de Matos Erica Rodrigues, Dos Santos Priscilla Nunes, Zweygarth Erich Peter, Jardim Talys Henrique Assumpção, Santos Huarrisson Azevedo, Cordeiro Matheus Dias, de Azevedo Baêta Bruna, Bell-Sakyi Lesley, da Fonseca Adivaldo Henrique, da Silva Claudia Bezerra
Department of Animal Parasitology, Federal Rural University of Rio de Janeiro (UFRRJ), Seropedica 23890-000, RJ, Brazil.
Course of Professional Master's Degree in Diagnosis in Veterinary Medicine, University of Vassouras, Vassouras 27700-000, RJ, Brazil.
Pathogens. 2025 Sep 7;14(9):901. doi: 10.3390/pathogens14090901.
The family Anaplasmataceae comprises etiological agents of infectious diseases of significant importance. This study aimed to achieve the in vitro isolation and propagation of an sp. using tick-derived cell lines. The study was realized in Seropédica municipality, Rio de Janeiro, Brazil. Blood smears from a naturally infected bovine revealed cytoplasmic inclusions in blood cells. To isolate and propagate the organism, IDE8 and ISE6 tick cell lines derived from were used. Two methods of inoculum preparation were employed: Histopaque density gradient and platelet-rich plasma separation. Following infection, cells were maintained in L-15B medium without antibiotics at 34 °C, and infection was monitored weekly by Giemsa-stained cytocentrifuge smears. After achieving ≥ 70% infection, bacteria were subcultured and successfully cryopreserved and resuscitated. PCR amplification and sequencing of 16S , 23S , , and genes were performed for molecular characterization. Phylogenetic analyses revealed that the isolated strain clustered within the . -like clade. This study reports the successful in vitro isolation, propagation, and cryopreservation of the '-like strain Natal' bacterium in tick cell lines and provides molecular evidence supporting its phylogenetic classification. These findings contribute to the understanding of genetic variability and host-cell interactions of spp., laying the groundwork for future research.
无形体科包含具有重要意义的传染病病原体。本研究旨在利用蜱源细胞系实现一种无形体的体外分离和增殖。该研究在巴西里约热内卢的塞罗佩迪卡市开展。一份来自自然感染牛的血涂片显示血细胞中有细胞质内包涵体。为了分离和增殖该病原体，使用了源自肩突硬蜱的IDE8和ISE6蜱细胞系。采用了两种接种体制备方法：Histopaque密度梯度法和富血小板血浆分离法。感染后，细胞在无抗生素的L-15B培养基中于34℃培养，并每周通过吉姆萨染色的细胞离心涂片监测感染情况。在感染率达到≥70%后，对细菌进行传代培养，并成功进行了冷冻保存和复苏。对16S、23S、16S-23S ITS和groEL基因进行了PCR扩增和测序以进行分子鉴定。系统发育分析表明，分离出的菌株聚集在嗜吞噬细胞无形体样进化枝内。本研究报告了在蜱细胞系中成功实现“嗜吞噬细胞无形体样菌株纳塔尔”的体外分离、增殖和冷冻保存，并提供了支持其系统发育分类的分子证据。这些发现有助于了解嗜吞噬细胞无形体属的遗传变异性和宿主-细胞相互作用，为未来的研究奠定了基础。
