Sannö Axel, Jacobson Magdalena, Sterner Sandra, Thisted-Lambertz Susanne, Aspán Anna
Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
School of Health Sciences, Örebro University, Örebro, Sweden.
J Microbiol Methods. 2018 Jul;150:32-38. doi: 10.1016/j.mimet.2018.05.014. Epub 2018 May 21.
Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n=354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.
沙门氏菌病和耶尔森氏菌病是须上报的人类疾病，通常与受污染的食物有关。家猪以及野猪和其他野生动物已被确定为这些细菌的宿主。沙门氏菌属的培养方法和分子流行病学调查方法已经很成熟，然而，致病性耶尔森氏菌属的培养耗时，且常用的分子流行病学调查方法——脉冲场凝胶电泳，鉴别能力不足。本研究的目的是开发和评估一种适用于野生动物样本和其他高度污染样本的筛查方案。该方法基于PCR筛查，然后在富集肉汤上进行多位点可变数目串联重复分析（MLVA），以获得致病性耶尔森氏菌属的分子流行病学数据，而无需纯分离株。使用来自90头瑞典野猪的野猪样本（n=354），包括扁桃体、粪便和淋巴结，对该方案的性能进行了评估。新方案在检测和培养小肠结肠炎耶尔森氏菌和沙门氏菌属方面的表现与既定的ISO标准相当或更好，然而，对于假结核耶尔森氏菌的培养，还需要进一步改进。选择具有运动性的菌株似乎有利于沙门氏菌属和小肠结肠炎耶尔森氏菌的富集。此外，PCR分析前的选择性富集消除了原始样本中存在的抑制因子。总共获得了10株不同生物血清型的小肠结肠炎耶尔森氏菌分离株，这些分离株的MLVA图谱与相应富集肉汤的图谱一致。此外，还获得了22株沙门氏菌属分离株，包括6种不同的血清型，其中富氏沙门氏菌、哈达尔沙门氏菌和单相鼠伤寒沙门氏菌最为常见。总之，所提出的筛查方案提供了一种快速有效的方法，可在短时间内从大量样本集中获得流行率数据以及MLVA数据。因此，这些结果可以提高对这些病原体的流行病学和分布及其对公共卫生重要性的认识。
