The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales.
ABSTRACT Footrot, caused by the strictly anaerobic bacterium Dichelobacter nodosus, is the most common cause of lameness in sheep in Great Britain but problems exist in association with its diagnosis and control. The fastidious nature of D. nodosus means that complex media and several weeks are required for characterisation. An alternative method to simplify and enhance the detection of D. nodosus in clinical samples is therefore highly desirable. In terms of control, anecdotal evidence from the farming community suggests that the commercially available vaccine, based on Australian isolates of D. nodosus, is not widely employed in this country due to its perceived inefficacy. Seven hundred and six isolates, collected from outbreaks in England and Wales, were therefore used to investigate these issues. A 16S rRNA PCR was adapted to detect D. nodosus in clinical material within 1 day of sampling; a 15% increase in detection compared with culture and less than 1% false negatives were achieved. This represents a major advance in the rapid diagnosis of footrot and will be of great value to practitioners and diagnostic laboratories. Bacterial virulence was tested using protease thermostability and zymogram assays, whilst serogrouping was performed by slide agglutination. All isolates demonstrated virulence patterns previously recorded in Australia and all nine serogroups of D. nodosus (A-I) were represented. Serogroup H was predominant. There was, therefore, no evidence for the presence of novel strains of D. nodosus compared with Australia suggesting the need for further investigation into farmers' views on the use of the commercial vaccine in Great Britain.
Article: Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories.[show abstract] [hide abstract]
ABSTRACT: Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. 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 D. nodosus 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 D. nodosus and to compare its performance with culturing and conventional PCR. A D. nodosus-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. The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus 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. The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.Acta Veterinaria Scandinavica 01/2012; 54:6. · 1.00 Impact Factor
Article: Serological diversity and virulence determination of Dichelobacter nodosus from footrot in India.[show abstract] [hide abstract]
ABSTRACT: One hundred and twenty-eight swab samples from footrot lesions of naturally infected sheep were examined for presence of Dichelobacter nodosus (D. nodosus). The detection of D. nodosus was carried out by polymerase chain reaction (PCR), directly from swabs or after isolation, using 16S rDNA specific primers. The isolation of the bacterium was carried out anaerobically on trypticase-arginine-serine (TAS) agar containing 4% hoof powder. Serogrouping of the D. nodosus was accomplished with multiplex PCR using nine (A-I) serogroup specific primers. The virulent and benign status of the isolates was ascertained by detection of virulence specific integrase A (intA) gene. Out of total 83 D. nodosus isolates, 62 (74.69%) belonged to serogroup B, 18 (21.68%) to serogroup E and three (3.62%) to serogroup I. Serogroup I was detected and isolated for the first time in India. All the positive samples revealed infection by single serogroup of D. nodosus except one which showed mixed infection of serogroups B and E. Sixty (72.28%) isolates possessed intA gene and thus were considered as virulent strains. Serogroupwise intA gene was found in 43 (69.35%) isolates of serogroup B, 14 (77.78%) of E and in all the three (100%) of I.Molecular and Cellular Probes 02/2009; 23(2):112-4. · 2.08 Impact Factor