Danesi Patrizia, Ravagnan Silvia, Johnson Lynelle R, Furlanello Tommaso, Milani Adelaide, Martin Patricia, Boyd Susan, Best Matthew, Galgut Bradley, Irwin Peter, Canfield Paul J, Krockenberger Mark B, Halliday Catriona, Meyer Wieland, Malik Richard
Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy.
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, California, USA.
Med Mycol. 2017 Nov 1;55(8):828-842. doi: 10.1093/mmy/myx007.
Pneumocystis pneumonia (PCP) is a life-threatening fungal disease that can occur in dogs. The aim of this study was to provide a preliminary genetic characterisation of Pneumocystis carinii f.sp.'canis' (P. canis) in dogs and thereby develop a reliable molecular protocol to definitively diagnose canine PCP. We investigated P. canis in a variety of lung specimens from dogs with confirmed or strongly suspected PCP (Group 1, n = 16), dogs with non-PCP lower respiratory tract problems (Group 2, n = 65) and dogs not suspected of having PCP or other lower respiratory diseases (Group 3, n = 11). Presence of Pneumocystis DNA was determined by nested PCR of the large and small mitochondrial subunit rRNA loci and by a real-time quantitative polymerase chain reaction (qPCR) assay developed using a new set of primers. Molecular results were correlated with the presence of Pneumocystis morphotypes detected in cytological/histological preparations. Pneumocystis DNA was amplified from 13/16 PCP-suspected dogs (Group 1) and from 4/76 dogs of control Groups 2 and 3 (combined). The latter four dogs were thought to have been colonized by P. canis. Comparison of CT values in 'infected' versus 'colonized' dogs was consistent with this notion, with a distinct difference in molecular burden between groups (CT ≤ 26 versus CT range (26 <CT < 35), respectively). Phylogenetic analyses showed that P. canis is specifically 'canine' associated, being separated from other mammalian Pneumocystis species, thereby confirming the accuracy of qPCR amplicon for Pneumocystis in dogs. Using qPCR, Pneumocystis DNA can be detected in specimens from the respiratory tract and a CT value can be interpreted to distinguish infection versus colonization.
卡氏肺孢子虫肺炎（PCP）是一种可发生于犬类的危及生命的真菌性疾病。本研究的目的是对犬类中的卡氏肺孢子虫f.sp.'犬型'（犬肺孢子虫）进行初步的基因特征分析，从而开发一种可靠的分子检测方法以明确诊断犬类PCP。我们对来自确诊或高度怀疑患有PCP的犬类的多种肺标本（第1组，n = 16）、患有非PCP下呼吸道问题的犬类（第2组，n = 65）以及未怀疑患有PCP或其他下呼吸道疾病的犬类（第3组，n = 11）中的犬肺孢子虫进行了调查。通过对线粒体大亚基和小亚基rRNA基因座进行巢式PCR以及使用一组新引物开发的实时定量聚合酶链反应（qPCR）检测法来确定肺孢子虫DNA的存在。分子检测结果与在细胞学/组织学制剂中检测到的肺孢子虫形态型的存在情况相关联。在13/16只疑似PCP的犬类（第1组）以及对照组第2组和第3组的4/76只犬类（合并计算）中扩增出了肺孢子虫DNA。后四只犬类被认为已被犬肺孢子虫定植。“感染”犬与“定植”犬的CT值比较与此观点一致，两组之间的分子负荷存在明显差异（分别为CT≤26与CT范围（26＜CT＜35））。系统发育分析表明，犬肺孢子虫与犬类具有特异性关联，与其他哺乳动物肺孢子虫物种分开，从而证实了犬类肺孢子虫qPCR扩增子的准确性。使用qPCR，可以在呼吸道标本中检测到肺孢子虫DNA，并且可以通过解读CT值来区分感染与定植情况。
