The virulence of Dichelobacter nodosus,measured by the elastase test,is an important predictor for virulent footrot diagnosis in New South Wales sheep flocks

Ovine footrot is a contagious bacterial disease that causes foot lesions, and depending on the virulence of the causative strains, may lead to severe underrunning of the hoof and lameness. Virulent footrot can be identified, treated and controlled more effectively than less virulent benign forms. The in vitro elastase test for virulence of the causative bacteria, Dichelobacter nodosus, has been used to support clinical diagnosis. However, not all laboratory-designated virulent D. nodosus strains cause clinical signs of virulent footrot. This study evaluated retrospectively how well the elastase test supported clinical footrot diagnosis in 150 sheep flocks examined for suspect footrot in New South Wales between August 2020 and December 2021. Flocks were included if measures of clinical disease, environmental conditions and the virulence of D. nodosus isolates were available. Variation in the elastase activity result between D. nodosus isolated from the same flock made bacterial virulence hard to interpret, but calculating the mean elastase rate for all isolates from the same flock made correlations between bacterial virulence and flock footrot diagnosis possible. Simplifying bacterial virulence into whether there were any elastase-positive D. nodosus isolates before 12 days increased the predictive value of elastase results for virulent diagnosis, compared with using the first day that any isolate was elastase positive or the percentage of elastase-positive isolates by 12 days, but not all clinically virulent flocks had isolates with elastase activity before 12 days. Logistic regression models were fitted to identify the minimum number of predictors for virulent footrot diagnosis, with models suggesting that virulent footrot diagnosis was best predicted by adding the elastase test result and environmental conditions to the prevalence of severe foot lesions (score 4 and 5). However, performing the same analysis with different breeds, ages of sheep and seasons might highlight other factors important in the diagnosis of virulent footrot.     

Determination of prevalence,serological diversity,and virulence of Dichelobacter nodosus in ovine footrot with identification of its predominant serotype as a potential vaccine candidate in J&K,India

The aim of this study was to determine the prevalence, serological diversity, and virulence of Dichelobacter nodosus in footrot lesions of sheep and identification of its predominant serotype as a potential vaccine candidate. The overall prevalence of footrot in sheep was 16.19%, and ranged from 13.69 to 19.71%, respectively. A total of 759 flocks with 22,698 sheep were investigated for footrot and 2374 clinical samples were collected from naturally infected sheep exhibiting footrot lesions. Of the 2374 samples collected, 1446 (60.90%) were positive for D. nodosus by polymerase chain reaction (PCR). These positive samples when subjected to serogroup-specific multiplex PCR, 1337 (92.46%) samples carried serogroup B, 247 (17.08%) possessed serogroup E, 86 (5.94%) serogroup I, and one (0.069%) serogroup G of D. nodosus. While mixed infection of serogroups B and E was detected in 127 (8.78%), B and I in 46 (3.18%) and B, E, and I in 26 (1.79%) samples, respectively. The serogroup B of D. nodosus was the predominant (92.47%) serogroup affecting sheep population with footrot followed by serogroup E (19.91%) and serogroup I (4.57%), respectively. Virulent status of D. nodosus strains were confirmed by presence of virulence-specific integrase A (intA) gene and the production of thermostable proteases. The intA gene was detected in 709 (72.79%) samples while gelatin gel test carried out on 246 representative isolates all positive for intA gene produced thermostable proteases, confirming their virulence nature. The PCR-restriction fragment length polymorphism (PCR-RFLP) of whole fimA gene of serogroup B revealed the predominance of serotype B5 (82.97%) of serogroup B. This information suggests that serotype B5 is the predominant serotype of D. nodosus associated with severe footrot lesions in sheep in Jammu & Kashmir (J&K), India. Hence, this serotype can be a potential vaccine candidate for the effective control and treatment of ovine footrot.

     

Prevalence of footrot in Swedish slaughter lambs

Background

Footrot 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.

Methods

A 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.

Results

The 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.

Conclusions

In a national context, the findings indicate that footrot is fairly common in Swedish slaughter lambs, and should be regarded seriously.     

An evaluation of the ability of Dichelobacter nodosus to survive in soil

Background

Dichelobacter nodosus is the causative agent of footrot in sheep. The survival of the bacterium in soil is of importance for the epidemiology of the disease. The investigation evaluates the survival of D. nodosus in soil with and without added hoof powder stored under different temperatures.

Results

An experimental setup was used with bacteriological culture and real-time polymerase chain reaction (PCR), and the results indicate that the bacteria can survive in soil for longer time than previously expected. The survival time was found to be dependent on temperature and the addition of hoof powder to the soil, with the longest survival time estimated to be 24 days in soil samples with hoof powder stored at 5°C.

Conclusion

Our findings indicate that the survival time of D. nodosus and its ability to infect susceptible sheep on pasture under different climatic conditions should be studied further.     

Molecular genetic analysis of Dichelobacter nodosus proteases AprV2/B2, AprV5/B5 and BprV/B in clinical material from European sheep flocks

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.     

ISOLATION AND CHARACTERISATION OF BACTEROIDES NODOSUS FROM FOOT LESIONS OF CATTLE IN WESTERN AUSTRALIA

SUMMARY 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.     

Characteristics of Bacteroides nodosus isolated from cattle

Fourteen isolates of Bacteroides nodosus were cultured from cattle on six farms and examined for colony morphology, pilation, agglutinability, proteolytic activity and pathogenicity for sheep. Where sheep were also present on the farms, isolates from these were compared with those from cattle.Colonies of the bovine isolates were moderately fimbriate. Cells from these colonies were pilated, but not to the extent observed on virulent sheep isolates. The proteolytic activity of the isolates was also less than that described for virulent ovine isolates. When inoculated into sheep, B. nodosus from cattle produced only mild interdigital inflammation. There was no separation of horn characteristic of virulent foot-rot. Preliminary studies, based on agglutination tests, suggest that B. nodosus with different surface antigens may occur in cattle in the same herd and in cattle and sheep on the same farm. On one of six farms isolates from cattle and sheep were indistinguishable by the agglutination test.     

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

Background

Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR.

Methods

A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.

Results

The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.

Conclusions

The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.     

The first nosocomial outbreak of methicillin-resistant Staphylococcus aureus in horses in Sweden

Background

Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak.

Method

All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak.

Results

Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010.Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE.Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days).

Conclusions

Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.     

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen <Emphasis Type="Italic">Dichelobacter nodosus</Emphasis>

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.     

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.     

The effects of antigenic competition on the efficacy of multivalent footrot vaccines

SUMMARY A multivalent footrot vaccine has been developed, containing pilus antigens produced in recombinant Pseudomonas aeruginosa and representing all nine serogroups of Dichelobacter (Bacteroides) nodosus commonly recognised in the field. The responses of sheep to the multivalent vaccine have been compared with those to monovalent vaccines representing only a single serogroup. Antigenie competition between serogroups occurred in sheep immunised with the multivalent formulation, but high levels of protection were still achieved. The study showed that in multivalent footrot vaccines, antigenic competition is predominantly due to the presence of a family of immunologically-related pilus antigens rather than to interference by extraneous proteins.     

Molecular analysis of Dichelobacter nodosus isolated from footrot in sheep in Malaysia

Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5′GGGCCC3′), SfiI (5′GGCCNNNNNGGCC3′)and SmaI (′5CCCGGG3′) enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.     

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.     

Reactivity of sera from sheep immunised with individual outer membrane proteins of Bacteroides nodosus against heterologous bacterial strains

In order to identify those bacterial antigens which might be involved in immunity against ovine footrot, antisera were raised in sheep to 6 proteins in the outer membrane complex (OMC) of one strain of Bacteroides nodosus. Examination of the specificity of these antisera by Western blotting, crossed immunoelectrophoresis (XIEP) and IEP, revealed that they recognized the homologous OMC protein, but did not precipitate either undenatured pili or OMC, nor could they agglutinate the homologous bacteria. In contrast, anti-OMC and anti-pili sera could precipitate OMC or pili respectively, and agglutinate whole bacteria. Subsequent analysis of these sera against 5 strains of B. nodosus from different serogroups revealed that Proteins 1, 3 and 4 had a similar antigenic structure in all strains examined. The reactivity of anti-pili sera was restricted to homologous bacteria whereas anti-pilin sera (raised against denatured pili) also reacted with pilin from 2 of 3 heterologous strains. However, none of the patterns of staining or absorption of any of these sera matched the spectrum of cross-protection afforded by vaccination of sheep with B. nodosus strain 198 cells. The results question the role of individual OMC proteins in cross-protective immunity and may imply that interactions between several bacterial components are involved in the phenomenon.     

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.     

Yersinia enterocolitica in sheep - a high frequency of biotype 1A

Background

Pigs are regarded as the main reservoir for human pathogenic Yersinia enterocolitica, which is dominated by bioserotype 4/O:3. Other animals, including sheep, have occasionally been reported as carriers of pathogenic strains of Y. enterocolitica. To our knowledge, this is the first study performed in the Nordic countries in which the presence of Y. enterocolitica in sheep is investigated.

Methods

Tonsils and faecal samples collected from sheep slaughtered on the island Gotland (Sweden) from September 2010 through January 2011 were analysed for presence of Y. enterocolitica. In an attempt to maximize recovery, several cultural strategies were applied. Various non-selective media were used and different temperatures and durations of the enrichment were applied before subculturing on Cefsulodin Irgasan Novobiocin (CIN) agar. Presumptive Y. enterocolitica colonies were subjected to urease, API 20E and agglutination test. Yersinia enterocolitica isolates were biotyped, serotyped, and tested for pathogenicity using a TaqMan PCR directed towards the ail-gene that is associated with human pathogenic strains of Y. enterocolitica.

Results

The samples collected from 99 sheep yielded 567 presumptive Y. enterocolitica colonies. Eighty urease positive isolates, from 35 sheep, were identified as Y. enterocolitica by API 20E. Thirty-four of 35 further subtyped Y. enterocolitica isolates, all from faecal samples, belonged to biotype 1A serotype O:5, O:6. O:13,7 and O:10. One strain was Yersinia mollaretii serotype O:62. No human pathogenic strains of Y. enterocolitica were found in the investigated sheep. Other species identified were Y. kristensenii (n = 4), Y. frederiksenii/intermedia (n = 3), Providencia rettgeri (n = 2), Serratia marcescens (n = 1) and Raoultella ornithinolytica (n = 1).

Conclusions

This study does not support the hypothesis that sheep play an important role in transmission of the known human pathogenic Y. enterocolitica in the studied geographical region. However, because there are studies indicating that some strains of Y. enterocolitica biotype 1A may cause disease in humans, the relative importance of sheep as carriers of human pathogenic strains of Y. enterocolitica remains unclear. Tonsils do not appear to be favourable sites for Y. enterocolitica biotype 1A in sheep.     

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.