Genomic heterogeneity of Dichelobacter nodosus within and between UK sheep flocks and between age groups within a flock.

BACKGROUND: Footrot and interdigital dermatitis are endemic infectious diseases in all sheep farming regions, impairing welfare and production. The development of efficacious vaccines against the primary causative pathogen has been hampered by the extensive antigenic diversity of Dichelobacter nodosus. Understanding the heterogeneity of the pathogen within and between flocks is essential if the feasibility of bespoke vaccine production is to be assessed for use in the U.K. RESULTS: In this study 56 ewe and lamb isolates from 9 flocks were compared by D. nodosus serogroup and Multi Locus Sequence Type which provides significantly enhanced discriminatory power for molecular epidemiology. Serogroup heterogeneity between flocks ranged from two to five unique serogroups per flock. Three flocks contained isolates of two serogroups, two flocks contained isolates of three serogroups and one flock included isolates of five serogroups. Analysis of 25 isolates from one flock with high prevalence of lameness, identified that serogroup and sequence type was significantly correlated with age. Significantly higher proportion of lambs were infected with serogroup B (principally ST85) as opposed to serogroup H (principally ST86), which predominated amongst adult sheep. CONCLUSIONS: Genomic heterogeneity of the pathogen was significantly lower within flock compared to heterogenicity observed between flocks. Furthermore, this study indicates that within a flock, the host-pathogen dynamics and susceptibility to particular D. nodosus strains may be age dependent.

Identifying maintenance hosts for infection with Dichelobacter nodosus in free-ranging wild ruminants in Switzerland: A prevalence study.

Footrot is a worldwide economically important, painful, contagious bacterial foot disease of domestic and wild ungulates caused by Dichelobacter nodosus. Benign and virulent strains have been identified in sheep presenting with mild and severe lesions, respectively. However, in Alpine ibex (Capra ibex ibex), both strains have been associated with severe lesions. Because the disease is widespread throughout sheep flocks in Switzerland, a nationwide footrot control program for sheep focusing on virulent strains shall soon be implemented. The aim of this cross-sectional study was to estimate the nationwide prevalence of both strain groups of D. nodosus in four wild indigenous ruminant species and to identify potential susceptible wildlife maintenance hosts that could be a reinfection source for domestic sheep. During two years (2017-2018), interdigital swabs of 1,821 wild indigenous ruminant species (Alpine ibex, Alpine chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus), red deer (Cervus elaphus)) were analysed by Real-Time PCR. Furthermore, observed interspecies interactions were documented for each sample. Overall, we report a low prevalence of D. nodosus in all four indigenous wild ruminants, for both benign (1.97%, N = 36, of which 31 red deer) and virulent (0.05%, N = 1 ibex) strains. Footrot lesions were documented in one ibex with virulent strains, and in one ibex with benign strains. Interspecific interactions involving domestic livestock occurred mainly with cattle and sheep. In conclusion, the data suggest that wild ungulates are likely irrelevant for the maintenance and spread of D. nodosus. Furthermore, we add evidence that both D. nodosus strain types can be associated with severe disease in Alpine ibex. These data are crucial for the upcoming nationwide control program and reveal that wild ruminants should not be considered as a threat to footrot control in sheep in this context.

Detection and Serogrouping of Dichelobacter nodosus Infection by Use of Direct PCR from Lesion Swabs To Support Outbreak-Specific Vaccination for Virulent Footrot in Sheep.

Virulent footrot is an economically significant disease in most sheep-rearing countries. The disease can be controlled with vaccine targeting the fimbriae of virulent strains of the essential causative agent, Dichelobacter nodosus However, the bacterium is immunologically heterogeneous, and 10 distinct fimbrial serogroups have been identified. Ideally, in each outbreak the infecting strains would be cultured and serogrouped so that the appropriate serogroup-specific mono- or bivalent vaccine could be administered, because multivalent vaccines lack efficacy due to antigenic competition. If clinical disease expression is suspected to be incomplete, culture-based virulence tests are required to confirm the diagnosis, because control of benign footrot is economically unjustifiable. Both diagnosis and vaccination are conducted at the flock level. The aims of this study were to develop a PCR-based procedure for detecting and serogrouping D. nodosus directly from foot swabs and to determine whether this could be done accurately from the same cultured swab. A total of 269 swabs from the active margins of foot lesions of 261 sheep in 12 Merino sheep flocks in southeastern Australia were evaluated. DNA extracts taken from putative pure cultures of D. nodosus and directly from the swabs were evaluated in PCR assays for the 16S rRNA and fimA genes of D. nodosus Pure cultures were tested also by the slide agglutination test. Direct PCR using extracts from swabs was more sensitive than culture for detecting and serogrouping D. nodosus strains. Using the most sensitive sample collection method of the use of swabs in lysis buffer, D. nodosus was more likely to be detected by PCR in active than in inactive lesions, and in lesions with low levels of fecal contamination, but lesion score was not a significant factor. PCR conducted on extracts from swabs in modified Stuart's transport medium that had already been used to inoculate culture plates had lower sensitivity. Therefore, if culture is required to enable virulence tests to be conducted, it is recommended that duplicate swabs be collected from each foot lesion, one in transport medium for culture and the other in lysis buffer for PCR.

Direct serogrouping of Dichelobacter nodosus from Victorian farms using conventional multiplex polymerase chain reaction.

OBJECTIVE: Dichelobacter nodosus is the causative agent of footrot in sheep. Ovine footrot is a major problem in Australia that results in large economic losses and a represents a very significant animal welfare issue. D. nodosus is divided into 10 serogroups (A-I, M), based on sequence variation in the type IV fimbriae gene, fimA. Control of the bacteria is possible through use of serogroup-specific vaccination, however traditional identification of the serogroups of D. nodosus on infected sheep is time-consuming and costly. With the aim of reducing time and cost, a PCR assay was used to identify serogroups of D. nodosus directly from foot swabs of infected sheep in Victoria. RESULTS: It was shown that serogroup B was most common (10 locations), followed by A, G and H (4 locations), I and C (2 locations), D, E and F (1 location). Infections with multiple serotypes were observed in 50% of farms, with the remaining 50% having only a single serogroup detected. The ability to identify serogroups quickly and cheaply direct from foot swabs will aid the understanding of the epidemiology of D. nodosus and support control programs.

Artificial infection of sheep with multiple strains of Dichelobacter nodosus to induce footrot.

OBJECTIVES: To establish multiple strains of Dichelobacter nodosus in two flocks of sheep and to assess the virulence of five of these strains. METHODS: In experiment 1, sheep were challenged with five D. nodosus strains, which varied in both virulence and serotype. In experiment 2, consisting of four replicates (paddock groups), sheep were challenged with seven different strains of D. nodosus. In experiment 3, sheep were challenged with one of five D. nodosus strains. RESULTS: In experiment 1, at 28 days post challenge, four of the five challenge strains were present. Multiple-strain infections were present in 27 feet, with isolates from three serogroups being recovered from 5 feet, and four serogroups from 1 foot. Challenged hind feet were more frequently affected (P < 0.001). In experiment 2, four of the seven strains were recovered from one replicate and three strains from the remaining three replicates. Significantly more hind feet were affected (262/471, 55.6%) than front feet (198/481, 42%) (P < 0.001). Clinically, in both experiments 1 and 2 the footrot resembled an intermediate form, despite the inclusion of a virulent strain of D. nodosus. In experiment 3, this virulent strain caused a higher prevalence of more severe footrot, a greater mean total foot score and, in Merino sheep, resulted in significantly lower weight gains (P < 0.05). Interaction between D. nodosus strain and breed occurred, with Polwarth sheep being significantly more severely affected by one strain than Merino sheep. DISCUSSION: The clinical expression of multiple-strain infections has implications for both research and control of footrot. A novel method of control is proposed.

Identification and characterization of Dichelobacter nodosus serogroup H from ovine footrot in India.

A total of 56 foot swabs were collected from inter digital spaces of sheep with footrot lesions were screened for 16 rRNA of Dichelobacter nodosus by PCR. Out of the 56 samples, 38(67.85%) were found to be positive. All the positive samples were subjected to multiplex PCR targeting fimA gene for identification of serogroups of D. nodosus. Serogroup H was found along with serogroup B in 12 (55.26%) samples and with serogroup I in 8 (22.2%) samples. The serogroup H was identified for the first time from the Indian subcontinent. The phylogenetic analysis of the present sequence with the available serogroup H sequences of GenBank revealed to be in close association with the serotype H1.

Survival of the ovine footrot pathogen Dichelobacter nodosus in different soils.

Dichelobacter nodosus (D. nodosus) is the causative agent of footrot in sheep; one of the most important health and welfare issues of sheep worldwide. For control programmes to be effective, it is essential that the transmission cycle of D. nodosus is understood and bacterial reservoirs in the environment are better defined. This study evaluated the survival of D. nodosus in different soils using soil microcosms. Cultivation independent and dependent methods were used to detect D. nodosus over 40 days from seeding in soil. A D. nodosus specific probe was used for quantification by qPCR and viability was assessed by cell permeability to an intercalating dye, PMA, and by culture. Survival varied dramatically depending on soil type, matric potential (MP) and temperature. Our findings indicate that D. nodosus survival was higher at 5 °C compared with 25 °C in all soils and significantly longer at both temperatures in clay soil (>44% clay) compared with other soil types. Survival under all conditions was longer than 30 days for both culture independent and dependent methods, this is substantially longer than previous studies and, if this is an infectious dose, longer than the current recommendation of resting a field for 14 days to prevent onward infection.

Footrot vaccines and vaccination.

Research on footrot in small ruminants, which is caused by Dichelobacter nodosus, has led to development of vaccines and their application for control, treatment and eradication of the disease in sheep. Footrot vaccines have evolved over decades to contain monovalent whole cell, multivalent recombinant fimbrial, and finally mono or bivalent recombinant fimbrial antigens. Initially whole cell vaccines made against the few known serogroups of D. nodosus were found to be inefficient in control of the disease in the field, which was attributed to the presence of other unidentified serogroups and also the use of inefficient adjuvants. Fimbriae or pili, which are the basis for antigenic variation, were found to be the major protective and also curative antigens but they are not cross protective between the different serogroups. Multivalent vaccines incorporating all the known serogroups have been proven to be of limited efficacy due to the phenomenon of antigenic competition. Recent studies in Nepal, Bhutan and Australia have shown that outbreak-specific vaccination which involves targeting identified serogroups with mono- or bivalent recombinant fimbrial vaccines, can be very effective in sheep and goats. Where multiple serogroups are present in a flock, antigenic competition can be overcome by sequentially targeting the serogroups with different bivalent vaccines every 3 months. A common antigen which would confer immunity to all serogroups would be the ideal immunogen but the initial studies were not successful in this area. Until universal antigen/s are available, flock specific mono or bivalent fimbrial vaccines are likely to be the most effective tool for control and eradication of footrot in sheep and goats. Future research in footrot vaccines should be focused on improving the duration of prophylaxis by incorporating new and emerging immunomodulators or adjuvants with modified delivery vehicles, discovering a common antigen and understanding the mechanisms of acquired immunity.

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.

Multiple locus VNTR analysis highlights that geographical clustering and distribution of Dichelobacter nodosus, the causal agent of footrot in sheep, correlates with inter-country movements.

Dichelobacter nodosus is a Gram-negative, anaerobic bacterium and the causal agent of footrot in sheep. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is a portable technique that involves the identification and enumeration of polymorphic tandem repeats across the genome. The aims of this study were to develop an MLVA scheme for D. nodosus suitable for use as a molecular typing tool, and to apply it to a global collection of isolates. Seventy-seven isolates selected from regions with a long history of footrot (GB, Australia) and regions where footrot has recently been reported (India, Scandinavia), were characterised. From an initial 61 potential VNTR regions, four loci were identified as usable and in combination had the attributes required of a typing method for use in bacterial epidemiology: high discriminatory power (D>0.95), typeability and reproducibility. Results from the analysis indicate that D. nodosus appears to have evolved via recombinational exchanges and clonal diversification. This has resulted in some clonal complexes that contain isolates from multiple countries and continents; and others that contain isolates from a single geographic location (country or region). The distribution of alleles between countries matches historical accounts of sheep movements, suggesting that the MLVA technique is sufficiently specific and sensitive for an epidemiological investigation of the global distribution of D. nodosus.

Non-specificity of primers used for PCR based serogrouping of Dichelobacter nodosus and identification of a novel D. nodosus strain.

The present study records the first case of non-specificity of typing primers developed by Dhungyel et al. A strain of Dichelobacter nodosus (JKS-20G) isolated from ovine footrot in Kashmir, India, showed specificity for serogroup C and G primers. The fimA sequence of the strain turned out to be closer to serogroup G than C. The nucleotide sequence showed maximum homology of 92% with that of serotype G1 strain 238 and 95% with partial sequence available for serotype G2 strain VCS 1004. However, the deduced amino acid sequence of the fimbrial subunit gene of JKS-20G differed from strain 238 by 16 amino acids and by four amino acids from that of partial sequence of strain VCS 1004. This variation indicates towards declaring this isolate as a new serotype (G3) but just insufficient to classify this into a new serogroup. Some of the amino acid substitutions were located within three hypervariable regions a characteristic of different serogroups. However, to ascertain whether this isolate deserves a new serotype status, there is a need to go for antigenic characterisation of this isolate using the tube and cross tube agglutination test.

Outbreak-specific monovalent/bivalent vaccination to control and eradicate virulent ovine footrot.

Footrot is a contagious disease of small ruminants which is caused by the bacterium Dichelobacter nodosus. In its virulent form there are severe economic losses and a very significant animal welfare issue. Sheep and goats can be vaccinated for treatment and prevention of the disease. There are 10 different serogroups of D. nodosus (A-I and M) and immunity is serogroup-specific. When all 10 serogroups are presented together in a vaccine, protection persists for only a few months due to "antigenic competition". Consequently we evaluated the use of sequential monovalent or bivalent vaccines to control/eliminate/eradicate virulent footrot in a longitudinal intervention study on 12 commercial farms in southeast Australia with flock sizes of approximately 1200-4200 sheep. Overall, virulent footrot was eradicated from 4 of the flocks, 2 of which had 2 serogroups, and the others 4 or 5 serogroups. Where there were only 1 or 2 serogroups (3 farms) the clinical response was rapid and dramatic; prevalence was reduced from 45 to 50% before vaccination to 0% (2 farms) or 0.4% (1 farm) after one round of vaccination. In the remaining 9 flocks there were more than 2 serogroups and successive bivalent vaccines were administered leading to eradication of virulent footrot on 2 farms over 4 years and control of the disease on all but 3 of the others. Of the latter farms, 1 discontinued, and 2 initially had poor response to vaccine due to misdiagnosis of serogroup 'M', which was previously unknown in Australia. Control was achieved after administration of a serogroup M vaccine. These results provide clear evidence for control, elimination and eradication of virulent footrot by outbreak-specific vaccination in Australia.

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.

Bovine digital dermatitis: possible pathogenic consortium consisting of Dichelobacter nodosus and multiple Treponema species.

Bovine digital dermatitis (DD) is a multifactorial disease involving at least one or more treponemal species. Virulent phylotypes of Treponema and other infectious agents contributing to disease etiology still remain to be identified. This study addressed these questions by analyzing the prevalence and distribution of seventeen phylotypes of Treponema in DD lesions by fluorescent in situ hybridization (FISH) applying species/phylotype-specific oligonucleotide probes. In situ hybridization for Dichelobacter nodosus, the cause of ovine footrot, was additionally performed. We sampled 90 biopsies of DD lesions originating from one Norwegian and six Danish dairy herds, and 24 tissue samples of healthy skin. All lesions revealed intermingled infections with multiple Treponema phylotypes (mean>7). In six herds, the mean number of phylotypes identified varied between 12 and 15. D. nodosus was present in forty-nine (51%) of the lesions and in three of the apparently healthy skin samples. Two "healthy" samples also contained Treponema spp. and D. nodosus, and were histopathologically categorized as subclinical DD. Another eighteen of the "healthy" skin samples showed serious epidermal hyperplasia but were not colonized by bacteria while only four samples were found normal. We hypothesise that external noxious stimuli allow D. nodosus to break down the epidermal barrier creating a suitable environment for the secondary invaders, Treponema species, which gradually take over the infection site. The variety and different distribution of treponemes in the DD lesions observed in this study, suggests that most of the Treponema phylotypes have the potential to be pathogenic.

Identification of two new serotypes within serogroup B of Dichelobacter nodosus.

The present study records the strain-specific molecular typing system for Dichelobacter nodosus (D. nodosus) based on genetic analysis of fimA locus. Based on the study two new serotypes B5 and B6 are reported within the serogroup B. Out of 200 swab samples collected randomly from foot lesions of footrot affected sheep from all the districts of Kashmir, India, 122 (61.0%) detected positive for D. nodosus. Serogroup B was predominantly prevalent in 83.60% of positive samples. Restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) amplified fimA gene of D. nodosus serogroup B revealed only two fingerprint patterns (FP) designated as FP1 and FP2. The FP1 was most prevalent and depicted by 82.35% of the samples with serogroup B while, FP2 was depicted by rest (17.65%) of the samples. Though the FP1 fimA sequence had the homology of 95% to D. nodosus fimA of serotype B4 isolate VRS 54, but there were 14 nucleotide differences and four nucleotide insertions/deletions in the coding sequence between these two strains resulting in eight amino acid substitutions in the fimbrial subunit. Similarly the FP2 fimA showed the sequence homology of 97% with D. nodosus fimA of serotype B2 isolate 183, with 10 nucleotide differences and three nucleotide insertions/deletions between these two sequences. This resulted in six amino acid substitutions, plus an amino acid length variation in the subunit protein. Thus it was presumed that these FP1 and FP2 strains represented new serotypes (B5 and B6, correspondingly) within the B serogroup as the degree of amino acid sequence difference with their nearest homologous strains was much greater than that within a serotype (0-5 amino acid differences), but comparable to that between serotypes (8-15 amino acid differences). This presumption was confirmed by cross tube agglutination test.

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

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

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

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

Diagnostic sampling strategies for virulent ovine footrot: simulating detection of Dichelobacter nodosus serogroups for bivalent vaccine formulation.

Dichelobacter nodosus is a slow-growing anaerobic bacterium that is the causative agent of virulent ovine footrot. Vaccination targeted at up to two specific serogroups can eliminate those serogroups from infected flocks, but requires identification of serogroups present in infected flocks. Serogroups can be identified using slide agglutination or polymerase chain reaction (PCR) methods. The objectives of this project were to use stochastic simulation modeling to estimate the efficacy of sampling strategies encompassing 5-40 sheep per flock and 2-4 colonies per sheep, and to compare efficacies based on slide agglutination or multiplex PCR test results. Foot swabs collected from sheep in 12 flocks were used as the basis for a sampling strategy simulation model. None of the evaluated sampling strategies identified the two most common serogroups in the flock, or all serogroups present in the flock, in 95% of iterations. However, a simulated sample of 22 sheep/flock and 2 colonies/sheep resulted in a simulated vaccine that protected 95% of the sheep that could be protected by a single bivalent vaccine, while a sample of 24 sheep/flock and 2 colonies/sheep resulted in a series of simulated bivalent vaccines that protected 95% of diseased infected sheep. The difference in outcome was due to the distribution and frequency of serogroups within certain flocks where some serogroups were uncommon and others dominant. A sampling strategy (>40 sheep/flock, 4 colonies/sheep) that will identify the two most common serogroups in a flock 95% of the time may not be cost effective. Evaluating efficacy based on the expected effect on the flock may be more useful than one which seeks to determine the most common serogroups. These findings are broadly applicable to diseases where more than one strain or type of pathogen may be present and must be represented in a vaccine.

Understanding the molecular epidemiology of the footrot pathogen Dichelobacter nodosus to support control and eradication programs.

The Gram-negative anaerobe Dichelobacter nodosus is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of D. nodosus have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson's index of discrimination value (D) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and D = 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11, D = 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17, D = 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.

Footrot on a sheep breeding farm in the Himalayan state of Jammu and Kashmir.

In the present study ovine footrot was detected clinically on a sheep farm in the Himalayan state of Jammu and Kashmir. Dichelobacter nodosus was confirmed by culture and polymerase chain reaction (PCR) using species-specific 16S ribosomal RNA primers. When cultured, the organism appeared as flat colourless colonies having a fine granulated structure with irregular margins, and showing characteristic Gram-negative rods with swollen ends. Detection by PCR from cultured bacteria resulted in amplification of a 783 base pairs (bp) product. Serogrouping by multiplex PCR using group (A-I)-specific primers revealed the presence of serogroup B-specific bands of 283 bp.