Santos Huarrisson A, Pires Marcus S, Vilela Joice A R, Santos Tiago M, Faccini João L H, Baldani Cristiane D, Thomé Sandra M G, Sanavria Argemiro, Massard Carlos L
Department of Animal Parasitology, Federal Rural University of Rio de Janeiro, Rio de Janeiro, Seropédica 23890-000, Brazil.
J Vet Diagn Invest. 2011 Jul;23(4):770-4. doi: 10.1177/1040638711406974. Epub 2011 Jun 15.
Anaplasma phagocytophilum was detected in dogs from Brazil in the municipalities of Seropédica and Itaguaí, Rio de Janeiro state, by real-time polymerase chain reaction (PCR) using SYBR Green to detect the amplification. Of 253 samples, 18 (7.11%) were positive, with a threshold cycle (Ct) ranging from 31 to 35 cycles. The PCR product from a positive sample was cloned and sequenced. The sequence obtained demonstrated 100% identity with other A. phagocytophilum sequences published in the GenBank database. The analytical sensitivity of RT-PCR using SYBR Green system was able to detect 3 plasmid copies when defined numbers of plasmid copies containing 122 base pairs from the msp2 gene were used. The assay was considered specific when DNA from bacteria (Anaplasma platys, Anaplasma marginale, Ehrlichia canis, Neorickettsia risticii, Rickettsia rickettsii) closely related to A. phagocytophilum was placed in the reaction. These results demonstrate that the canine granulocytic anaplasmosis agent is present in regions in which dogs could be a source of infection for tick vectors. The current study reports the detection of A. phagocytophilum, a zoonotic agent responsible for Human granulocytic anaplasmosis, in Brazilian dogs.
在巴西里约热内卢州塞罗佩迪卡市和伊塔瓜伊市的犬类中，通过使用SYBR Green的实时聚合酶链反应（PCR）检测扩增情况，发现了嗜吞噬细胞无形体。在253份样本中，18份（7.11%）呈阳性，阈值循环（Ct）范围为31至35个循环。对一份阳性样本的PCR产物进行了克隆和测序。获得的序列与GenBank数据库中公布的其他嗜吞噬细胞无形体序列显示出100%的同一性。当使用含有来自msp2基因的122个碱基对的特定数量的质粒拷贝时，使用SYBR Green系统的RT-PCR的分析灵敏度能够检测到3个质粒拷贝。当将与嗜吞噬细胞无形体密切相关的细菌（血小板无形体、边缘无形体、犬埃立克体、里氏新立克次体、立氏立克次体）的DNA放入反应中时，该检测方法被认为具有特异性。这些结果表明，犬粒细胞无形体病病原体存在于犬类可能成为蜱传播媒介感染源的地区。当前的研究报告了在巴西犬类中检测到嗜吞噬细胞无形体，这是一种导致人类粒细胞无形体病的人畜共患病原体。
