The Novel Zoonotic Pathogen, Anaplasma capra, Infects Human Erythrocytes, HL-60, and TF-1 Cells In Vitro

Abstract

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295-518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.

Keywords: Anaplasma capra; HL-60; TF-1; erythrocyte; zoonotic pathogen.

Figure 1

Wright–Giemsa-stained human erythrocytes: (A) an uninoculated human erythrocyte smear; and (B) the erythrocyte smear inoculated and infected with A. capra. The red arrows indicate A. capra.

Figure 2

Wright–Giemsa-stained HL-60 and TF-1 cells, where the arrows indicate positive HL-60 and TF-1 cells: (A1,A2) the negative controls; (B1–D1) the A. capra-positive HL-60 cells, where the white arrow indicates a morula of A. capra and the black arrow indicates vacuole formation in the cytoplasm of the pathogen; and (B2–D2) A. capra-positive TF-1 cells, where the black arrow indicates a morula of A. capra and the red arrows in the figures indicate A. capra.

Figure 3

Immunocytochemistry of the erythrocytes incubated with positive goat serum: (A) an uninoculated human erythrocyte smear; and (B) the erythrocyte smear inoculated and infected with A. capra. The arrows indicate A. capra.

Figure 4

Immunocytochemistry of the HL-60 and TF-1 cells incubated with A. capra-positive goat serum: (A1,A2) the uninoculated cells; (B1–D1) the HL-60 cells inoculated and infected with A. capra, where the red arrows indicate vacuole formation in the cytoplasm of the pathogen; and (B2–D2) the TF-1 cells inoculated and infected with A. capra, where the red arrows indicate the morulae of the pathogen in the cytoplasm.

Figure 5

CISH analyses of the uninfected and infected human erythrocytes. The probe was designed based on the groEL gene sequence of A. capra and labeled with digoxigenin (DIG): (A) an uninoculated human erythrocyte smear; and (B) the erythrocyte smear inoculated and infected with A. capra. The red arrow indicates one of the A. capra-positive cell.

Figure 6

TEM photomicrographs of the HL-60 and TF-1 cells. Uranyl acetate and Reynolds’ lead citrate stain were used. (A1,A2) The negative controls. The black and white triangular symbols indicate the different states of the lysosomes. (B1,C1) The A. capra-positive HL-60 cells. Red arrows depict the morulae. Circular or elliptical bodies indicated by the black arrow in (C1) are seen inside the morulae. (B2,C2) The A. capra-positive TF-1 cells. Red arrows indicate the morulae.