Frosth Sara, König Ulrika, Nyman Ann-Kristin, Aspán Anna
Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, P. O. Box 7036, SE-750 07, Uppsala, Sweden.
Department of Microbiology, National Veterinary Institute (SVA), SE-751 89, Uppsala, Sweden.
Vet Res Commun. 2017 Sep;41(3):189-193. doi: 10.1007/s11259-017-9686-9. Epub 2017 Mar 25.
Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.
坏死梭杆菌是羊腐蹄病的主要病因，菌株毒力是影响疾病严重程度的重要因素。因此，坏死梭杆菌的检测和毒力测定对于该病的正确诊断至关重要。如今，通过实时荧光定量PCR分析可以实现这一点。分析大量样本成本高且费力；因此，在监测项目中通常会将个体样本合并。然而，合并样本会降低该方法的灵敏度。本研究的目的是开发一种用于实时荧光定量PCR分析的合并样本方法，该方法能够灵敏地检测坏死梭杆菌并同时测定其毒力。2014年，在瑞典腐蹄病防控项目中，使用ESwabs对来自17个羊群的225只绵羊进行了采样。样本首先单独进行分析，然后通过针对坏死梭杆菌16S rRNA和aprV2/B2基因的实时荧光定量PCR检测，以五个样本为一组进行分析。每组由四个坏死梭杆菌阴性样本和一个含有不同数量该细菌的阳性样本组成。在个体分析中，61个（27.1%）样本在16S rRNA和aprV2/B2 PCR检测中呈阳性，164个（72.9%）样本呈阴性。所有在aprV2/B2 PCR检测中呈阳性的样本均为aprB2变体。合并样本分析表明，所有41组样本在坏死梭杆菌16S rRNA和aprB2变体检测中也呈阳性。因此，合并样本和个体样本的诊断灵敏度相似。我们的方法包括在DNA提取前对细菌进行浓缩。这可能是保持诊断灵敏度的原因。合并样本实时荧光定量PCR检测的诊断灵敏度与个体分析样本获得的灵敏度相当。即使是亚临床感染在合并的PCR样本中也能够被检测到，这对于疾病的控制很重要。因此，这种方法可以应用于腐蹄病防控项目中，取代个体样本分析。
