Good diagnostic tools are essential to identify the presence of D. nodosus
and to study its epidemiology. Such knowledge is important to limit and control footrot, a disease that constitutes a major animal welfare problem. A correct diagnosis is a prerequisite to distinguish footrot from other diseases or conditions that can affect the feet of sheep such as contagious ovine digital dermatitis (CODD), white line disease, granulomas and toe and pedal joint abscesses [13
Footrot is often introduced into a sheep flock by the purchase of an infected animal and transmission within a flock occurs mainly from sheep to sheep via the environment [14
]. The environment can also be a source for introduction of footrot as reported by Whittington et al. [15
] where a flock became infected after using the same yard used by an infected flock some hours earlier. The risk of introduction or re-introduction of footrot into a flock can be reduced by some principal preventive strategies as described by Abbott and Lewis [16
]: animals should only be purchased from footrot-free flocks, purchased animals should be quarantined, or the flock should be sequestered from outside introduction.
In this study, a TaqMan-based real-time PCR assay targeting the 16S rRNA gene for the detection of D. nodosus in clinical samples was developed in collaboration between SVA, NVI and DTU-VET. The approach was chosen with the aim of improving detection of D. nodosus compared to traditional culturing and conventional PCR. The real-time PCR assay was compared to culturing for 126 Swedish sheep by the SVA and to conventional PCR for 224 Norwegian sheep by the NVI. Its specificity (inclusivity/exclusivity) was tested at SVA and its sensitivity was tested and compared at all three laboratories. It is an advantage that the same detection method with the same sensitivity can be used in the three different Scandinavian countries, so that results can be easily compared.
Another real-time PCR targeting D. nodosus
has recently been published by Calvo-Bado et al. [17
] but its emphasis is on quantification rather than detection. This assay is based on the rpoD
gene which is a single copy gene in the D. nodosus
genome while the developed real-time PCR in this study is based on the 16S rRNA gene which exists in three copies [18
]; this is an advantage when sensitive detection is required.
There was a significant difference between the real-time PCR assay and culturing with the real-time PCR method being three times more sensitive in detecting positive samples. This is not surprising because D. nodosus
is a fastidious organism that can be difficult to culture, particularly when samples are not plated immediately. It was, however, somewhat surprising that the total number of real-time PCR positive samples was not higher (81.7%) as all samples were from sheep with clinical signs of footrot (score ≥2 foot lesions). One explanation could be that sampling, which took place in the field and by different persons, was not optimal and that sample quality deteriorated in the post. In a footrot prevalence study by König et.al. [19
], in which sampling took place at the laboratory and by the same persons, 97% of the samples from sheep with score 2 footrot lesions were found positive with the same real-time PCR developed in this study and 79% by culturing.
When weak bands in the conventional PCR were sequenced there was good agreement between conventional PCR and the developed real-time PCR method. However, the real-time PCR method detected 8% more positive samples compared to the conventional PCR.
In the 13 samples found to be negative by the real-time PCR, the conventional PCR gave a faint band of approximately the correct size. Without sequencing, these samples could have been incorrectly interpreted as D. nodosus
positive. At the NVI this was a severe problem when D. nodosus
diagnostics were first implemented there. Of approximately 6000 samples analysed by the conventional PCR in a screening study in 2008 [20
], 11% had to be sequenced due to diffuse bands. Of the sequenced samples, 75% of these were found not to be D. nodosus
after a BLAST search (Jannice S Slettemeås, personal communication).
A main advantage with the probe-based, real-time PCR over conventional PCR is that it eliminates non-specific amplicons and faint bands of the correct product size. A great proportion (21%) of the conventional PCR products had to be sequenced. The real-time PCR is more sensitive, less time consuming and laborious, and does not involve post-PCR processing. A greater sensitivity of real-time PCR compared to conventional PCR has also been shown in previous studies [21
] and [22
]. Real-time PCR is a good tool for identifying slow-growing bacteria like D. nodosus
. Probe-based real-time PCR provides specificity, i.e. it limits some of the nonspecific fluorescence signals toward the end of the reaction [23
There were no signs of inhibition in the developed real-time PCR assay in this study, but inhibitors can vary with sample material. Inhibition is also dependent on the DNA purification method used, so one should run an IAC simultaneously with the samples or in a subsequent assay.