de Graaf Dirk C, De Vos Paul, Heyndrickx Marc, Van Trappen Stefanie, Peiren Nico, Jacobs Frans J
Laboratory of Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
J Invertebr Pathol. 2006 Feb;91(2):115-23. doi: 10.1016/j.jip.2005.10.010. Epub 2005 Dec 20.
This study was initially aimed at developing a PCR-test to differentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the "insert of clone 9" (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian field strains revealed five isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conflicting data, this carbohydrate acidification was not conclusive. Therefore, the exact taxonomic position of the five retained strains was determined by a polyphasic approach using SDS-PAGE, AFLP, and ERIC-based PCR. Four iC9-negative field strains could be identified as P. larvae subsp. larvae; the taxonomic position of the fifth field strain remained ambiguous. The latter was provisionally classified as a subspecies pulvifaciens strain on the basis of SDS-PAGE. The present paper demonstrates the existence of field strains that do not fit well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.
本研究最初旨在开发一种聚合酶链反应（PCR）检测方法，以区分蜜蜂美洲幼虫腐臭病的病原体（幼虫芽孢杆菌幼虫亚种）和粉介壳病的病原体（幼虫芽孢杆菌粉状亚种）。该检测基于“克隆9插入片段”（iC9），指的是一个克隆的1.9 kB HaeIII片段，该片段仅存在于幼虫芽孢杆菌幼虫亚种参考菌株中，可能与美洲幼虫腐臭病的毒力相关。结果表明，基于iC9的PCR检测能够区分这两个亚种的比利时微生物保藏中心/比利时微生物遗传资源保藏中心（BCCM/LMG）参考菌株。然而，对179株比利时田间菌株的筛选发现，有5株分离株未产生基于iC9的扩增子，因此更类似于幼虫芽孢杆菌粉状亚种。此外，它们都能从甘露醇中产酸，这是粉状亚种先前具有的一个特征。由于参考菌株给出了相互矛盾的数据，这种碳水化合物酸化并不具有决定性。因此，通过使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、扩增片段长度多态性（AFLP）和基于肠杆菌基因间重复共有序列（ERIC）的PCR的多相方法，确定了这5株保留菌株的确切分类地位。4株iC9阴性田间菌株可鉴定为幼虫芽孢杆菌幼虫亚种；第5株田间菌株的确切分类地位仍不明确。根据SDS-PAGE结果，后者暂时被归类为粉状亚种菌株。本文证明了存在一些田间菌株，它们并不完全符合将幼虫芽孢杆菌物种细分为两个亚种的分类方式。由于已知两个亚种中只有一个代表美洲幼虫腐臭病的病原体这一情况，这对这种蜜蜂疾病的诊断来说是一个严重障碍。
