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

Experimental infection of sheep with ovine and bovine Dichelobacter nodosus isolates

AbstractThe aim of this study was, under experimental conditions, to investigate infection of Norwegian White sheep with ovine and bovine isolates of Dichelobacter nodosus of varying virulence. In addition, the efficacy of gamithromycin as a treatment for the experimentally induced infections was examined. The study was performed as a single foot inoculation using a boot. Four groups, each with six lambs, were inoculated with four different challenge strains (Group 1: benign bovine strain; Group 2: virulent bovine strain; Group 3: benign ovine strain; Group 4: virulent ovine strain). The main criterion to determine that infection was transferred was that D. nodosus isolate was obtained by culture. After the trial all lambs were treated with gamithromycin. Clinical symptoms of footrot developed in all groups, and when removing the boots two weeks after challenge, D. nodosus was isolated from 5 of 24 experimental lambs. All lambs tested negative for D. nodosus by PCR within six weeks after treatment with gamithromycin. This study strongly indicates that D. nodosus isolates from both sheep and cattle can be transferred to sheep under experimental conditions. The study also indicates that gamithromycin may be effective against D. nodosus

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

DirtyGenes: testing for significant changes in gene or bacterial population compositions from a small number of samples

High throughput genomics technologies are applied widely to microbiomes in humans, animals, soil and water, to detect changes in bacterial communities or the genes they carry, between different environments or treatments. We describe a method to test the statistical significance of differences in bacterial population or gene composition, applicable to metagenomic or quantitative polymerase chain reaction data. Our method goes beyond previous published work in being universally most powerful, thus better able to detect statistically significant differences, and through being more reliable for smaller sample sizes. It can also be used for experimental design, to estimate how many samples to use in future experiments, again with the advantage of being universally most powerful. We present three example analyses in the area of antimicrobial resistance. The first is to published data on bacterial communities and antimicrobial resistance genes (ARGs) in the environment; we show that there are significant changes in both ARG and community composition. The second is to new data on seasonality in bacterial communities and ARGs in hooves from four sheep. While the observed differences are not significant, we show that a minimum group size of eight sheep would provide sufficient power to observe significance of similar changes in further experiments. The third is to published data on bacterial communities surrounding rice crops. This is a much larger data set and is used to verify the new method. Our method has broad uses for statistical testing and experimental design in research on changing microbiomes, including studies on antimicrobial resistance

Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.

Funder: Department of Large Animal Clinical SciencesFunder: USDA‐NIFA AgriLife and the Link Equine Research EndowmentFunder: Texas A&M University; Id: http://dx.doi.org/10.13039/100007904Funder: FormasFunder: Intervacc ABFunder: The Horse TrustFunder: SEIBFunder: Paul Mellon FoundationFunder: Alice Noakes Memorial Charitable TrustFunder: Ivo TrustFunder: TattersallsFunder: The Elise Pilkington Charitable TrustFunder: The European Breeders FundFunder: The Serth and Gates CharityFunder: Margaret Giffen Charitable TrustFunder: The Payne Gallwey Charitable TrustFunder: The Stafford TrustFunder: Marjorie Coote Animal Charity TrustFunder: Beryl Evetts and Robert Luff Animal Welfare TrustFunder: The Anne Duchess of Westminster’s Charitable TrustBACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi