Garcia Amanda Barbosa, Jusi Márcia Mariza Gomes, Freschi Carla Roberta, Ramos Inalda Angélica Souza, Mendes Natália Serra, Bressianini do Amaral Renan, Gonçalves Luiz Ricardo, André Marcos Rogério, Machado Rosangela Zacarias
Postgraduate Program in Agricultural Microbiology, Universidade Estadual Paulista (FCAV / UNESP), Jaboticabal, SP, Brazil; Immunoparasitology Laboratory, Department of Veterinary Pathology, Faculdade de Ciências Agrárias e Veterinárias - Universidade Estadual Paulista (FCAV / UNESP), Jaboticabal, SP, Brazil.
Immunoparasitology Laboratory, Department of Veterinary Pathology, Faculdade de Ciências Agrárias e Veterinárias - Universidade Estadual Paulista (FCAV / UNESP), Jaboticabal, SP, Brazil.
Ticks Tick Borne Dis. 2022 Jan;13(1):101829. doi: 10.1016/j.ttbdis.2021.101829. Epub 2021 Sep 23.
Anaplasma marginale is an obligate intracellular Gram-negative bacterium that is parasitic to erythrocytes and is the main agent of bovine anaplasmosis. This disease causes severe anemia and reduces weight gain and milk production, thus giving rise to major economic losses relating to livestock worldwide. The genetic diversity of this bacterium has been characterized based on sequences of major surface proteins (MSPs), especially MSP1α. This has enabled identification of several geographical strains, according to different amino acid sequences. The aim of this study was to investigate the genetic diversity of A. marginale in naturally infected Angus beef cattle during a disease outbreak in southeastern Brazil. Four blood samples were collected over a four-month period from each of 20 animals on a cattle farm in Itú, São Paulo, Brazil. Serum samples were subjected to indirect ELISA to detect anti-A. marginale IgG antibodies. The 80 whole-blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1β gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing using the Sanger method. The sequences obtained were analyzed for genetic diversity using the RepeatAnalyzer software. Both iELISA tests, using recombinant MSP5 and the Anaplasma antibody test kit (VMRD), revealed high seroprevalence: 91.25% and 97.5%, respectively. In qPCR, 100% of the samples were positive, with between 10 and 10 DNA copies/μL. In the snPCR based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis on the 36 sequences obtained showed the presence of genotypes H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). The RepeatAnalyzer software identified 36 strains in the study region, among which some had not previously been described in the literature (13 27 13 27 13 F; 16 FF; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91). High genetic diversity of A. marginale bacteria was found on this farm in Itú, São Paulo.
边缘无形体是一种专性细胞内革兰氏阴性菌，寄生于红细胞，是牛无形体病的主要病原体。这种疾病会导致严重贫血，并降低体重增加和产奶量，从而在全球范围内给畜牧业带来重大经济损失。基于主要表面蛋白（MSP）的序列，特别是MSP1α的序列，对这种细菌的遗传多样性进行了表征。这使得能够根据不同的氨基酸序列鉴定出几种地理菌株。本研究的目的是调查巴西东南部疾病暴发期间自然感染安格斯肉牛的边缘无形体的遗传多样性。在巴西圣保罗州伊图的一个养牛场，在四个月的时间里从20头牛中的每头牛身上采集了四份血样。对血清样本进行间接ELISA检测抗边缘无形体IgG抗体。对获得的80份全血样本进行DNA提取、针对msp1β基因的定量实时PCR（qPCR）、针对msp1α基因的半巢式PCR（snPCR）、目标片段的克隆以及使用桑格法进行测序。使用RepeatAnalyzer软件对获得的序列进行遗传多样性分析。使用重组MSP5和无形体抗体检测试剂盒（VMRD）进行的两种间接ELISA检测均显示出高血清阳性率，分别为91.25%和97.5%。在qPCR中，100%的样本呈阳性，每微升有10至10个DNA拷贝。在基于msp1α基因的snPCR中，57.5%（46/80）的样本呈阳性。对获得的36个序列进行的微卫星分析显示存在基因型H（58.3%）、F（25%）、E（19.4%）、C（2.7%）和G（2.7%）。RepeatAnalyzer软件在研究区域鉴定出36个菌株，其中一些菌株以前在文献中未曾描述过（13 27 13 27 13 F；16 FF；τ 27；63 29 104 29；LJ1 13 LJ1 13；16 F 17；16 F 91）。在圣保罗州伊图的这个农场发现了边缘无形体细菌的高遗传多样性。
