Project description:Type IV fimbriae are essential virulence factors of Dichelobacter nodosus, the principal causative agent of ovine foot rot. The fimA fimbrial subunit gene is required for virulence, but fimA mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the pilT or pilU gene eliminated the ability to carry out twitching motility. However, the pilT mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the pilU mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in D. nodosus. It was postulated that sufficient fimbrial retraction must occur in the pilU mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a pilU mutant of D. nodosus, aberrant motion was detected in an equivalent mutant of Pseudomonas aeruginosa. These observations explain how in D. nodosus protease secretion can occur in a pilU mutant but not in a pilT mutant. In addition, virulence studies with sheep showed that both the pilT and pilU mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.
Project description:Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (sigma 54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and sigma 54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and sigma 54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and sigma 54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a sigma 54-dependent PilR/S system that also indirectly controls protease secretion.
Project description:Footrot is one of the major causes of lameness in sheep and leads to decreased animal welfare and high economic losses. The causative agent is the Gram-negative anaerobic bacterium Dichelobacter nodosus. The prevalence of D. nodosus in 207 sheep flocks across Germany was 42.9%. Based on the sequence variation in the type IV fimbrial gene fimA, D. nodosus can be subdivided into ten serogroups (A-I and M). There are commercially available vaccines covering nine serogroups, but the efficacy is low compared to bivalent vaccines. The aim of this study was to investigate the diversity of serogroups in Germany at the flock and animal levels. In total, we detected at least one serogroup in 819 samples out of 969 D. nodosus-positive samples from 83 flocks using serogroup-specific singleplex PCR for the serogroups A-I. Serogroup A was most prevalent at the animal level, followed by serogroups B, H and C. At the flock level, serogroups A and B had the highest prevalence, each with 64%, but only 40% of flocks had both. The average number of serogroups per animal was 1.42 (range one to five) and, per flock, 3.10 (range one to six). The serogrouping showed within-flock specific clusters but were widely distributed, with 50 different combinations across the flocks. The factors associated with the number of serogroups per animal and single serogroups were the load of D. nodosus, footrot score, sheep breed and flock. Our results indicate that efficient vaccination programs would benefit from tailor-made flock-specific vaccines and regular monitoring of circulating serotypes in the flock to be able to adjust vaccine formulations for nationwide progressive control of footrot in Germany.
Project description:Footrot causes 70-90% of lameness in sheep in Great Britain. With approximately 5% of 18 million adult sheep lame at any one time, it costs the UK sheep industry £24-84 million per year. The Gram-negative anaerobe Dichelobacter nodosus is the causative agent, with disease severity influenced by bacterial load, virulence, and climate. The aim of the current study was to characterize strains of D. nodosus isolated by culture of swabs from healthy and diseased feet of 99 ewes kept as a closed flock over a 10-month period and investigate persistence and transmission of strains within feet, sheep, and the flock. Overall 268 isolates were characterized into strains by serogroup, proline-glycine repeat (pgr) status, and multi-locus variable number tandem repeat analysis (MLVA). The culture collection contained 87 unique MLVA profiles and two major MLVA complexes that persisted over time. A subset of 189 isolates tested for the virulence marker aprV2 were all positive. The two MLVA complexes (76 and 114) comprised 62 and 22 MLVA types and 237 and 28 isolates, respectively. Serogroups B, and I, and pgrB were associated with MLVA complex 76, whereas serogroups D and H were associated with MLVA complex 114. We conclude that within-flock D. nodosus evolution appeared to be driven by clonal diversification. There was no association (P > 0.05) between serogroup, pgr, or MLVA type and disease state of feet. Strains of D. nodosus clustered within sheep and were transmitted between ewes over time. D. nodosus was isolated at more than one time point from 21 feet, including 5 feet where the same strain was isolated on two occasions at an interval of 1-33 weeks. Collectively, our results indicate that D. nodosus strains persisted in the flock, spread between sheep, and possibly persisted on feet over time.
Project description:The bacterium Dichelobacter nodosus (D. nodosus) is the causative agent of ovine footrot. The aim of this field study was to determine the prevalence of D. nodosus in German sheep flocks. The sheep owners participated voluntarily in the study. More than 9000 sheep from 207 flocks were screened for footrot scores using a Footrot Scoring System from 0 to 5 and sampling each sheep using one interdigital swab for all four feet of the sheep. The detection and discrimination between benign and virulent strains was done employing a real-time PCR. Our results showed a mean prevalence of 42.93% of D. nodosus in German sheep on an animal level. Underrunning of hoof horn on at least one foot (Scores 3-5) was detected in 567 sheep (6.13%). Sheep with four clinically healthy feet were found through visual inspection in 47.85% of all animals included in this study. In total, 1117 swabs from sheep with four clinically healthy feet tested positive for D. nodosus. In 90.35% of the positive swabs, virulent D. nodosus were detected. Benign D. nodosus were detected in 4.74% of the D. nodosus-positive swabs while 4.91% tested positive for both, benign and virulent D. nodosus. In 59 flocks D. nodosus were not detected and in 115 flocks only virulent D. nodosus were found while seven flocks tested positive for benign strains.