Detection of Dirofilaria immitis using microscopic, serological and molecular techniques among dogs in Cabo Frio, RJ, Brazil

Abstract Heartworm disease is a health problem for dogs and cats, especially in tropical and subtropical coastal regions of the world. Some studies have compared the efficacy of the diagnostic techniques used to detect this parasitosis. Therefore, the aim of this study was to compare parasitological optical microscopy (POM), serological and molecular techniques for diagnosing canine heartworm infection. Samples were collected between July 2015 and April 2016 from 103 dogs in Cabo Frio, RJ, Brazil. The wet fresh blood, thick smears, thin smears and modified Knott’s test were used to detect microfilariae. ELISA (Snap™ 4Dx ® IDEXX) was used to detect antigens and the polymerase chain reaction (PCR) was used to detect DNA and enable sequencing for species differentiation and confirmation. 19.4% of samples were positive according to microscopy. Through PCR, 15.5% of the total were positive. Using ELISA, the positivity rate was 29.1%. Occult heartworm infection was detected in 11.6% of the samples. ELISA sensitivity was shown to be higher than PCR or microscopy (P = 0.001). Sequencing of samples confirmed the presence of Dirofilaria immitis and Acanthocheilonema reconditum . ELISA was more effective for serological diagnosis canine heartworm and should be used in clinical and epidemiological studies.

摘要 心丝虫病是犬与猫的常见健康问题，在全球热带及亚热带沿海地区尤为高发。已有多项研究针对该寄生虫病的各类诊断检测技术的效能展开对比分析。为此，本研究旨在对比寄生虫光学显微镜检法（parasitological optical microscopy, POM）、血清学及分子生物学技术在犬心丝虫感染诊断中的应用效果。本研究于2015年7月至2016年4月间，从巴西里约热内卢州卡布弗里奥的103只受试犬只身上采集检测样本。实验采用新鲜湿血涂片、厚血膜涂片、薄血膜涂片及改良Knott试验检测微丝蚴；使用ELISA试剂盒（Snap™ 4Dx ® IDEXX）检测血清抗原；采用聚合酶链式反应（polymerase chain reaction, PCR）扩增样本DNA，用于物种鉴定与确认的测序分析。经显微镜检，19.4%的样本呈阳性；经PCR检测，总阳性率为15.5%；采用ELISA检测的阳性率则为29.1%。在11.6%的样本中检出隐匿性心丝虫感染。统计学分析表明，ELISA的灵敏度显著高于PCR与显微镜检（P=0.001）。对阳性样本的测序结果确认了犬恶丝虫（Dirofilaria immitis）与刚棘唇线虫（Acanthocheilonema reconditum）的存在。综上，ELISA在犬心丝虫血清学诊断中具有更优的应用效能，适用于临床诊疗及流行病学相关研究。