Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal

Abstract The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1β gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.

摘要 边缘无浆体（Anaplasma marginale，A. marginale）的msp4基因（major surface protein 4）具有单密码子特性、稳定性强且整体同源性较高，被视为该细菌系统地理学鉴定的有效标记基因。本研究旨在以巴西潘塔纳尔湿地肉牛体内的msp4基因为基础，分析边缘无浆体的系统地理学特征，并与全球其他地区的分离株进行对比。本研究采集了该区域5个粗放型养殖牧场中饲养的400头肉牛的血液样本（其中母牛200头、犊牛200头）。检测结果显示，经定量聚合酶链式反应（qPCR）靶向msp1β基因（major surface protein 1β）检测，受试样本中56.75%（227/400）为边缘无浆体阳性；在上述阳性样本中，经常规聚合酶链式反应（conventional PCR）检测，8.37%（19/227）的样本msp4基因呈阳性。系统发育网络距离分析结果表明，来自巴西潘塔纳尔湿地的14条msp4基因序列，与泰国、印度、西班牙、哥伦比亚、巴西巴拉那州、墨西哥、葡萄牙、阿根廷、中国、委内瑞拉、澳大利亚、意大利以及巴西米纳斯吉拉斯州的对应序列聚为同一类群。在全球范围内的68条巴西及其他国家的序列中，共存在15种基因型，其中1号基因型（#1）在全球分布最为广泛。分裂树（Splitstree）分析与网络分析均显示，巴西潘塔纳尔湿地肉牛体内检出的边缘无浆体msp4基因序列多态性较低，且形成了一个与南美、中美洲、欧洲及亚洲反刍动物分离株系统发育亲缘性较高的基因群。