The Gametocytes of Leucocytozoon sabrazesi Infect Chicken Thrombocytes, Not Other Blood Cells

Abstract

Leucocytozoon parasites infect a large number of avian hosts, including domestic chicken, and cause significant economical loss to the poultry industry. Although the transmission stages of the parasites were observed in avian blood cells more than a century ago, the specific host cell type(s) that the gametocytes infect remain uncertain. Because all the avian blood cells, including red blood cells (RBCs), are nucleated, and the developing parasites dramatically change the morphology of the infected host cells, it has been difficult to identify Leucocytozoon infected host cell(s). Here we use cell-type specific antibodies to investigate the identities of the host cells infected by Leucocytozoon sabrazesi gametocytes. Anti-RBC antibodies stained RBCs membrane strongly, but not the parasite-infected cells, ruling out the possibility of RBCs being the infected host cells. Antibodies recognizing various leukocytes including heterophils, monocytes, lymphocytes, and macrophages did not stain the infected cells either. Antisera raised against a peptide of the parasite cytochrome B (CYTB) stained parasite-infected cells and some leukocytes, particularly cells with a single round nucleus as well as clear/pale cytoplasm suggestive of thrombocytes. Finally, a monoclonal antibody known to specifically bind chicken thrombocytes also stained the infected cells, confirming that L. sabrazesi gametocytes develop within chicken thrombocytes. The identification of L. sabrazesi infected host cell solves a long unresolved puzzle and provides important information for studying parasite invasion of host cells and for developing reagents to interrupt parasite transmission.

Fig 1. Gametocytes of Leucocytozoon sabrazesi.

(A and B) Mature female and male gametocytes, showing elongated host cells (or parasites) with two long extensions. (C and D) Putative young female and male gametocytes within host cells having round nuclei that are slightly larger and stained darker than those of red blood cells. Arrows point to putative parasite nuclei. The blood smears were stained with Giemsa stain and observed under a microscope. hn, host cell nucleus; pn, parasite nucleus. (E) A flow chart of study design, antibodies used, and staining results from the antibodies. RBCs, red blood cells; WBCs, white blood cells.

Fig 2. Confocal images of infected chicken blood cells stained with anti-red blood cell antibodies.

Anti-RBC, images stained with rabbit anti-chicken red blood cell antibody 103–4139; DIC, differential interference contrast images; Hoechst, Hoechst dye staining of nuclei (DNA); Merged, merged images of anti-RBC, DIC, and DAPI. (A-H) Two cells infected with L. sabrazesi gametocytes have some red dots (yellow arrowheads) that appear to be within the cytoplasm of the host cells. (E-H) A strongly stained white cell (grey arrowheads) that has two nuclei and rough granules in the cytoplasm, suggesting heterophils, monocytes, macrophages, or eosinophils. (I-L) A small cell that has a round nucleus and red dots similar (green arrowheads) to those seen in the infected cells. The small size of the cell suggests that it is likely a thrombocyte. The red ruler in each image indicates 10 μm.

Fig 3. Images of infected chicken blood cells stained with anti-red blood cell antibodies and observed under a fluorescent microscope.

Anti-RBC, images stained with anti-RBC antibodies; Hoechst, images stained by Hoechst dye; DIC, bright light images; Merged, merged images of the three. All staining and labeling are the same as those in Fig 2. (C, G, and K) Images under bright light show four cells (numbered 1–4) with relatively round nuclei and pale large cytoplasm with a few black dots under bright light or red dots after anti-RBC staining (yellow arrows). Number 5 indicates a cell that is likely a lymphocyte; number 6 is a cell possibly representing a heterophil; “p” indicates a gametocyte. (D) Red arrows point to red dots in a gametocyte-infected cell. Green arrows in J point to three nuclei of a white cell stained by anti-RBC (I and L). Images M-P show two mature gametocytes and one immature gametocyte (p = parasite). Host cell membrane (hm), parasite membrane (pm), and the host cell nucleus (hn) can be clearly seen (O and P), but not stained by the antibodies.

Fig 4. Confocal images of infected chicken blood cells after staining with anti-CYTB antibodies.

Anti-CYTB, images stained with mouse anti-L. sabrazesi CYTB antibodies; DIC, differential interference contrast images; Hoechst, Hoechst dye staining cell nuclei (DNA); Merged, merged images of anti-RBC, DIC, and hoechst staining. The yellow arrowheads in (D, H, and L) point to three cells infected with L. sabrazesi gametocytes. Four types of recognizable host white cells are stained by the antibodies and are marked as 1–4: 1, likely mature thrombocyte; 2, monocyte/heterophil/neutrophil; 3, lymphocyte; 4, immature thrombocyte. The red ruler in each image indicates 10 μm.

Fig 5. Images of infected chicken blood cells that are stained with anti-CYTB antibodies and observed under a regular fluorescent microscope.

Anti-CYTB, Images stained with mouse anti-L. sabrazesi CYTB antibodies; Hoechst, image of Hoechst staining for nuclei (DNA); Bright light, bright light images; Merged, merged image of anti-CYTB, Hoechst, and bright light. Images (A-D) the yellow arrows point to a gametocyte; the green arrows indicate a white cell likely to be a heterophil with granules; the red arrows point to multiple putative mature thrombocytes; and number 1 and 2 mark two putative immature thrombocytes, with clear cytoplasm under bright light but stained by the antibodies. (E-P) images taken at 40X magnification. Yellow arrowheads point to putative immature thrombocytes; green arrowheads point to mature thrombocytes; grey arrowheads point to gametocytes. Note that a large number of putative thrombocytes were stained.

Fig 6. Confocal images of infected chiceken blood cells stained with cell lineage-specific antibodies.

Green, images stained with specific antibodies (as labeled); DIC, differential interference contrast images; Hoechst, images of Hoechst dye staining for nuclei (DNA); Merged, merged images of anti-RBC, DIC, and Hoechst. (A-D) Cells stained with Anti-monocyte antibody that stains mostly monocytes and macrophages. (E-H) Cells stained with anti-Bu-1b (anti-chicken Bu-1b) that stains most bursal cells, thymocytes, and peripheral monocytes and macrophages. (I-P) Cells stained with anti-CD4 antibody (mouse anti-chicken CD4) that stains thymocytes, spleen cells, and peripheral lymphocytes. Cells infected with L. sabrazesi gametocytes (red arrowheads) are not stained by any of the three antibodies.

Fig 7. Images of infected chicken blood cells that are stained with anti-CD51/CD61 monoclonal antibody and observed under a regular fluorescent microscope.

(A-D) stained cells with characteristic of thrombocytes having round nuclei and small pale cytoplasm (green arrows) and a weakly stained parasite-infected cell (white arrows). (E-H) A cluster of five thrombocytes stained red by the anti-CD51/CD61 antibody. (I-L) A stained parasite-infected cell and a stained thrombocyte (small red dot). (M-P) Another stained parasite-infected cell (red). Inset images of enlarged gametocytes in L and P show host cell membrane (arrows) and nuclei of host cells (marked ‘N’). Image A-D are from formaldehyde fixed cells; images E-P are from ‘wet’ IFA without fixation. The white scale bar is 10 μM.