Update on footrot in south-west Germany

82 Dichelobacter nodosus strains isolated from 9 footrot affected sheep flocks in south west Germany were serotyped and tested for virulence. Serovar B was present in all flocks, representing 64.4% of all isolated D. nodosus field strains. Other serovares found were type A, C, E, G and H. Virulent strains were identified in 5 flocks, while intermediate strains were isolated from all 9 flocks. All serological untypeable strains proved to be avirulent. Based on these epidemiological findings the use of currently available commercial footrot vaccines is appropriate in south west German sheep populations.     

Experimental evaluation of a commercial footrot vaccine against native Canadian strains of Dichelobacter nodosus.

Two serotypes of the anaerobic bacterium Dichelobacter nodosus were used to experimentally infect young sheep resulting in infectious pododermatitis or footrot characteristic of the natural disease in sheep. The specific serotypes of D. nodosus were reisolated from the feet and identified using immunofluorescent microscopy of hoof scrapings. Prior immunization of sheep with a commercially available bacterin containing whole cell preparations of ten strains of D. nodosus resulted in serum IgG reactive to a serotype of D. nodosus common to the vaccine. Immunization also produced serum IgG reactive to a serotype of D. nodosus not incorporated in the vaccine. A less severe infection occurred in the immunized sheep than in the controls regardless of the serotype of bacteria used to infect them. Clinical lameness and lesion severity were milder in sheep infected with the serotype of D. nodosus common to the vaccine. Western blot analysis of sera from convalescent sheep showed cross-reactive antibodies to nonfimbrial cell surface proteins, as well as bacterial lipopolysaccharide. Such cross-reactivity may explain the partial protection seen in animals infected with a serotype distinctive from the ones in the vaccine. Despite the historical emphasis of fimbrial immunogens in ovine footrot this study using a new model of experimental ovine footrot suggests other surface antigens may also be important in protective immunity.     

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 microg 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.     

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 of Nepal. From 1993 to 1996 vaccination against two endemic virulent strains of Dichelobacter nodosus was tested for its potential to contribute to the eradication of footrot from the region. Only sheep and goats which had been free of signs of footrot at three inspections at monthly intervals before their annual migration to alpine pastures were eligible for inclusion. From November 1992, the treatment of cases identified during inspections included the injection of specific vaccine. Successfully treated cases migrated with their flocks but were excluded from the vaccine trial. Non-responding cases were culled. Forty combined flocks of sheep and goats (approximately 9500 animals) were used initially to compare three vaccination regimens. Eleven flocks (sheep and goats) were treated with two doses of specific vaccine (group A), nine (sheep and goats) were treated with commercial vaccine followed by specific vaccine (group B) and 10 (sheep and goats) were treated with two doses of commercial vaccine (group C) in March to April 1993 before the annual migration; 10 flocks (sheep and goats) remained unvaccinated (group D). Only sheep and goats free of signs of footrot were allowed to migrate. Nevertheless, virulent footrot recurred in many flocks three months later. However, its prevalence was significantly lower in group A than in the other three groups combined. Groups A, B and C then received the specific vaccine before their migrations in 1994 to 1996; group D remained unvaccinated. The annual programme of inspection and identification and treatment of cases continued for seven years, but the vaccinations ceased after four years. There was no recurrence of virulent footrot after November 1993. After the first season the virulent strains of D nodosus used in the specific vaccine could no longer be isolated, although antigenically distinct, benign strains of the organism persisted in cases of benign 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 a t 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.     

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.     

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.     

Novel fimbrial subunit genes of Dichelobacter nodosus: recombination in vivo or in vitro?

Polymerase chain reaction (PCR) was used to amplify the variable region of the fimbrial subunit encoding gene (fimA) of Dichelobacter nodosus from sheep and goats infected with footrot. Two amplimers (designated X and Y) generated single-strand conformation polymorphism (SSCP) patterns different to those of previously identified serogroups and serotypes. DNA sequencing revealed that these two fragments were novel. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1, whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype F1 and the upstream primer specific for serotype M or E1, generated PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artifacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It, therefore, seems more likely that fragments X and Y reflect genuine fimA genes of D. nodosus which have resulted from in vivo DNA recombination rather than from a PCR recombination artifact.     

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.     

Distribution and prevalence of footrot in Bhutan

The first cases of footrot in Bhutan were reported in sheep in 1990 at the National Sheep Breeding Centre (NSBC), which supplies breeding animals to village sheep flocks throughout Bhutan. Despite the presence of footrot at the Centre the distribution of apparently disease-free sheep continued. Cases of footrot were reported in village flocks soon after the disease was diagnosed at NSBC. A national survey was designed to establish the distribution and prevalence of footrot in Bhutan. This detected footrot in 19/94 village sheep flocks surveyed. The 19 affected flocks were distributed among nine different administrative districts whereas the villages selected were in 13 of a total of 16 sheep growing districts. The highest within-flock prevalences were among the seven flocks sampled in Bumthang district (mean 20.4%). The prevalence of the disease within flocks was generally much lower in other affected districts and in three districts a single affected animal was identified in the sample of 14 sheep examined in each village. Nationally, footrot prevalence was estimated to be 3.1% (95% CI 2.16-4.04%). There was a positive association between the receipt of animals from NSBC and the presence of footrot. The prevalence of the disease was higher in flocks with a migratory system of management than in those using a sedentary system. The relative risk of there being footrot in a migratory flock was nine-times higher than in a non-migratory flock. Only one strain of Dichelobacter nodosus (serogroup B) was identified among the 234 isolates obtained from the 19 affected flocks. Sheep with footrot healed quickly when treated with a vaccine made from this strain.     

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.     

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.     

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.     

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.     

Aetiology of ovine footrot in the Portuguese region of Alto Alentejo

In this work, we found it appropriate to carry out a study directed towards isolating and identifying the entailed microorganisms which trigger off footrot in sheep, placing special emphasis on the serotipification of the different Dichelobacter nodosus species. With this goal in mind four flocks from the Portuguese region of 'Alto Alentejo' were selected, all of them had one common feature: their main health problem was ovine footrot. We also set out to determine the elastolitic capacity of isolated strict-anaerobic bacteria, in order to be able to clarify the direct involvement of these microorganisms in the outbreak of this infectious process.     

Observations on the indirect transmission of virulent ovine footrot in sheep yards and its spread in sheep on unimproved pasture

SUMMARY: Virulent ovine footrot was transmitted accidentally to a group of 23 adult Merino sheep (flock B) after holding for 1 hour in sheep yards, which earlier the same day had contained another flock (flock A) with < 1% prevalence of sheep with footrot lesions. Sheep in flock B were rendered susceptible to virulent footrot by grazing 600 mm high unimproved pasture dominated by paspalum ( Paspalum dilatatum ) and kangaroo grass ( Themeda australis ) during warm, humid and wet weather. In addition to moisture, interdigital abrasions caused by the pasture might have predisposed the interdigital skin to infection with Dichelobacter nodosus .     

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.     

Transmission of virulent footrot between sheep and goats

OBJECTIVE: To determine the infectivity of ovine and caprine strains of Dichelobacter nodosus for both sheep and goats. DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.     

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.     

Comparative antigenic analysis of extracellular proteins of Bacteroides nodosus isolated from virulent and benign ovine footrot

Antigens in the extracellular protein (ECP) complexes of Bacteroides nodosus, isolated from sheep with either benign or virulent footrot, were studied by immunoelectrophoresis (IEP). Rabbit antisera against ECP from virulent and benign strains, were used in homologous and heterologous crossed IEP. Four precipitin peaks unique to the virulent strain, and five peaks unique to the benign strain were identified. In an attempt to characterize the different antigens in ECP, rabbit antisera were raised against an outer membrane protein (OMP, mol. wt. 35 000 daltons), pili and various proteases of virulent and benign strains of B. nodosus. No precipitin band was observed when ECP from both B. nodosus strains were reacted against anti-OMP and anti-pilus antisera. However, single precipitin bands unique to one protease from the benign strain and one protease from the virulent strain were identified. The results suggest that specific antigens other than proteases or pili are important in determining whether a B. nodosus isolate is virulent or benign.