Om P. Dhungyel

The type IV fimbrial subunit gene (fimA) of Dichelobacter nodosus is essential for virulence, protease secretion, and natural competence.

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.

The Subtilisin-Like Protease AprV2 Is Required for Virulence and Uses a Novel Disulphide-Tethered Exosite to Bind Substrates

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

Twitching Motility Is Essential for Virulence in Dichelobacter nodosus

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.

Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination

Programmes based on the identification and treatment of cases and the culling of animals refractory to treatment had failed to eradicate virulent footrot from two districts in the western region of Nepal. From 1993 to 1996 vaccination against two endemic virulent strains of Dichelobader nodosus was tested for its potential to contribute to the eradication of footrot from the region. Only sheep and goats which had been free of signs of footrot at three inspections at monthly intervals before their annual migration to alpine pastures were eligible for inclusion. From November 1992, the treatment of cases identified during inspections included the injection of specific vaccine. Successfully treated cases migrated with their flocks but were excluded from the vaccine trial. Non-responding cases were culled. Forty combined flocks of sheep and goats (approximately 9500 animals) were used initially to compare three vaccination regimens. Eleven flocks (sheep and goats) were treated with two doses of specific vaccine (group A), nine (sheep and goats) were treated with commercial vaccine followed by specific vaccine (group B) and 10 (sheep and goats) were treated with two doses of commercial vaccine (group C) in March to April 1993 before the annual migration; 10 flocks (sheep and goats) remained unvaccinated (group D). Only sheep and goats free of signs of footrot were allowed to migrate. Nevertheless, virulent footrot recurred in many flocks three months later. However, its prevalence was significantly lower in group A than in the other three groups combined. Groups A, B and C then received the specific vaccine before their migrations in 1994 to 1996; group D remained unvaccinated. The annual programme of inspection and identification and treatment of cases continued for seven years, but the vaccinations ceased after four years. There was no recurrence of virulent footrot after November 1993. After the first season the virulent strains of D nodosus used in the specific vaccine could no longer be isolated, although antigenically distinct, benign strains of the organism persisted in cases of benign footrot.

Genomic Evidence for a Globally Distributed, Bimodal Population in the Ovine Footrot Pathogen Dichelobacter nodosus

ABSTRACT Footrot is a contagious, debilitating disease of sheep, causing major economic losses in most sheep-producing countries. The causative agent is the Gram-negative anaerobe Dichelobacter nodosus. Depending on the virulence of the infective bacterial strain, clinical signs vary from a mild interdigital dermatitis (benign footrot) to severe underrunning of the horn of the hoof (virulent footrot). The aim of this study was to investigate the genetic relationship between D. nodosus strains of different phenotypic virulences and between isolates from different geographic regions. Genome sequencing was performed on 103 D. nodosus isolates from eight different countries. Comparison of these genome sequences revealed that they were highly conserved, with >95% sequence identity. However, single nucleotide polymorphism analysis of the 31,627 nucleotides that were found to differ in one or more of the 103 sequenced isolates divided them into two distinct clades. Remarkably, this division correlated with known virulent and benign phenotypes, as well as with the single amino acid difference between the AprV2 and AprB2 proteases, which are produced by virulent and benign strains, respectively. This division was irrespective of the geographic origin of the isolates. However, within one of these clades, isolates from different geographic regions generally belonged to separate clusters. In summary, we have shown that D. nodosus has a bimodal population structure that is globally conserved and provide evidence that virulent and benign isolates represent two distinct forms of D. nodosus strains. These data have the potential to improve the diagnosis and targeted control of this economically significant disease. IMPORTANCE The Gram-negative anaerobic bacterium Dichelobacter nodosus is the causative agent of ovine footrot, a disease of major importance to the worldwide sheep industry. The known D. nodosus virulence factors are its type IV fimbriae and extracellular serine proteases. D. nodosus strains are designated virulent or benign based on the type of disease caused under optimal climatic conditions. These isolates have similar fimbriae but distinct extracellular proteases. To determine the relationship between virulent and benign isolates and the relationship of isolates from different geographical regions, a genomic study that involved the sequencing and subsequent analysis of 103 D. nodosus isolates was undertaken. The results showed that D. nodosus isolates are highly conserved at the genomic level but that they can be divided into two distinct clades that correlate with their disease phenotypes and with a single amino acid substitution in one of the extracellular proteases. The Gram-negative anaerobic bacterium Dichelobacter nodosus is the causative agent of ovine footrot, a disease of major importance to the worldwide sheep industry. The known D. nodosus virulence factors are its type IV fimbriae and extracellular serine proteases. D. nodosus strains are designated virulent or benign based on the type of disease caused under optimal climatic conditions. These isolates have similar fimbriae but distinct extracellular proteases. To determine the relationship between virulent and benign isolates and the relationship of isolates from different geographical regions, a genomic study that involved the sequencing and subsequent analysis of 103 D. nodosus isolates was undertaken. The results showed that D. nodosus isolates are highly conserved at the genomic level but that they can be divided into two distinct clades that correlate with their disease phenotypes and with a single amino acid substitution in one of the extracellular proteases.

Pilot trials in Australia on eradication of footrot by flock specific vaccination.

Footrot is a contagious disease of ruminants requiring strains of Dichelobacter nodosus that possess virulence factors including proteases and fimbriae. Sheep can be immunised against footrot using vaccine-containing fimbriae, either native or recombinant. The fimbriae are responsible for the serological K-agglutination reaction, which has been used to classify field isolates into nine major serogroups. The range of protection conferred by vaccination is largely restricted to the serogroup involved, but antigenic competition precludes effective vaccination with multivalent vaccines that contain all serogroups. However, vaccination with specific bivalent recombinant fimbrial vaccine led to eradication of virulent footrot from small ruminants in Nepal and the same result was obtained in Bhutan using a specific whole cell vaccine. In the study reported here two pilot trials have been conducted in Australian sheep flocks, one with a virulent form of footrot caused by a single serogroup F, and the other with an intermediate form also caused by a single serogroup C. In trial 1 pre-vaccination prevalence of clinical footrot in a group of randomly selected animals was 44%. This reduced to 2% at 3 months and 0.5% at 4 months, and there were no clinical cases at 5 months or at 16 months post-vaccination in the whole flock. Similarly in trial 2 pre-vaccination whole flock prevalence was 8.5%, while it was 2% at 3 months, 0.3% at 6 months and zero at 18 months post-vaccination. Use of flock specific monovalent whole cell vaccines over whole flocks for only one season and culling of the few non-responders has been a successful approach in eradication of the disease from both these flocks. This is the first study to report the successful use of specific vaccine for the intermediate form of footrot.

Characterisation of Dichelobacter nodosus isolated from footrot in sheep and goats in Nepal

Abstract Cases of footrot from sheep and goats in 45 village flocks in Nepal were cultured for Dichelobacter nodosus . Six hundred and eighty-two isolates were obtained from 25 flocks, of which 527 were derived from field isolation, and 155 from a vaccination trial. Of the 527 field isolates, 477 (90.5%) were provisionally classified as serogroup E. The remainder of those classified were either serogroup B (20) or serogroup C (14). Sixteen isolates were not classifiable by slide agglutination tests with antisera to any of the nine recognised serogroups of D. nodosus . Representative serogroup E isolates were all elastase positive and produced thermostable proteases. Those of serogroup B were either elastase positive and thermostable or elastase negative and thermolabile while those of serogroup C and the untypable isolates were elastase negative and thermolabile. Initially, all D. nodosus isolates from the field were of serogroup E. To increase the likelihood of isolating other serogroups, 21 Baruwal sheep affected with footrot were treated twice 30 days apart with a vaccine based on the Nepalese variant of serogroup E. Thirty days after receiving a second dose of vaccine, 17 of 20 affected animals were free of clinical footrot compared with 5 of 17 unvaccinated animals. Whereas at the beginning of this experiment only serogroup E could be recovered, at its conclusion although serogroup E persisted in the unvaccinated animals, isolates of serogroups B and C, and one untypable isolate were obtained from vaccinates. The demonstration of two virulent strains of D. nodosus (serogroup B and E) in these investigations provided an incentive to proceed with specific vaccination as a method of footrot management in these flocks.

Characterisation of Dichelobacter nodosus isolates from Norway.

An outbreak of ovine footrot in Norway in 2008, the first reported since 1948, prompted action to investigate Norwegian isolates of Dichelobacter nodosus. A total of 579 isolates from 124 different farms were characterised. These included 519 isolates from sheep, 52 isolates from cattle and 8 isolates from goats. The potential virulence of the isolates was assessed by the gelatin gel test (GG-test) and the elastin agar test, that test the heat stability and elastase activity of bacterial proteases, respectively. The isolates were also tested for the presence of intA by PCR, and allocated to serogroups by differentiation of fimA variants using multiplex PCR or sequencing. Thirty of the isolates were also serogrouped by slide agglutination. Three hundred and five isolates were defined as virulent by the GG-test. All these were from sheep from 52 farms located in the county of Rogaland in the south west of Norway. All isolates from cattle and goats were defined as benign by the GG-test. IntA was only detected in 6 (2.0%) of the virulent isolates. All serogroups except D and F were detected. Three hundred and seventy-two (64.3%) of the isolates belonged to serogroup A, and 96% of the virulent isolates belonged to this serogroup. On the grounds that virulent isolates were only found in one county, and that the majority belonged to the same serogroup (A), it is believed that a virulent D. nodosus strain was introduced to Norway relatively recently and that so far it has only spread locally.

Risk factors for Escherichia coli O157 shedding and super-shedding by dairy heifers at pasture.

We undertook a longitudinal study within a cohort of 52 dairy heifers maintained under constant management systems and sampled weekly to investigate a comprehensive range of risk factors which may influence shedding or super-shedding of E. coli O157 (detected by direct faecal culture and immunomagnetic separation). E. coli O157 was detected from 416/933 (44.6%) samples (faeces and recto-anal mucosal swabs) and 32 (3.4%) samples enumerated at >10000 c.f.u./g. Weekly point prevalence ranged from 9.4% to 94.3%. Higher temperature (P < 0.001), rainfall (P = 0.02), relative humidity (P < 0.001), pasture growth (P = 0.013) and body score (P = 0.029) were positively associated with increased shedding. Higher rainfall (P < 0.001), hide contamination (P = 0.002) and increased faecal consistency (P = 0.023) were positively associated with super-shedding. Increased solar exposure had a negative effect on both shedding and super-shedding within bivariate analyses but in the final multivariate model for shedding demonstrated a positive effect (P = 0.017). Results suggest that environmental factors are important in E. coli O157 shedding in cattle.

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot (Dichelobacter nodosus) vaccines in sheep.

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.