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PubMed2006

Current understanding of the aetiology and laboratory diagnosis of footrot.

Footrot is a highly contagious disease of the feet of ruminants caused by the synergistic action of certain bacterial species of which Dichelobacter nodosus (D. nodosus) is the main transmitting agent. The infection is specific to sheep and goats, although it has also been reported in cattle, horses, pigs, deer and mouflon. The antigenic diversity of D. nodosus is due to variations in the DNA sequence of its fimbrial subunit gene (fimA) and provides the basis for classification of the organism into at least 10 major serogroups (A-I and M), the distribution of which varies with different geographical locations. Host immune response to vaccination is serogroup specific. There are three different clinical forms of disease caused by virulent, intermediate and benign strains of D. nodosus, respectively. In order to facilitate rapid and reliable clinical diagnosis, virulence determination, strain differentiation and serogroup identification for effective control measures, immunological tests, DNA probes and PCR based techniques have been introduced. This review summarises the current understanding of the mechanisms of antigenic diversity of D. nodosus as well as advances made in its strain differentiation and diagnosis.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

The use of melatonin as a vaccine agent.

Molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. Melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. In seasonally-breeding animals, the indolamine is able to improve reproductive performance. With the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in the humoral response after a vaccination. We reported here a new feature of melatonin as an adjuvant-like system towards Dichelobacter nodosus (A1 and C serotypes)--the bacterium which cause ovine footrot--the most important cause of lameness in sheep. Antibody titres determined by both agglutination and ELISA techniques were substantially higher and were sustained for a longer duration than non-implanted animals. Remarkably, the effect of melatonin was completely dependent on the presence of aluminium hydroxide. The finding that melatonin enhances a defined immune response in vivo opens new perspectives for the improvement of Th2-biased immune responses by alum adjuvants.

Adjuvants, Immunologic↗
PubMed2005

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

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

Animals↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

The occurrence of treponemes in contagious ovine digital dermatitis and the characterisation of associated Dichelobacter nodosus.

Contagious ovine digital dermatitis (CODD) is a recently recorded, apparently new infection of the ovine hoof, which differs clinically from footrot caused by Dichelobacter nodosus and which fails to respond well to accepted treatment practices for footrot. Despite the welfare implications of such an infection, very little research has been performed on CODD to date and the aetiology remains confused. Suggestions have been made that there is a potential role for treponemes in the pathogenesis of CODD but that D. nodosus is apparently not involved. Six farms were therefore targeted in this study to provide a more in-depth investigation into the bacterial flora of CODD lesions. Dark ground microscopy, culture and PCR techniques were used, concentrating on the presence of D. nodosus and spirochaetes, particularly those of the genus Treponema. The results demonstrated that isolates of D. nodosus were indeed present in a high percentage (74%) of CODD lesions compared with 31% of apparently healthy feet. The isolates were shown to be of similar virulence type to those reported previously in cases of footrot, and the range of serogroups was also found to be similar to footrot, with serogroup H being prevalent. Treponemes were present in 70% of CODD lesions and 38% of apparently healthy feet, supporting a possible association between CODD and treponemes. However, any further progress on the aetiology of CODD and the potential for novel, effective treatment will depend on an improved ability to culture these organisms routinely in the laboratory thereby enabling their complete characterisation.

Animals↗
PubMedDate not supplied

Development of gene probes of Dichelobacter nodosus for differentiating strains causing virulent, intermediate or benign ovine footrot.

Seven Dichelobacter nodosus genomic DNA clones including six specific for virulent and one for benign strains were identified. A collection of 96 footrot isolates, which in turn comprised 27 virulent isolates showing elastase activity at 7 days, 25 high intermediate isolates with elastase activity at 14 days, 24 low intermediate isolates with elastase activity at 21-28 days and 20 benign isolates with no elastase activity at up to 28 days, were used to assess these clones. Of the six virulent specific clones, five (pV238-83, pV405-239, pV470-65, pV470-145 and pV470-178) reacted with 27 virulent isolates, and 12 of 25 high intermediate isolates, but none of 24 low intermediate isolates and 20 benign isolates in dot blot hybridization. The other virulent-specific clone (pV470-13) recognized all 27 virulent and 25 intermediate isolates, and 22 of 24 low intermediate isolates and three of 20 benign isolates in dot blot hybridization. By contrast, the benign-specific clone (pB645-335) detected all 20 benign isolates and 24 low intermediate isolates, and also 13 of 25 high intermediate isolates, but none of 27 virulent isolates in dot blot hybridization. Southern hybridization analysis indicated that whereas clones pV238-83, pV405-239 and pV470-178 bound a Sau3A band of 0.5 kb, clones pV470-65 and pV470-145 recognized two Sau3A bands of 0.7 and 0.5 kb in virulent strains of serogroups A to I. However, clone pV470-13 detected a Sau3A band pattern in virulent strains different from those recognized by the other five virulent specific-clones. Besides showing a distinct Sau3A band pattern in intermediate strains, pV470-13 also reacted with three benign strains that showed binding with it in dot blot hybridization. The benign-specific clone pV645-335 detected a Sau3A band of 0.5 kb in both intermediate and benign strains of serogroups A to I. Thus the combination of pV470-13 and pB645-335, or any other virulent-specific clone, would clearly differentiate among D. nodosus strains causing virulent, intermediate or benign footrot.

Animals↗
PubMedDate not supplied

The use of long-acting oxytetracycline for the treatment of ovine footrot.

The use of long-acting oxytetracycline for the treatment of ovine footrot was investigated under different experimental conditions. In sheep with artificially induced footrot housed under dry conditions, treatment with long-acting oxytetracycline produced a cure in 6/6 affected feet (a cure rate of 100%); foot-bathing in zinc sulphate produced a cure in 2/8 affected feet (a cure rate of 25%). This difference was statistically significant (P < 0.05). In sheep with naturally occurring footrot, housed under dry conditions, treatment with long-acting oxytetracycline produced a cure in 8/9 affected feet (a cure rate of 89%); foot-bathing in zinc sulphate produced a cure in 7/10 affected feet (a cure rate of 70%). This difference was not statistically significant. In sheep with naturally occurring footroot, kept under field conditions, treatment with a combination of long-acting oxytetracycline and foot-bathing in zinc sulphate produced a cure in 49/52 affected feet (a cure rate of 94%); treatment by foot-bathing alone produced a cure in 39/51 affected feet (a cure rate of 77%). This difference was statistically significant (P < 0.05).

Animals↗
PubMed1996

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 humoral 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 micrograms. 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.

Aging↗
PubMedDate not supplied

Association between alleles of the ovine major histocompatibility complex and resistance to footrot.

Variation in natural resistance to footrot may be genetically derived, implying that genetic markers for resistance may exist and allow selection of superior animals. In this study association between variation within the ovine MHC class II region and resistance to footrot was investigated in two trials. Half-sib progeny were subjected to a field challenge with footrot and their condition subsequently recorded. The animals were then typed at their MHC class II loci to investigate associations between inherited paternal haplotype and footrot status. In the first trial an association between MHC haplotype and footrot status was observed across all animals (P = 0.005), when the self-curing and resistant animals were combined (P = 0.002) and when the self-curing animals were excluded from the analysis (P = 0.001). No association was observed in the second trial, a result attributed to the dry weather conditions which led to poor disease transmission and unreliable disease classification.

Alleles↗
PubMed1999

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

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

Agglutination Tests↗
PubMed2000

A spirochete isolated from a case of severe virulent ovine foot disease is closely related to a Treponeme isolated from human periodontitis and bovine digital dermatitis.

The isolation of spirochetes from severe ovine foot disease has been reported recently by our research group. In this study we describe the preliminary classification of this spirochete based on nucleotide sequence analysis of the PCR-amplified 16S rRNA gene. Phylogenetic analysis of this sequence in comparison with other previously reported 16S rRNA gene sequences showed that the spirochete belonged to the treponemal phylotype Treponema vincentii which has been associated with bovine digital dermatitis and human periodontal disease. Further work is required to define the common virulence determinants of these closely related treponemes in the aetiology of these tissue destructive diseases.

Animals↗
PubMed2000

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation.

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping). The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.

Amino Acid Sequence↗