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.     

A DEGRADING PROTEINASE TEST TO DISTINGUISH BENIGN AND VIRULENT OVINE ISOLATES OF BACTEROIDES NODOSUS

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

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.     

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.     

Serological classification and virulence determination of Dichelobacter nodosus isolated from Alberta and British Columbia sheep.

Ovine footrot is a contagious disease of sheep that occurs in temperature climates. It is caused by the strict anaerobe, Dichelobacter nodosus. Benign and virulent organisms are differentiated according to serotype and protease production. This study was conducted to identify the presence of virulent serotypes of D. nodosus in sheep flocks in Alberta and British Columbia. Dichelobacter nodosus was detected in lame sheep from 11 of 15 (73%) flocks in Alberta and in 4 of 5 (80%) British Columbia flocks. It was recovered from 57 of 107 (53%) lame sheep. In Alberta, 4 distinct serotypes were isolated from the 11 positive flocks while in British Columbia a total of 6 different serotypes were isolated. One British Columbia isolate could not be classified into existing serotypes. Of the 19 field strains tested, all but 3 were defined as virulent based upon the rapid rise in protease activity in vitro which was maintained between 3 and 5 d. The knowledge of the serotype and virulence of the D. nodosus isolated from affected animals can assist in the control and prevention of ovine footrot.     

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.     

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.     

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.     

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.     

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

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

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.     

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.     

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.     

Temporal relationships and characterisation of extracellular proteases from benign and virulent strains of Bacteroides nodosus as detected in zymogram gels

Extracellular proteases produced by Bacteroides nodosus in a peptone rich modified trypticase-arginine-serine broth medium were separated and characterised by relative mobility (Rf) in electrophoretic zymogram gels. One benign and two virulent protease banding patterns were established with isolates from sheep, cattle and goats. They correlated with other laboratory tests for virulence but were independent of serogroup. The electrophoretic zymogram method was unable to differentiate intermediate from virulent strains. The time required for the production of maximum levels and numbers of protease bands was four to five days for benign and five to six days for virulent B nodosus. Elevated temperatures (above 45 degrees C) and pH extremes (below pH 6 and above pH 9) modified the electrophoretic banding patterns. The molecular weights of the proteases ranged from 8000 to 43,000 daltons and the isoelectric points from pH 4.90 to 5.90. They are serine proteases and this property can be utilised in affinity purification of these molecules.     

A recently introduced Dichelobacter nodosus strain caused an outbreak of footrot in Norway

Background

In 2008, an outbreak of ovine footrot occurred in Norway. Dichelobacter nodosus isolates collected between 2008 and 2011 have been characterised. Isolates defined as virulent by the gelatin gel test (GG-test) were only found in sheep in Rogaland County, where the severe cases of footrot were registered. The majority (96%) of the virulent isolates belonged to serogroup A. It is suspected that they represent a newly introduced strain, and the aim of the present study was to investigate whether they are genetically similar. Sixty-one virulent isolates from sheep and 116 benign isolates from sheep, cattle and goats were included. Four GG-test virulent isolates from Danish sheep were also included. All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and by PCR for pgr variant determination.

Results

The Norwegian virulent isolates were assigned to 8 pulsotypes (PTs), while the benign isolates were assigned to 66 PTs. Thirty-seven (68.5%) of the 54, virulent, serogroup A isolates belonged to the same PT, and included isolates from 2008 through 2011. Isolates belonging to this PT were defined as the outbreak strain. The remaining virulent serogroup A isolates belonged to 4 PTs differing by ≤3 bands from the outbreak strain. Two virulent, Danish, serogroup A isolates differed by 2 bands from the Norwegian outbreak strain. All but 3 (95%) of the virulent isolates had the pgrA variant while 85% of the benign isolates had the pgrB variant.

Conclusion

This study provides evidence that the footrot outbreak in Norway in 2008 most likely was caused by new introduction and local spread of one virulent D. nodosus strain.     

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.     

Characterisation of virulent and benign strains of Bacteroides nodosus

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

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.     

Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats

Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand. DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms. The predominant serogroup was B which contained six strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Eleven out of the 15 D. nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples). Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.