DIRECT DETECTION OF ACTINOBACILLUS PLEUROPNEUMONIAE IN SUIDAE ORGANS IN SÃO PAULO STATE, BRAZIL, BY POLYMERASE CHAIN REACTION (NESTED-PCR)

ABSTRACT Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an important respiratory disease, responsible for economic losses and reduced productivity. The aim of this study was to determine occurrence of A. pleuropneumoniae in field samples, using an adapted nested-PCR reaction targeting the Apx IV gene. Different DNA concentrations (from 30 µg/mL to 0.01 ng/mL) of A. pleuropneumoniae serotype III reference strain were used to determine the level of sensitivity of first generation and nested-PCR reactions. Thirty-seven field samples sent to Instituto Biológico from 1995 to 2007 were tested by PCR and nested-PCR. Determination of the level of sensitivity showed that PCR could amplify to 2 ng/mL of extracted DNA and nested-PCR to 0.4 ng/mL. Since the nested reaction exhibited a level of sensitivity 5 times greater than the PCR reaction to detect a reference strain, using nested-PCR could minimize the occurrence of false-negative results. Among tested samples, 10 of them were nested-PCR positive, showing occurrence of A. pleuropneumoniae in 9 different animals (including one wild boar). This nested-PCR reaction can be used for direct detection of A. pleuropneumoniae in field samples, even after frozen storage for long periods, without the need for previous bacterial isolation.

摘要：由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，下称A. pleuropneumoniae）引起的猪传染性胸膜肺炎是一种重要的呼吸道传染病，可造成显著经济损失并降低生产效率。本研究旨在以A. pleuropneumoniae的Apx IV基因为靶标，采用优化的巢式聚合酶链式反应（nested-PCR），检测该菌在野外样本中的感染情况。本研究使用浓度范围为30 μg/mL至0.01 ng/mL的A. pleuropneumoniae血清型III参考菌株基因组DNA，以测定常规聚合酶链式反应（PCR）与巢式PCR的灵敏度阈值。1995年至2007年间送往生物研究所（Instituto Biológico）的37份野外样本，均通过PCR与巢式PCR完成了检测。灵敏度测定结果显示，常规PCR的最低可检测DNA浓度为2 ng/mL，而巢式PCR可低至0.4 ng/mL。由于巢式PCR的灵敏度较常规PCR提升5倍，采用该方法可有效降低假阴性结果的发生概率。在受试的37份样本中，共有10份呈巢式PCR阳性，涉及9头不同动物（包含1头野猪），证实了A. pleuropneumoniae在野外样本中的存在。本研究所用的巢式PCR可直接用于野外样本中A. pleuropneumoniae的检测，即便样本经过长期冷冻保存，也无需预先进行细菌分离培养。