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）的msp4基因（major surface protein 4 gene）具有单拷贝、稳定性强且同源性较高的特征，被视为该病原菌系统地理学特征分析的有效分子标记。本研究旨在以巴西潘塔纳尔湿地肉牛体内的边缘无浆体msp4基因为基础，分析其系统地理学特征，并与全球其他地区的分离株序列进行对比。本研究采集的血样来自该区域5个粗放型肉牛养殖场的400头肉牛（其中200头母牛、200头犊牛）。结果显示，经定量聚合酶链式反应（quantitative PCR, qPCR）检测，基于msp1β基因（major surface protein 1β gene），400份受检样本中有56.75%（227/400）呈边缘无浆体阳性；在上述227份阳性样本中，经常规聚合酶链式反应检测，有8.37%（19/227）的msp4基因呈阳性。在系统发育网络距离分析中，来自巴西潘塔纳尔湿地的14条序列与泰国、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被归为同一分支。在来自巴西及全球的68条序列中，共鉴定出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）分析与系统发育网络分析均显示，从巴西潘塔纳尔湿地肉牛体内检测到的边缘无浆体msp4基因序列多态性较低，且形成了一个与南美、中美、欧洲及亚洲反刍动物体内分离株系统发育相关的基因群。