Isolation, Molecular Characterization and In Vitro Propagation of an Anaplasma platys-Like Bacterium in Tick Cells.

Abstract

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 Anaplasma 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 Ixodes scapularis 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 rDNA, 23S rDNA, rpoB, and groEL genes were performed for molecular characterization. Phylogenetic analyses revealed that the isolated strain clustered within the A. platys-like clade. This study reports the successful in vitro isolation, propagation, and cryopreservation of the 'A. platys-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 Anaplasma spp., laying the groundwork for future research.