Identification of Streptococcus dysgalactiae using matrix-assisted laser desorption/ionization-time of flight mass spectrometry; refining the database for improved identification.

Matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) has revolutionized bacterial identification. However, the phylogenetic resolution is still insufficient for discerning several ß-haemolytic streptococcal species. We aimed to improve the diagnostic performance of MALDI-ToF through manual curation of the reference spectra in Brukers Compass Library DB-7854. Before intervention, only 133 out of 217 (62%) Streptococcus dysgalactiae isolates were successfully identified to the species level, 83 isolates were identified to the genus level as either S. dysgalactiae, S. pyogenes or S. canis, and one S. dysgalactiae isolate was wrongly identified as S. canis. All 109 S. canis isolates were successfully identified to the species level. Removal of three reference spectra from the database significantly improved the identification of S. dysgalactiae to 94%, without compromising identification of S. canis. This illustrates the advantage of refinement of the reference database in order to improve the analytic precision of MALDI-ToF.

Genomic evidence for a globally distributed, bimodal population in the ovine footrot pathogen Dichelobacter nodosus.

UNLABELLED: 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.

A recently introduced Dichelobacter nodosus strain caused an outbreak of footrot in Norway.

BACKGROUND: In 2008, an outbreak of ovine footrot occurred in Norway. Dichelobacter nodosus isolates collected between 2008 and 2011 have been characterised. Isolates defined as virulent by the gelatin gel test (GG-test) were only found in sheep in Rogaland County, where the severe cases of footrot were registered. The majority (96%) of the virulent isolates belonged to serogroup A. It is suspected that they represent a newly introduced strain, and the aim of the present study was to investigate whether they are genetically similar. Sixty-one virulent isolates from sheep and 116 benign isolates from sheep, cattle and goats were included. Four GG-test virulent isolates from Danish sheep were also included. All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and by PCR for pgr variant determination. RESULTS: The Norwegian virulent isolates were assigned to 8 pulsotypes (PTs), while the benign isolates were assigned to 66 PTs. Thirty-seven (68.5%) of the 54, virulent, serogroup A isolates belonged to the same PT, and included isolates from 2008 through 2011. Isolates belonging to this PT were defined as the outbreak strain. The remaining virulent serogroup A isolates belonged to 4 PTs differing by ≤3 bands from the outbreak strain. Two virulent, Danish, serogroup A isolates differed by 2 bands from the Norwegian outbreak strain. All but 3 (95%) of the virulent isolates had the pgrA variant while 85% of the benign isolates had the pgrB variant. CONCLUSION: This study provides evidence that the footrot outbreak in Norway in 2008 most likely was caused by new introduction and local spread of one virulent D. nodosus strain.

Cross-infection of virulent Dichelobacter nodosus between sheep and co-grazing cattle.

Dichelobacter nodosus is the main aetiological agent of ovine footrot and the bacterium has also been associated with interdigital dermatitis is cattle. The aim of this study was to investigate possible cross-infection of virulent D. nodosus between sheep and co-grazing cattle. Five farms, where sheep previously diagnosed with virulent D. nodosus were co-grazing with cattle for different periods of time, were included. The study sample consisted of 200 cows and 50 sheep. All cows were examined for the presence of interdigital dermatitis, and ten ewes, preferably with symptoms of footrot, had the footrot scores recorded. On each farm, the same ten ewes and ten cows were chosen for bacterial analyses. Swabs were analysed for D. nodosus by PCR and culturing. D. nodosus isolates were virulence-tested and assigned to serogroups by fimA variant determination. Biopsies were evaluated histopathologically and analysed by fluorescent in situ hybridization for D. nodosus, Treponema spp. and Fusobacterium necrophorum. D. nodosus defined as virulent by the gelatin gel test were isolated from 16 sheep from four farms and from five cows from two of the same farms. All five cows had interdigital dermatitis. Two of the cows stayed infected for at least eight months. By pulsed-field gel electrophoresis (PFGE), the isolates from the five cows were found to be genetically indistinguishable or closely related to isolates from sheep from the same farm. This indicates that cross-infection between sheep and cows have occurred.

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.