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

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

<p>Abstract</p> <p>Background</p> <p>Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium <it>Dichelobacter nodosus</it>. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but <it>D. nodosu</it>s should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of <it>D. nodosus </it>and to compare its performance with culturing and conventional PCR.</p> <p>Methods</p> <p>A <it>D. nodosus-</it>specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.</p> <p>Results</p> <p>The developed assay had a detection limit of 3.9 fg of <it>D. nodosus </it>genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the <it>D. nodosus </it>genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.</p> <p>Conclusions</p> <p>The developed real-time PCR assay has good specificity and sensitivity for detection of <it>D. nodosus</it>, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.</p

Ovine pedomics : the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H), interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified due to mismatches in the 16S rRNA universal forward primer (27F). A specific real time PCR assay was used to demonstrate the presence of D. nodosus which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (104-109 cells/g tissue) than those with H or VFR feet

Sites of persistence of Fusobacterium necrophorum and Dichelobacter nodosus: a paradigm shift in understanding the epidemiology of footrot in sheep

Sites of persistence of bacterial pathogens contribute to disease dynamics of bacterial diseases. Footrot is a globally important bacterial disease that reduces health and productivity of sheep. It is caused by Dichelobacter nodosus, a pathogen apparently highly specialised for feet, while Fusobacterium necrophorum, a secondary pathogen in footrot is reportedly ubiquitous on pasture. Two prospective longitudinal studies were conducted to investigate the persistence of D. nodosus and F. necrophorum in sheep feet, mouths and faeces, and in soil. Molecular tools were used to detect species, strains and communities. In contrast to the existing paradigm, F. necrophorum persisted on footrot diseased feet, and in mouths and faeces; different strains were detected in feet and mouths. D. nodosus persisted in soil and on diseased, but not healthy, feet; similar strains were detected on both healthy and diseased feet of diseased sheep. We conclude that D. nodosus and F. necrophorum depend on sheep for persistence but use different strategies to persist and spread between sheep within and between flocks. Elimination of F. necrophorum would be challenging due to faecal shedding. In contrast D. nodosus could be eliminated if all footrot-affected sheep were removed and fade out of D. nodosus occurred in the environment before re-infection of a foot

A distinct bacterial dysbiosis associated skin inflammation in ovine footrot

Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1β and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression

Chapitre 14: Phytopathogènes et stratégies de contrôle en aquaponie

peer reviewedAmong the diversity of plant diseases occurring in aquaponics, soil-borne pathogens, such as Fusarium spp., Phytophthora spp. and Pythium spp., are the most problematic due to their preference for humid/aquatic environment conditions. Phytophthora spp. and Pythium spp. which belong to the Oomycetes pseudo-fungi require special attention because of their mobile form of dispersion, the so-called zoospores that can move freely and actively in liquid water. In coupled aquaponics, curative methods are still limited because of the possible toxicity of pesticides and chemical agents for fish and beneficial bacteria (e.g. nitrifying bacteria of the biofilter). Furthermore, the development of biocontrol agents for aquaponic use is still at its beginning. Consequently, ways to control the initial infection and the progression of a disease are mainly based on preventive actions and water physical treatments. However, suppressive action (suppression) could happen in aquaponic environment considering recent papers and the suppressive activity already highlighted in hydroponics. In addition, aquaponic water contains organic matter that could promote establishment and growth of heterotrophic bacteria in the system or even improve plant growth and viability directly. With regards to organic hydroponics (i.e. use of organic fertilisation and organic plant media), these bacteria could act as antagonist agents or as plant defence elicitors to protect plants from diseases. In the future, research on the disease suppressive ability of the aquaponic biotope must be increased, as well as isolation, characterisation and formulation of microbial plant pathogen antagonists. Finally, a good knowledge in the rapid identification of pathogens, combined with control methods and diseases monitoring, as recommended in integrated plant pest management, is the key to an efficient control of plant diseases in aquaponics.Cos

A longitudinal study of the role of Dichelobacter nodosus and Fusobacterium necrophorum load in initiation and severity of footrot in sheep

Footrot is an infectious bacterial disease of sheep that causes lameness. The causal agent is Dichelobacter nodosus. There is debate regarding the role of Fusobacterium necrophorum in disease initiation. This research used an observational longitudinal study of footrot, together with quantitative PCR (qPCR) of bacterial load of D. nodosus and F. necrophorum, to elucidate the roles of each species in the development of disease. All feet of 18 a priori selected sheep were monitored for five weeks assessing disease severity (healthy, interdigital dermatitis (ID) and severe footrot (SFR)) and bacterial load. A multinomial model was used to analyse these data. Key results were that D. nodosus was detected more frequently on feet with ID, whereas F. necrophorum was detected more frequently on feet with SFR. In the multinomial model, ID was associated with increasing log10 load of D. nodosus the week of observation (OR = 1.28 (95% CI = 1.08-1.53)) and the week prior to development of ID (OR = 1.20 (95% CI = 1.01-1.42). There was no association between log10 load^2 of F. necrophorum and presence of ID (OR = 0.99 (95% CI = 0.96-1.02))). SFR was associated with increasing log10 load of D. nodosus the week before disease onset (OR = 1.42 (95% CI = 1.02-1.96)) but not once SFR had occurred. SFR was positively associated with log10 load^2 of F. necrophorum once disease was present (OR = 1.06 (95% CI = 1.01-1.11)). In summary, there was an increased risk of increasing D. nodosus load the week prior to development of ID and SFR and during an episode of ID. In contrast, F. necrophorum load was not associated with ID before or during an episode, and was only associated with SFR once present. These results contribute to our understanding of the epidemiology of footrot and highlight that D. nodosus load plays the primary role in disease initiation and progression, with F. necrophorum load playing a secondary role. Further studies in more flocks and climates would be useful to confirm these findings. This study identifies that D. nodosus load is highest during ID. This supports previous epidemiological findings, which demonstrate that controlling ID is the most effective management strategy to prevent new cases of ID and SFR