A metagenomics approach to characterize the footrot microbiome in Merino sheep.

In the Portuguese Alentejo region, Merino sheep breed is the most common breed, reared for the production of meat, dairy, and wool. Footrot is responsible for lameness, decreased animal welfare, and higher production losses, generating a negative economic impact. The disease is caused by Dichelobacter nodosus that interacts with the sheep foot microbiome, to date largely uncharacterized. In fact, Dichelobacter nodosus is not able to induce footrot by itself being required the presence of a second pathogen known as Fusobacterium necrophorum. To understand and characterize the footrot microbiome dynamics of different footrot lesion scores, a whole metagenome sequencing (WMGS) approach was used. Foot tissue samples were collected from 212 animals with different degrees of footrot lesion scores, ranging from 0 to 5. Distinct bacterial communities were associated with feet with different footrot scores identifying a total of 63 phyla and 504 families. As the severity of footrot infection increases the microorganisms' diversity decreases triggering a shift in the composition of the microbiome from a dominant gram-positive in mild stages to a dominant gram-negative in the severe stages. Several species previously associated with footrot and other polymicrobial diseases affecting the epidermis and provoking inflammatory responses such as Treponema spp., Staphylococcus spp., Streptococcus spp. and Campylobacter spp. were identified proliferating along with the lesions' severity. Although these bacteria are not able to initiate footrot, several evidences have been described supporting their association with the severity and incidence increase of footrot lesions caused by Dichelobacter nodosus and Fusobacterium necrophorum. Further investigation is required to establish the roles of particular taxa and identify which of them play a role in the disease process and which are opportunistic pathogens.

The impact of glutaraldehyde based footbaths on Dichelobacter nodosus prevalence and the antimicrobial resistant community of the ovine interdigital skin.

Ovine footrot, is a highly contagious polymicrobial bacterial infection, primarily caused by Dichelobacter nodosus. Preventative bactericidal footbaths are commonly used in the sheep industry to reduce the spread of bacteria. However, their effect on the bacterial community is poorly understood. This is the first study to investigate the impact of 2% Digicur (ProGiene,UK) footbath on the bacterial community of the ovine interdigital skin following a common UK footbathing routine. Swab samples were analysed by qPCR to determine prevalence and load of D. nodosus and numerated on MacConkey agar in the presence or absence of tetracycline and ampicillin to determine phenotypic antimicrobial resistance. Metagenomics were used to determine the impact of a single footbath on the bacterial community and genotypic antimicrobial resistance. The results suggest 2% Digicur is ineffective at reducing the load of D. nodosus when applied as a one off or weekly footbath, however sheep may act as a reservoir for multi-drug resistant bacteria creating opportunities to spread antimicrobial resistance to other sheep and their environment.

Risk factors associated with the infection of sheep with Dichelobacter nodosus.

Ovine footrot is a highly contagious foot disease caused by the gram-negative bacterium Dichelobacter nodosus (D. nodosus). In a recent report, we showed a prevalence of 42.9% D. nodosus positive swabs across Germany. In this follow-up study, we used real-time PCR results for D. nodosus and footrot scores of 9297 sheep from 208 flocks and collated these data with survey data on herd and animal characteristics and herd management. The aims of the present study were to investigate herd and animal factors associated with D. nodosus infection and footrot scores in individual sheep. Multivariable analyses with generalized mixed models showed that month of recording, breed, herdbook membership, use of antibiotics, and footbaths in the past 3-10 years, signs of footrot in the past 12 months and flock environment of the sheep, modelled as a random farm effect within region, were significant risk factors. Among the 21 different breeds, Romney had the lowest risk of D. nodosus infection, while Swifter had the highest risk and German Merino and German White Heath were the next breeds at highest risk of D. nodosus infection. The variance between farms in the prevalence of D. nodosus was large and accounted for 84% of the total variance in the mixed model analysis. We conclude that specific and as yet unknown effects influencing D. nodosus infections in flocks, as well as breed and weather, are the most important effects on D. nodosus infection in sheep, pointing towards the need to establish adequate infection control at farm level.

Development of breeding values for susceptibility to virulent footrot in sheep: A strategy to accommodate variable disease progression at time of scoring.

Genetic evaluations utilising footrot scores from industry flocks in their essence, incorporate data from a wide range of challenge environments, resulting in potentially large differences in means, variances and distribution of scores across challenges. The date that commencement of infection occurs is generally unknown, and progression of the infection varies with the prevailing environmental and management conditions, virulence of the bacterium Dichelobacter nodosus, as well as the genetic potential and (permanent) environmental ability of animals to resist footrot. In practice, animals are unlikely to be repeatedly scored to identify the best time for comparison, or monitor development of disease progression. Furthermore, field challenges are limited by the need to treat animals before their welfare is compromised. Therefore, the duration and intensity of infection varies and this affects comparisons between animals for their susceptibility. Diseases such as footrot are characterised by multiple categorical scores reflecting clinical stages that describe the progression and relative impact of the disease. This provides the opportunity for the transformation of the data to a standardised prevalence. Scoring events from multiple footrot field challenges under a standardised protocol were used to establish a series of transition matrices to describe disease progression between scores over time. These transition matrices were used to standardise challenge events to the more severe scoring events, observed later in the challenge. The accuracy of the transition technique was tested by comparing the ranking of animals and sires against the observed scores. Transitioning the data from low disease prevalence to the higher prevalence at the subsequent scoring event improved the correlations between the scoring events, at the animal level, by upwards of 0.10 across challenges. The utilisation of a transition matrix to transform low prevalence disease challenges by taking into account the natural biological rate of progression through the clinical stages of the disease provides a more accurate technique to account for variation in disease prevalence. The transition technique increases the acceptable range of disease expression targeted by producers when scoring virulent footrot challenges reducing the need for repeat scoring and allowing earlier treatment and reducing the impact of the disease on the host animal.

Ovine footrot in Southern Portugal: Detection of Dichelobacter nodosus and Fusobacterium necrophorum in sheep with different lesion scores.

The Mediterranean climate region of Alentejo in the Southern of Portugal is an important sheep production centre but little is known about the presence and characteristics of Dichelobacter nodosus in association with Fusobacterium necrophorum in the different footrot lesion scores. DNA from 261 interdigital biopsy samples, taken from 14 footrot affected flocks and from three non-affected flocks, were analysed for the presence of D. nodosus and F. necrophorum by real-time PCR. Both virulence and serogroup were determined for 132 and 53 D. nodosus positive biopsy samples, respectively. The co-infection with both bacteria was the commonest epidemiological finding associated with a greater disease severity. There was a statistically significant association (p = 0.002) between footrot-affected flocks and the presence of D. nodosus. Most D. nodosus positive samples were virulent (96.2 %) and belonged to serogroup B (90 %).

Genome sequence of Dichelobacter nodosus JKS-07B isolate from J&K, India associated with virulent footrot of sheep.

INTRODUCTION: Virulent footrot of sheep caused by Dichelobacter nodosus is associated with tremendous economic losses due to recurrent treatment costs and increased culling rates. This organism being a fastidious anaerobe is difficult to isolate on ordinary media that does not support its growth. The D. nodosus serogroup B isolate described in the present study has been used in the preparation of the whole-cell killed vaccine against footrot in India. D. nodosus serogroup B is the predominant serogroup involved in virulent footrot (lesion score 4) in India as well as in many sheep-rearing countries of the globe. METHODS: Genomic DNA was extracted using wizard Genomic DNA purification kit. The whole genome of the D. nodosus strain B was sequenced using an Illumina HiSeq 2500 platform and annotated according to functional gene categories. Annotations were performed using in-house developed Perl scripts using Nr/Nt database, uniprot, Pfam, KEGG, Panther DB, and GO database. RESULT: The assembled genome size is 1.311,533 Mb and GC content is 44.38. A total of 1215 protein-coding genes, 44tRNA and 7 rRNA were identified. The genome shows 98.63% sequence homology with the reference genome. However, 21 new genes have been identified in this genome. The information will provide insights into the various genes and regulators necessary for D. nodosus growth and survival. DISCUSSION: The genome information of this serogroup B of D. nodosus isolate involved in 85-90% cases of virulent footrot of sheep in India provides further insights for improvement of the killed vaccine (B serogroup) developed recently in India. For the development of an efficacious vaccine against virulent footrot, it is essential to know the serological diversity as well as the virulent status of the strains of the D. nodosus. This serogroup isolate is a potential vaccine candidate to mitigate ovine footrot in India as the majority of virulent footrot cases belong to serogroup B of D. nodosus.

Serogroups of Dichelobacter nodosus, the cause of footrot in sheep, are randomly distributed across England.

We present the largest and most representative study of the serological diversity of Dichelobacter nodosus in England. D. nodosus causes footrot and is one of the top five globally important diseases of sheep. The commercial vaccine, containing nine serogroups, has low efficacy compared with bivalent vaccines. Our aim was to investigate the prevalence and distribution of serogroups of D. nodosus in England to elucidate whether a bivalent vaccine could protect the national flock. Farmers from 164 flocks submitted eight interdigital swabs from eight, preferably diseased, sheep. All serogroups, A-I, were detected by PCR in 687/1150 D. nodosus positive swabs, with a prevalence of 2.6-69.3% of positive swabs per serogroup. There was a median of two serogroups per flock (range 0-6). Serogroups were randomly distributed between, but clustered within, flocks, with 50 combinations of serogroups across flocks. H and B were the most prevalent serogroups, present in > 60% of flocks separately but in only 27% flocks together. Consequently, a bivalent vaccine targeting these two serogroups would protect 27% of flocks fully (if only H and B present) and partially, if more serogroups were present in the flock. We conclude that one bivalent vaccine would not protect the national flock against footrot and, with 50 combinations of serogroups in flocks, flock-specific vaccines are necessary.

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.

Antimicrobial Resistance in Clostridium and Brachyspira spp. and Other Anaerobes.

This article describes the antimicrobial resistance to date of the most frequently encountered anaerobic bacterial pathogens of animals. The different sections show that antimicrobial resistance can vary depending on the antimicrobial, the anaerobe, and the resistance mechanism. The variability in antimicrobial resistance patterns is also associated with other factors such as geographic region and local antimicrobial usage. On occasion, the same resistance gene was observed in many anaerobes, whereas some were limited to certain anaerobes. This article focuses on antimicrobial resistance data of veterinary origin.

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.

Evaluation of loop-mediated isothermal amplification (LAMP) assay for detection of aprV2 positive Dichelobacter nodosus in-field by secondary users.

OBJECTIVE: Dichelobacter nodosus is the primary aetiological agent of footrot in sheep. Ovine footrot causes considerable economic losses and substantial animal welfare issues in the Australian sheep industry. Current methods for detecting D. nodosus are difficult, laborious and time-consuming. Recently, we developed a robust LAMP assay (VDN LAMP) that was able to identify aprV2 positive D. nodosus in-field. A major advantage of LAMP technology is the ability of the assay to be performed by non-specialists with minimal training. We aimed to assess the performance of the VDN LAMP in-field in comparison to a laboratory-based aprV2/aprB2 rtPCR when used by secondary users after training by the authors. RESULTS: Two animal health officers (termed secondary users) from Department of Primary Industries and Regions, South Australia (PIRSA) were trained in the use of VDN LAMP, before carrying out in-field testing on several locations in South Australia. The performance of VDN LAMP assay by secondary user 1 was shown to successfully detect 73.91% (n = 53) aprV2 positive samples, while secondary user 2 detected 37.93% (n = 30) aprV2 positive samples. Overall, the ability to identify virulent D. nodosus by VDN LAMP by secondary users was mixed for various reasons, however, this could be rectified by additional training and commercial production of the LAMP kits to increase stability. We envisaged in the future VDN LAMP will able to be used by non-specialists to aid control programs.

Detection of specific Treponema species and Dichelobacter nodosus from digital dermatitis (Mortellaro's disease) lesions in Swiss cattle.

INTRODUCTION: The aim of this study was to determine the prevalence of the three Treponema species as well as D. nodosus in Digital dermatitis (DD) and slurry of Swiss cattle using PCR. A total of 86 specimens from 24 farms were enrolled in the study. Slurry samples from 21 DD-affected and one unaffected farm were collected to assess the potential of environmental transmission. Nested and real-time PCR were performed from the specimens to detect Treponema species and D. nodosus, respectively. The DD-stages were positive for at least one or more of the DD-associated Treponema species in 50 of 61 cases (82.0%) and in 9 of 25 cases (36.0%) in unaffected animals. Infected animals with small focal active lesions showed a significantly lower prevalence (14.8%) compared to the other DD stages (67.2%; P=0.011). Most prevalent was T. phagedenis (65.1%). D. nodosus was detected in 51.8% of clinical DD lesions and 24.1% in unaffected cases, but its presence was not significantly associated with the various DD-stages. All samples positive for D. nodosus contained the acid protease gene aprB2 but were negative for aprV2, the latter associated with virulence in sheep foot rot. Control farms were negative for all DD-associated Treponema species while positive for aprB2 and negative for aprV2. The presence of aprB2 suggests it is ubiquitous in the animal environment. With respect to the slurry samples, three out of 21 specimens (14.3%) were positive for one or more of the DD-associated Treponema species and eleven out of 21 specimens (52.4%) were positive for aprB2 and negative for aprV2 of D. nodosus. In conclusion, an association was found between the presence of clinical DD and specific Treponema species, while for D. nodosus no such link with DD lesions could be observed.

INTRODUCTION: La Dermatite digitée (DD) chez les bovins est une maladie infectieuse podale multifactorielle, qui est devenue un problème émergent pour le bien-être animal et pour l'économie au niveau mondial. Trois espèces de Treponema, T. pedis, T. medium et T. phagedenis, sont associées avec la DD. Cependant, leur prévalence est inconnue en Suisse. Il a également été rapporté que Dichelobacter nodosus pouvait contribuer au développement de la DD. Le but de cette étude a été de déterminer la prévalence des trois espèces de Treponema ainsi que de D. nodosus dans des lésions de DD et du lisier de vaches suisses, en utilisant des techniques basées sur la PCR. Vingt-deux exploitations avec de la DD clinique et deux exploitations sans signes cliniques de DD ont été inclues dans l'étude. Un total de 86 échantillons de cas de DD ont été prélevés (M1, n=15; M2, n=19; M3, n=9; M4, n=2, M4.1, n=16 and M5, n=25) en utilisant des coton-tiges secs et stériles. De plus, afin d'évaluer le potentiel de transmission par l'environnement, des échantillons de lisier ont été prélevés sur des exploitations atteintes de DD (n=21) et sur une exploitation à stabulation libre exempte de DD. La PCR nichée et la PCR en temps réel ont ensuite été utilisées sur l'ADN extrait des échantillons afin de détecter les espèces de Treponema et D. nodosus, respectivement. Les associations entre la présence d'espèces de Treponema et de D. nodosus avec le statut DD des animaux ont été évaluées avec le test Pearson's Chi-Square. Les stades de DD (M1 à M4.1) et M5 (peau saine) étaient positifs pour au moins une ou plusieurs des espèces de Treponema associées à la DD dans 50 de 61 cas (82.0%) et 9 de 25 cas (36.0%), respectivement. Les lésions M1 ont montré une prévalence nettement inférieure (14.8%) comparé aux autres stades de DD (M2, M3, M4 et M4.1; 67.2%; P=0.011). T. phagedenis était prédominant (65.1%). D. nodosus a été détecté dans 51.8% des lésions cliniques de DD (M1 à M4) et 24.1% des échantillons M5, mais sa présence n'était pas associée significativement avec les divers stades de DD. Tous les échantillons positifs pour D. nodosus contenaient le gène de la protéase acide aprB2, mais étaient négatifs pour aprV2, un gène associé à la virulence dans le piétin des moutons. Les exploitations de contrôle étaient négatives pour toutes les espèces de Treponema associées à la DD, mais positives pour aprB2 et négatives pour aprV2. La présence du gène aprB2 suggère qu'il est ubiquitaire dans l'environnement des animaux et n'est pas une association en soi avec le piétin des moutons. En ce qui concerne les échantillons de lisier, trois des 21 échantillons (14.3%) étaient positifs pour au moins une ou plusieurs espèces de Treponema associées à la DD et onze des 21 échantillons (52.4%) étaient positifs pour aprB2 et négatifs pour aprV2 de D. nodosus. En conclusion, une association a été trouvée entre la présence de DD clinique et des espèces de Treponema spécifiques, alors que pour D. nodosus aucun lien avec des lésions de DD n'a pu être observé. Cette étude démontre la présence des trois espèces de Tréponèmes chez les bovins suisses et facilite la compréhension de l'implication de Treponema spécifiques dans les lésions de DD.

Draft genome sequence of Dichelobacter nodosus JKS-07 serogroup E from India.

OBJECTIVES: Dichelobacter nodosus is an anaerobic bacterium with fastidious growth requirements that is the principal cause of footrot associated with lameness in sheep and goats. In India, D. nodosus serogroups B and E have been recorded as major causes of footrot. Here we report the draft genome sequence of a D. nodosus serogroup E strain (JKS-07) from a case of virulent footrot in India. METHODS: The whole genome of the D. nodosus JKS-07 serogroup E was sequenced using an Illumina HiSeq 2500 platform and was annotated according to functional gene categories. De novo genome assembly and annotation were performed using Perl scripts developed in-house using the Nr/Nt and UniProt databases. RESULTS: The assembled genome is 1389350bp and contains 1301 genes. The genome has 45 tRNAs and 9 rRNAs. The draft genome sequence will provide insight into the various genes and regulators involved in D. nodosus growth and survival. CONCLUSION: Information on the genome of the D. nodosus serogroup E strain is important bearing in mind the fact that both serogroups B and E are associated with virulent footrot, either alone or frequently together. In order to develop an efficacious vaccine against virulent footrot, it is essential to know the serological diversity as well as the virulence status of the D. nodosus strains. Serogroups B and E are potential vaccine candidates to mitigate ovine footrot in India.

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

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

The development and deployment of a field-based loop mediated isothermal amplification assay for virulent Dichelobacter nodosus detection on Australian sheep.

Dichelobacter nododus is the causative agent of footrot, a major disease of sheep that creates welfare concerns and large economic loss. The virulence of D. nododus depends on the presence of extracellular proteases, AprV2 and AprB2, which differ by one amino acid. Strains possessing AprV2 can cause clinically virulent disease, while AprB2 may cause clinically benign disease. Current methods for detecting D. nodosus are difficult, laborious and time consuming. New techniques capable of rapidly detecting and typing D. nodosus are needed to aid control programs. Molecular methods, like real-time polymerase chain reaction (rtPCR) can detect aprV2 and aprB2, however, this assay is not field-deployable and cannot support local decision-making during an outbreak. Here we present a field-based molecular assay for detecting aprV2, using loop mediated isothermal amplification (LAMP). The aprV2 LAMP (VDN LAMP) assay was optimised to reliably detect aprV2 from laboratory purified genomic (gDNA) of virulent D. nodosus down to 5x10(-3) ng µL-1, with time to positive (Tp) ≤ 16 minutes, while aprB2 was unreliably detected at 5 ng µL-1 from 16-20 minutes. The use of field collected samples that were rtPCR positive for aprB2 resulted in no amplification, while aprV2 positive field samples by VDN LAMP assay are defined as having Tps' of < 20 minutes and melting temperature between 88.0-88.9°C. When compared to rtPCR, the VDN LAMP was shown to have a diagnostic specificity of 100% and sensitivity of 83.33%. As proof of concept, the VDN LAMP was taken on farm, with all processing occurring in-field. The on farm VDN LAMP successfully detected 91.67% aprV2 positive samples, no aprB2 positive samples (n = 9) or D. nodosus negative (n = 23) samples, with a kappa agreement of 'almost perfect' to rtPCR. This highlights the potential of the assay to inform local treatment decisions for management.

Assessment of a rtPCR for the detection of virulent and benign Dichelobacter nodosus, the causative agent of ovine footrot, in Australia.

BACKGROUND: Ovine footrot is a highly contagious bacterial disease of sheep, costing the Australian sheep industry millions of dollars annually. Dichelobacter nodosus, the causative agent of footrot, is a gram-negative anaerobe classed into virulent and benign strains as determined by thermostability of their respective protesases. Current methods for detection of D. nodosus are difficult and time-consuming, however new molecular techniques capable of rapidly detecting and typing D. nodosus have been reported. RESULTS: A competitive real-time PCR (rtPCR) method, based on the ability to detect a 2 nucleotide difference in the aprV2 (virulent) and aprB2 (benign) extracellular protease gene has been tested on Australian samples for determining detection rates, along with clinically relevant cut-off values and performance in comparison to the traditional culturing methods. The rtPCR assay was found to have a specificity of 98.3% for virulent and 98.7% for benign detection from samples collected. Sheep with clinical signs of footrot showed a detection rate for virulent strains of 81.1% and for benign strains of 18.9%. A cut-off value of a Ct of 35 was found to be the most appropriate for use in Victoria for detection of sheep carrying virulent D. nodosus. CONCLUSIONS: In summary, the rtPCR assay is significantly more capable of detecting D. nodosus than culturing, while there is no significant difference seen in virotyping between the two methods.

Potential transmission routes of Dichelobacter nodosus.

Footrot caused by Dichelobacter nodosus is a highly contagious bacterial disease affecting the claw of sheep and the main cause of lameness in these animals. It is not only an economic burden but also a serious animal welfare issue. More information about the transmission of D. nodosus is needed for effective footrot control programs. We therefore determined the prevalence of D. nodosus in sheep presented at shows and markets where commingling of animals occurs. Furthermore, possible transmission vectors during foot trimming were investigated and trimming knife decontamination protocols evaluated. Sheep at six markets and four shows were sampled and tested for the presence of D. nodosus by real-time PCR. Different vectors, such as trimming knives were tested by real-time PCR and for viable D. nodosus by culture. The prevalence of virulent D. nodosus in sheep presented at shows and markets ranged from 1.7% to 100%. Regions with an ongoing control program showed significantly lower prevalence. After trimming, positive real-time PCR and culture results were obtained from the knives, the hands of the claw trimmers as well as removed claw horn material whereas boots were only positive by real-time PCR. In conclusion, markets and shows pose a risk for transmission of D. nodosus. The risk of transmission is particularly high during claw trimming and recommended measures to limit this risk include wiping the knife with a disinfection towel, wearing and changing gloves after every sheep, as well as proper disposal of trimmed and infectious horn.

Pathology and bacteria related to digital dermatitis in dairy cattle in all year round grazing system in Brazil.

Digital dermatitis (DD) is one of the main causes of lameness in dairy cattle worldwide, and it is frequently reported in high-yielding, free stall dairy herds from regions with a temperate climate. However, DD is also observed with high prevalence in grazing cattle with a low milk yield in tropical regions. To clarify whether these differences have an impact on the etiology of the disease, we studied DD lesions from all year round grazing cattle of mixed breed in Brazil using high-throughput 16S rRNA gene sequencing and fluorescent in situ hybridization. The study included samples from 66 skin lesions and 5 healthy skins collected from five farms. Both techniques showed Treponema spp. to be the most abundant bacteria, present in all but one of the samples with minimal epidermal alterations. We identified eleven different Treponema strains belonging to the six major phylotypes of Treponema which have all previously been identified in DD lesions. Furthermore, we identify Dichelobacter nodosus in DD lesions by gene sequencing and also by fluorescent in situ hybridization in almost half of biopsy specimens in areas with mild epithelial damage and together with Treponema. The present data support the hypothesis that Treponema constitutes the main pathogen responsible for DD, independent of the environment and region where cows are kept, and it further suggests D. nodosus as another potentially important pathogen.

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.

Pooling of interdigital swab samples for PCR detection of virulent Dichelobacter nodosus.

Virulent ovine foot rot is a contagious foot disease. Given the development and validation of a real-time PCR to detect Dichelobacter nodosus isolates that contain the virulence-associated protease genes aprV2 and aprB2, the diagnosis of foot rot has made considerable progress. We evaluated pooling methods to reduce the number of samples during a foot rot control program. Samples of individual feet were compared to a 4-feet sample of the same sheep. All further analyses based on 4-feet samples (pools-of-5 and pools-of-10 4-feet samples) were compared to samples of individual sheep, and a risk-based herd sampling was evaluated and compared to the whole flock. The sensitivity and specificity of the 4-feet samples for detection of aprV2-positive strains was 93.8% (CI: 87.6-97.5%) and 98.3% (CI: 96.5-99.3%), respectively. The sensitivity and specificity of the pools-of-10 was 86.7% (CI: 78.4-92.7%) and 100.0% (CI: 97.4-100%), respectively. Pools-of-5 were not significantly more sensitive than pools-of-10. The pooling of 4 individual foot samples into one 4-feet sample is an adequate method to reduce the number of samples of individual sheep. The sensitivity of pools-of-5 and pools-of-10 is too imprecise for a control program. Risk-based sampling allowed for a substantial reduction of samples to be tested, had a sensitivity of 95.8% (CI: 78.9-99.9%) and specificity of 100.0% (CI: 88.1-100.0%) when determining the foot rot flock status, and represents an adequate methodology to predict within-flock freedom from infection.