Isolation and attempted cultivation of an Anaplasma marginale strain from Brazilian brown brocket deer (Mazama gouazoubira, Fisher, 1814) in the tick cell line IDE8

The aim of the study was to isolate and establish an Anaplasma marginale strain from Brazilian brown brocket deer, Mazama gouazoubira, in the Ixodes scapularis cell line IDE8. Blood from a free-living adult female M. gouazoubira naturally infected with A. marginale (MGI5) was inoculated intravenously into a splenectomized calf. When A. marginale rickettsemia was 2.5%, blood was collected and cryopreserved in liquid nitrogen with dimethylsulfoxide (DMSO). IDE8 cell cultures were infected with calf blood inoculated with the A. marginale (MG15) isolate. The cultures were monitored by examination of Giemsa-stained cytocentrifuge smears. Light microscopy of stained IDE8 samples revealed the first inclusions of A. marginale (MGI5) at 48 days post-inoculation (d.p.i). The IDE8-infected cells contained parasitophorous vacuoles with amorphous material and a few cocci-like organisms. A sample from IDE8-infected cells from the 16th subculture (336 d.p.i.) was analyzed by nPCR, nucleotide sequencing, electron microscopy, and an indirect fluorescent antibody test (IFAT). The IFAT highlighted some IDE8-infected cells with intense fluorescence in the parasitophorous vacuole, while in other cells, fluorescence was observed only at the periphery. DNA from a culture of the MG15 isolate was amplified with A. marginale msp4 gene primers, and nucleotide sequencing of the PCR product and BLAST software analysis further confirmed 100% identity with the MGI5 blood isolate (GenBank no. JN022558.1). Electron microscopy revealed increased numbers of lysosomes in the cytoplasm of IDE8 cells. Several cells exhibited large vacuoles containing cellular debris and amorphous material. After the 29th subculture, it was not possible to detect compatible Anaplasma structures by light microscopy, and subculture samples tested negative in nPCR. Despite the failure of the attempt to establish A. marginale (MGI5) in IDE8 cells, the results demonstrated the isolate's ability to infect, survive and multiply, although in limited numbers, in IDE8 cells.