Dipartimento di Scienze Biomediche, Università di Sassari, Sassari, Italy.
Istituto Zooprofilattico della Sardegna, Sassari, Italy.
Sci Rep. 2022 Jan 10;12(1):475. doi: 10.1038/s41598-021-04451-w.
Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.
副结核病是一种无法治愈的反刍动物胃肠炎，由鸟分枝杆菌亚种副结核分枝杆菌（MAP）引起，是一种耐酸分枝杆菌。为了加速对活病原体的检测，优化了一种常规（肽介导磁分离：PMS）和新型（噬菌体-珠 qPCR：PBQ）基于噬菌体的检测方法。与 PMS-噬菌体检测方法的 100 个细胞/10 毫升相比，PBQ 对 MAP 的检测限（LOD）为 10 个 MAP/10 毫升。通过 PBQ，在 41 份未经巴氏消毒的绵羊和山羊奶样本中发现了 48.78%的活 MAP。通过 PMS-噬菌体检测方法检测的绵羊奶样本中未发现活 MAP。在为期两周的间隔后，通过 PBQ 也证实了来自 21 只近期绵羊的奶样中没有活 MAP。尽管两种噬菌体检测方法在奶样中均未检测到活 MAP，但在其中两次采样中，一些动物的奶和血清中均检测到了 MAP DNA 和针对 MAP 的抗体，表明一些绵羊动物是 MAP 的排放者。总之，PBQ 和 PMS-噬菌体检测方法有望用于评估奶样中的 MAP 活性。然而，与 PMS-噬菌体检测方法相比，PBQ 具有较低的 LOD、快速性、更高的敏感性、不需要分枝杆菌和相应的病毒灭活处理，这些在 PMS-噬菌体检测方法中分别是制作草坪和灭活外源性分枝杆菌噬菌体所必需的，因此 PBQ 更具优势。
