of Dichelobacter nodosus was identified among 40 isolates cultured from affected sheep. A vaccine prepared from this strain was used in a pilot trial to compare the response of 14 treated and 14 untreated sheep. All affected, vaccinated animals in this trial healed quickly and were protected against re-infection while additional cases developed among untreated sheep during a period favourable for the spread of footrot. The serogroup B vaccine was administered to the whole flock for two successive years. No other footrot treatment was given during these or subsequent years. The whole flock was examined three times, foot by foot, for two years and twice yearly for another two years. When vaccination began there were 88 affected sheep in the flock, an affected sheep being defined as an animal with a foot-score of 2 or greater in one or more feet. There were neither affected sheep in the flock 30 days after the first dose of vaccine nor were any identified in later inspections. Virulent footrot, originating from the farm under investigation, persisted in neighbouring village flocks during this period. It was concluded that whole flock specific D. nodosus vaccination made a major contribution to the elimination of all clinical signs of footrot from the flock of 605 sheep where the condition had previously persisted for 10 years.

Effect of climatic region on the clinical expression of footrot of lesser clinical severity (intermediate footrot) in sheep.

To determine if the clinical classification of intermediate footrot (IFR) is changed to virulent footrot (VFR) by a transfer of the infected flock to a region where climatic conditions are more favourable for the transmission of the disease. Clinical examination of two groups of Merino wethers infected with IFR; one group of 309 in a region considered less favourable for footrot and another group of 343 at a second site considered more favourable. After characterising the form of footrot at the first site, infection was established at the second site by mixing 142 wethers from the first site with 201 unrelated wethers considered to be free of IFR and VFR. Observations of clinical characteristics were made over a 16 month period during which an outbreak of footrot occurred. Clinical assessments were made by inspecting every foot of every sheep at regular intervals and allocating a footscore. Evidence that the same clonal lines of D. nodosus were responsible for the footrot at both sites was provided by serotyping of isolates and using omp gene RFLP as a molecular epidemiological tool. The disease at the first site was classified as IFR because 7% of the sheep developed a maximum footscore (MFS) of 4, the most severe category, despite relatively low rates of transmission. When the outbreak occurred at the second site, which was more suitable for footrot transmission, the maximum proportion of the flock that developed a MFS of 4 was 3.6%, confirming the initial classification of IFR. When a flock infected with IFR was moved to a region where climatic conditions were more favourable for footrot transmission, the clinical classification of the disease remained the same in both the original flock and in sheep exposed to the infection for the first time.

Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination

Programmes based on the identification and treatment of cases and the culling of animals refractory to treatment had failed to eradicate virulent footrot from two districts in the western region...

Pilus ELISA and an anamnestic test for the diagnosis of virulent ovine footrot and its application in a disease control program in Nepal

The immunological memory (anamnestic) responses in sheep recovered from virulent footrot (VFR) can be aroused by subcutaneous injection of outer membrane protein (OMP) antigens of Dichelobacter nodosus. The magnitude of this response is directly correlated to the highest antibody response attained during infection and memory lasts at least a year after recovery from VFR. However, some older animals show non-specific responses to OMP antigens. In this study an evaluation of D. nodosus pilus antigen for the anamnestic diagnosis of footrot in sheep was undertaken. The results indicated that the primary and anamnestic responses to pilus were similar in character to OMP antigen but were highly specific. The sensitivity of the procedure for detection of sheep with a history of VFR was approximately 80%. A low proportion of sheep with mild lesions due to virulent strains of D. nodosus reacted to anamnestic challenge. Anamnestic challenge with 10 μg pilus was used in a VFR surveillance program in migratory sheep flocks in Nepal. Conventional diagnostic methods could not be applied during the disease transmission periods in these flocks because of their migration to alpine pastures far away from human habitation. The results supported clinical and bacteriological findings suggesting that virulent strains of D. nodosus have apparently been eliminated from these flocks in Nepal.

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

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. 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. Each fortnight, all feet were examined, their lesion scores were recorded and samples of lesion material were taken for laboratory tests. 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. 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.

Transmission of virulent footrot between sheep and goats.

To determine the infectivity of ovine and caprine strains of Dichelobacter nodosus for both sheep and goats. Pen experiments in which 20 sheep and 19 goats were challenged directly with the two strains, and transmission experiments on pasture, using donors infected by experimental challenge. Sheep and goat strains of D nodosus infected both animal species in experimental challenges. Animals so infected transmitted footrot to both sheep and goats on pasture plots. A significantly smaller proportion of goats than sheep was infected when challenged with either strain. The interval between exposure and development of footrot in goats was longer than in sheep when recipient animals were exposed to infected donors on pasture. The disease was less invasive in goats than in sheep. With the strains of D nodosus used there was no evidence of host specificity. Direct transmission of footrot can occur between sheep and goats in the same environment. There is a need to include goats in ovine footrot eradication programs and vice versa.

Effectiveness of small workshops for improving farmers' knowledge about ovine footrot.

To determine sheep farmers' attitudes to and beliefs about ovine footrot, and to improve their knowledge about the diagnosis, control and eradication of this disease. Eighteen workshops, involving 291 farmers, were conducted across Victoria in the spring of 1996. The workshops were designed as small-group discussions with a maximum attendance of 20 farmers to encourage active participation. All participants completed questionnaires before each workshop and 12 to 40 weeks after the last workshop. Before the workshops the farmers had a poor understanding of the principles of diagnosis, control and eradication of footrot. For example, only 50% knew the footrot organism survived in soil for less than 7 days, over two-thirds did not know the reason for paring sheep's feet during an eradication program, and only 31% realised cattle were a potential source of footrot infection for sheep. After the workshops, understanding about footrot was significantly improved; 87% said Dichelobacter nodosus survived in soil less than 7 days, 71% knew the reason for paring sheep's feet and 64% realised that cattle were a potential source of footrot infection. As well as improved knowledge, change of attitude among farmers is fundamentally important if virulent footrot is to be successfully controlled and eradicated. The workshops successfully initiated this process; 40% of farmers thought the workshops changed their attitudes to footrot, while 37% said they gained an increased understanding of other people's opinions about the disease. Farmers' poor understanding of ovine footrot is a constraint to the programs aimed at controlling this disease. Small group workshops may be an effective way to influence farmers' attitudes and beliefs, and could facilitate the effectiveness of regulatory disease control programs.

Comparison of erythromycin and oxytetracycline for the treatment of virulent footrot in grazing sheep

Comparison of erythromycin and oxytetracycline for the treatment of virulent footrot in grazing sheep

A survey of sheep flocks on Kangaroo Island for virulent footrot.

A survey of sheep flocks on Kangaroo Island for virulent footrot.

Further observations on the primary and anamnestic humoral responses to Dichelobacter nodosus in sheep in relation to the diagnosis of footrot

An anamnestic serological test for ovine footrot was evaluated. Footrot-free lambs were infected with Dichelobacter nodosus and treated four, six or eight weeks later. There were strong linear correlations between the severity of the lesions and both the primary/response and the anamnestic response evoked by the subcutaneous injection of an antigen from D nodosus 16 weeks after the treatment of the lambs; the latter correlation was stronger than the correlations reported elsewhere in mature sheep. Similar anamnestic responses were elicited six and 12 months after the treatment of mature sheep which had had severe lesions. Natural anamnestic responses were demonstrable in sheep which had had recurrent clinical episodes of virulent footrot. The non-specific hummoral responses after the anamnestic challenge of footrot-free sheep increased with age and did not depend on the dose of the antigen between 10 and 200 pg. Using the pooled data from sheep of all ages and a positive-negative cut-off which was selected to obtain a sensitivity of 75 per cent, the specificity of the anamnestic test was 90 per cent, similar to that reported for the primary response when it was used to diagnose footrot. The anamnestic test can be applied to determine the presence and severity of footrot in young sheep.