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.

The prevalence of Dichelobacter nodosus in clinically footrot-free sheep flocks: a comparative field study on elimination strategies.

BACKGROUND: Ovine footrot caused by Dichelobacter nodosus (D nodosus) is an infectious disease affecting sheep worldwide. Switzerland plans a nationwide footrot eradication program, based on PCR-testing of interdigital swab samples. The aim of this study was to test for the presence of D nodosus in clinically footrot-free sheep flocks which had been subjected to different treatment strategies, to assess whether they were feasible for the eradication process, especially focussing on antimicrobial flock treatments. Clinical scoring and PCR-results were compared. Ten farms had used hoof bathing and hoof trimming without causing bleeding, ten had used individual treatments and flock vaccines to gain the free status and ten had become free through whole-flock systemic macrolide treatment. For every farm, three risk-based collected pool samples were analysed for the occurrence of virulent and benign D nodosus by PCR detection of aprV2/aprB2. RESULTS: Six flocks from any treatment group tested positive for aprB2 in all pools. Clinical signs were absent at the time of sampling, but some flocks had experienced non-progressive interdigital inflammation previously. Two flocks tested aprV2-positive in the high-risk pool. One of them underwent a progressive footrot outbreak shortly after sampling. Individual retesting indicated, that virulent D nodosus most likely was reintroduced by a recently purchased ram. In the second flock, a ram was tested positive and treated before clinical signs occurred. CONCLUSIONS: All treatment strategies eliminated the causative agent and were found to be suitable for implementation in the PCR-based eradication process. PCR-testing proved to be more sensitive than visual scoring, as it also detected clinically healthy carriers. It will be of benefit as a diagnostic tool in elimination and surveillance programs.

The Influence of Food Intake Specificity in Children with Autism on Gut Microbiota.

Autism spectrum disorder (ASD) is a complex of neurodevelopmental conditions with increasing incidence. The microbiota of children with ASD is distinct from neurotypical children, their food habits are also different, and it is known that nutrient intake influences microbiota in a specific way. Thus, this study investigates the food habits of children with ASD and their association with the gut microbiota. Children with ASD had their dietary energy intakes similar to controls, but they more often demonstrated food selectivity, which seemed to result in deficiency of micronutrients such as vitamins K, B6, C, iron, cooper, docosahexaenoic and docosapentanoic acid. Using high-throughput sequencing, a DNA library of intestinal microbiota was performed. Core microbiota was similar in children with and without ASD, but Dichelobacter, Nitriliruptor and Constrictibacter were found to be putative markers of ASD. The changes in gut microbiota that we observed in connection to food selectivity, intake of fats and omega-3 in particular, fermented milk products and animal/plant protein consumption had similar character, independent of diagnosis. However, high fibre intake was connected with a decreased α-diversity only in children with ASD. High carbohydrate and fibre intake influenced ß-diversity, changing the abundance of Bacteroides and other genera, many of them members of the Clostidiaceae. Modulating food habits of ASD children can influence their gut microbiota composition.

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.

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.

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.

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep.

Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%).

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.

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.

High-level association of bovine digital dermatitis Treponema spp. with contagious ovine digital dermatitis lesions and presence of Fusobacterium necrophorum and Dichelobacter nodosus.

Contagious ovine digital dermatitis (CODD) is an important foot disease in sheep, with significant animal welfare and economic implications. It is thought that CODD emerged from bovine digital dermatitis (BDD) via treponemal bacteria. With wildlife species such as elk now suffering a CODD-like disease, it is imperative to clarify these disease etiologies. A large investigation into treponemal association with CODD is warranted. CODD lesions (n = 58) and healthy sheep foot tissues (n = 56) were analyzed by PCR for the three BDD-associated Treponema phylogroups and two other lameness-associated bacteria, Dichelobacter nodosus and Fusobacterium necrophorum. Spirochete culture was also attempted on CODD lesions. "Treponema medium/Treponema vincentii-like," "Treponema phagedenis-like," and Treponema pedis spirochetes were identified in 39/58 (67%), 49/58 (85%), and 41/58 (71%) of CODD lesions, respectively. One or more BDD-associated Treponema phylogroups were detected in 100% of CODD lesions. Healthy foot tissues did not amplify BDD-associated Treponema phylogroup DNA. D. nodosus and F. necrophorum were present in 34/58 (59%) and 41/58 (71%) of CODD lesions and 22/56 (39%) and 5/56 (9%) of healthy foot tissues, respectively. Thirty-two spirochetes were isolated from CODD lesions, with representatives clustering with, and indistinguishable from, each of the three BDD-associated Treponema phylogroups based on 16S rRNA gene comparisons. This study for the first time demonstrates a high-level association for BDD treponeme phylogroups in CODD and their absence from healthy tissues, supporting the hypothesis that BDD treponemes play a primary causative role in CODD and confirming that the specific PCR assays are an effective differential diagnostic tool for CODD.

The potential spread of severe footrot in Norway if no elimination programme had been initiated: a simulation model.

When severe footrot was detected in Norway in 2008, a surveillance programme was initiated and followed by an elimination programme. By 2013 the disease had spread to two of 19 counties and a total of 119 (1%) sheep flocks had been diagnosed with severe footrot. A simulation model was developed to estimate the potential spread of severe footrot in Norway and to estimate the relative importance of the different spreading routes. The model parameters were based on the rate of spread of the first 38 diagnosed cases and the management and climatic factors particular for Norway. The model showed that by 2013, severe footrot would have spread to six counties and infected 16% of the sheep flocks if no elimination programme had been initiated. If this is compared with the 1% of flocks that were diagnosed in Norway by 2013, there seems to be a large effect of the implemented footrot elimination programme. By 2035, it was estimated that severe footrot would have spread to 16 counties and 64% of the sheep flocks. Such an extensive spread would probably impose a large negative impact on the sheep industry and welfare of the sheep. The most effective way to curb the spread of severe footrot was by decreasing the within county infection rate. This could be achieved by decreasing the contact between flocks or by decreasing the environmental load of D. nodosus, for example by footbathing sheep, culling diseased sheep or eliminating severe footrot in the flock.

The detection of Dichelobacter nodosus and Fusobacterium necrophorum from ovine footrot in Kashmir, India.

In a study conducted, a total of 450 swab samples from footrot lesions of naturally infected sheep were collected in all the ten districts of the Kashmir valley and were examined for the presence of Dichelobacter nodosus (D. nodosus) and Fusobacterium necrophorum (F. necrophorum), in order to determine if F. necrophorum was associated with ovine footrot. The detection of F. necrophorum and D. nodosus was carried out by polymerase chain reaction targeting the leukotoxin (lktA) and 16S rRNA genes, respectively. In this study, only less than 50% of positive samples contained both the bacteria, so it is not possible to conclude with certainty that both bacteria are together required for the disease manifestation.

Outbreak of severe foot rot associated with benign Dichelobacter nodosus in an Alpine ibex colony in the Swiss Prealps.

An outbreak of foot rot occurred in the ibex colony "Vanil Noir" in Switzerland from May to December 2014. This article describes field observations and the analyses carried out on the limbs of 3 animals submitted for postmortem examination. Disease signs observed in the field included lameness, poor body condition and overgrown hooves. Macroscopic examination of selected limbs revealed severe lesions in all of them, including interdigital inflammation with ulceration and malodorous exudation. Histological changes were consistent with chronic laminitis with bone resorption, which was not detected at radiographical examination. Grocott-positive organisms compatible with Dichelobacternodosus were detected in the lesions. Samples collected from the lesions were positive by polymerase chain reaction for benign D. nodosus, which is typically associated with only mild lesions in domestic sheep. Whether D. nodosus is endemic in the colony or had previously been transmitted from sympatric domestic livestock is unclear. The unusual warm and humid weather conditions in 2014 may well have contributed to the outbreak.

Ribonucleases in bacterial toxin-antitoxin systems.

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea and play important roles in a diverse range of cellular activities. TA systems have been broadly classified into 5 types and the targets of the toxins are diverse, but the most frequently used cellular target is mRNA. Toxins that target mRNA to inhibit translation can be classified as ribosome-dependent or ribosome-independent RNA interferases. These RNA interferases are sequence-specific endoribonucleases that cleave RNA at specific sequences. Despite limited sequence similarity, ribosome-independent RNA interferases belong to a limited number of structural classes. The MazF structural family includes MazF, Kid, ParE and CcdB toxins. MazF members cleave mRNA at 3-, 5- or 7-base recognition sequences in different bacteria and have been implicated in controlling cell death (programmed) and cell growth, and cellular responses to nutrient starvation, antibiotics, heat and oxidative stress. VapC endoribonucleases belong to the PIN-domain family and inhibit translation by either cleaving tRNA(fMet) in the anticodon stem loop, cleaving mRNA at -AUA(U/A)-hairpin-G- sequences or by sequence-specific RNA binding. VapC has been implicated in controlling bacterial growth in the intracellular environment and in microbial adaptation to nutrient limitation (nitrogen, carbon) and heat shock. ToxN shows structural homology to MazF and is also a sequence-specific endoribonuclease. ToxN confers phage resistance by causing cell death upon phage infection by cleaving cellular and phage RNAs, thereby interfering with bacterial and phage growth. Notwithstanding our recent progress in understanding ribonuclease action and function in TA systems, the environmental triggers that cause release of the toxin from its cognate antitoxin and the precise cellular function of these systems in many bacteria remain to be discovered. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Simultaneous detection and discrimination of virulent and benign Dichelobacter nodosus in sheep of flocks affected by foot rot and in clinically healthy flocks by competitive real-time PCR.

Ovine foot rot caused by Dichelobacter nodosus is affecting sheep worldwide. The current diagnostic methods are difficult and cumbersome. Here, we present a competitive real-time PCR based on allelic discrimination of the protease genes aprV2 and aprB2. This method allows direct detection and differentiation of virulent and benign D. nodosus from interdigital skin swabs in a single test. Clinically affected sheep harbored high loads of only virulent strains, whereas healthy sheep had lower loads of predominantly benign strains.

Resistance of zebu cattle (Bos indicus) to colonization by major ruminant hoof pathogens.

Zebu cattle (Bos indicus) is reported to be more resistant towards harmful environmental factors than taurine cattle (Bos taurus). A few hundred zebu cattle are kept in Switzerland and in contrast to the Swiss indigenous breeds, infectious hoof disease in zebu is not observed. Therefore, we compared the prevalence of three ruminant hoof pathogens in zebu and taurine cattle. These included Treponema spp., Fusobacterium necrophorum and Dichelobacter nodosus which are associated with bovine digital dermatitis (BDD), different bovine hoof diseases and ovine footrot, respectively. Interdigital swabs and punch biopsies from hind feet of slaughter animals were tested for the three pathogens by PCR. Sixty zebu from eight farms were compared to a convenience sample of 20 taurine cattle from 17 farms. Treponema spp. associated with BDD were not detected in zebu while 23 % of animals and 50 % of farms were positive for benign D. nodosus, with results indicating environmental contamination rather than colonization. Taurine cattle showed 35 % of animals and 41 % of farms positive for T. phagedenis while 90 % of animals and 94 % of farms were colonized by D. nodosus as indicated by a 500-fold higher bacterial load than in zebu. The difference in prevalence of the two pathogens between zebu and taurine cattle was highly significant. F. necrophorum was as well only detected in taurine cattle with values of 15 % of animals and 17.7 % of farms, being significantly different at the animal level. Furthermore, genetic analysis of Swiss zebu indicates high genomic diversity and clear separation from taurine cattle. This is the first evidence that zebu show resistance towards colonization by bacterial hoof pathogens in contrast to taurine cattle.

Prevalence of Dichelobacter nodosus in western Austrian sheep flocks: Comparison of bacterial cultures, clinical foot rot and lameness with PCR and analysis of sample pooling for PCR diagnosis.

INTRODUCTION: Ovine foot rot is a highly contagious and multifactorial claw disease, caused by Dichelobacter nodosus (D. nodosus) and is the main cause of lameness in sheep. The aim of this cross-sectional study was to determine the prevalence of D. nodosus in western Austria both at animal and farm levels. Real-time PCR was evaluated in comparison with clinical and bacteriological investigations from interdigital foot swabs to detect D. nodosus-infected animals. In addition, the use of pooled four-foot swabs to detect foot rot was determined. In course of the study a total of 3156 sheep from 124 farms were examined for lameness and clinical signs of foot rot. The found flock prevalence of D. nodosus was 30,65 % with bacterial culture showing a sensitivity of 75,0 % and a specificity of 100,0 % (p < 0,001) respectively, compared with PCR. Furthermore, clinical foot rot scores (Ckorr = 0,87; p < 0,001) and lameness scores (Ckorr = 0,71; p < 0,001) highly correlated with the detection of D. nodosus by PCR. The result showed that the clinical examination can be used to identify animals infected with D. nodosus in flocks, but PCR must be used to confirm the diagnosis. D. nodosus could be detected equally well with risk-based pools-of-five samples as with undiluted samples (p < 0,001), suggesting that a pool-of-five samples might be a suitable and cost-effective method for detecting D. nodosus in sheep flocks. This study provides an overview of foot rot in Tyrolean sheep flocks and outlines the possibilities and limitations of the various diagnostic tools for D. nodosus. Further studies to investigate possible influencing factors, including alpine pasturing, management factors and biosecurity predisposing to foot rot are necessary for the design of effective future control programs in alpine regions.

INTRODUCTION: Le piétin ovin est une maladie des onglons hautement contagieuse et multifactorielle, causée par Dichelobacter nodosus (D. nodosus) qui constitue la principale cause de boiterie chez les ovins. L'objectif de cette étude transversale était de déterminer la prévalence de D. nodosus dans l'ouest de l'Autriche, tant au niveau de l'animal que de l'exploitation. La PCR en temps réel a été évaluée en comparaison avec les examens cliniques et bactériologiques effectués à partir d'écouvillons des espaces interdigités pour détecter les animaux infectés par D. nodosus. En outre, l'utilisation d'un pool d'écouvillons des quatre membres pour détecter le piétin a été déterminée. Au cours de l'étude, un total de 3156 moutons provenant de 124 fermes ont été examinés pour détecter des boiteries et des signes cliniques de piétin. La prévalence de D. nodosus dans les troupeaux était de 30,65 %, la culture bactérienne montrant une sensibilité de 75 % et une spécificité de 100 % (p < 0,001), respectivement, par rapport à la PCR. En outre, les scores cliniques de piétin (Ckorr = 0,87; p < 0,001) et les scores de boiterie (Ckorr = 0,71; p < 0,001) étaient fortement corrélés avec la détection de D. nodosus par PCR. Les résultats montrent que l'examen clinique peut être utilisé pour identifier les animaux infectés par D. nodosus dans les troupeaux mais que la PCR doit être utilisée pour confirmer le diagnostic. D. nodosus a pu être détecté aussi bien avec des pools de cinq échantillons basés sur le risque qu'avec des échantillons non dilués (p < 0,001), ce qui suggère qu'un pool de cinq échantillons pourrait être une méthode appropriée et rentable pour détecter D. nodosus dans les troupeaux de moutons. Cette étude donne un aperçu du piétin dans les troupeaux de moutons tyroliens et souligne les possibilités et les limites des différents outils de diagnostic pour D. nodosus. D'autres études visant à examiner les facteurs d'influence possibles, y compris les pâturages alpins, les facteurs de gestion et la biosécurité prédisposant au piétin, sont nécessaires pour la conception de futurs programmes de contrôle efficaces dans les régions alpines.

Prevalence of ovine footrot in the tropical climate of southern India and isolation and characterisation of Dichelobacter nodosus.

The present communication records the first determination of the prevalence of footrot in the unexpected situation of the tropical climate of Andhra Pradesh and Tamil Nadu, two states in southern India where the maximum temperature rises to 42 degrees C. In total, 73 outbreaks of footrot in Nellore brown sheep were investigated in 11 districts of Andhra Pradesh and one district of Tamil Nadu during the period March 2009 to March 2011.The overall prevalence of ovine footrot was 15%, with severity scores of 2 to 4 (lesion severity scale 0 to 4). The outbreaks occurred mostly during the rainy season, which is usually from June to December. From a total of 1,050 samples of lesions in naturally infected sheep, 478 (45.5%) were positive for Dichelobacter nodosus. Serogrouping of the isolates revealed six serogroups: A, B, C, E, F and I. Among the positive samples, 448 (93.7%) were a single serogroup and 30 (6.3%) carried a mixed infection with two serogroups. Taking single and mixed infections together, serogroup B was most frequent at 50.4% and was found in all districts, followed by serogroup I in 29.3% of samples, A in 14%, F in 6.7% and C in 5.6%. Serogroup E was detected in only one sample. Serogroups A and F were detected for the first time in India. All of 58 D. nodosus isolates in a sub-sample representing different serogroups were found to be virulent, based on the production of thermostable proteases and the presence of the integrase A gene intA. Thus, the present paper reporting isolation and characterisation of D. nodosus confirms the occurrence of virulent footrot in the tropical climate of southern India.

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.

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.