Blood samples were collected from clinically healthy camels (Camelus dromedaries) at the Camel Research Center, King Faisal University, Saudi Arabia. The involved animals included 23 newborn camel calves (aged < four weeks) and 62 adult camels aged between 4 and 10 years. Blood was obtained by venepuncture of the vena jugularis externa into vacutainer tubes containing EDTA (Becton Dickinson, Heidelberg, Germany). All experimental procedures and management conditions used in this study were approved by the Ethics Committee at King Faisal University, Saudi Arabia (Permission number DSR 1811001).
2.2 Hypotonic lysis and separation of whole blood leukocytes
Separation of whole camel leukocytes was done after hypotonic lysis of blood erythrocytes . Briefly, blood was suspended in distilled water for 20 sec and double concentrated PBS was added to restore tonicity. This was repeated until complete erythrolysis. Separated cells were finally suspended in MIF buffer (PBS containing bovine serum albumin (5 g/L) and NaN3 (0.1 g/L)) at 5 x 106 cells/ml. The mean viability of separated cells was evaluated flow cytometrically by dye exclusion (propidium iodide; 2 µg/ml, Calbiochem, Germany) and consistently > 95%.
2.3 Monoclonal antibodies
Monoclonal antibodies used in this study are listed in table 1.
2.4 Flow cytometric analysis of camel blood monocytes
The identification of camel monocyte subsets and the analysis of the subset-specific expression pattern of monocytic markers and adhesion molecules were performed after direct and indirect labeling of cells with surface molecule-specific antibodies and flow cytometrical analysis . Separated camel leukocytes (5 x 106 cells / ml) were incubated in 96 well round-bottom microtiter plates (1 x 106 / well; 20 min; 4°C), in a three-step staining process, with monoclonal antibodies specific for CD172a, MHCII and CD163 (in the following two combinations: CD172a; MHCII; CD14 and CD163; MHCII; CD14 ) or with isotype control antibodies in PBS containing bovine serum albumin (5 g / L) and NaN3 (0.1 g / L). After incubation, cells were washed twice and incubated with mouse secondary antibodies IgG1, IgG2a (BD) labelled with different fluorochromes. In a third labelling step, after washing the cells, directly labeled monoclonal antibodies to CD14, CD11a, CD11b, and CD18 were added. Finally, cells were washed and analyzed by flow cytometry. For each measurement 100 000 events were acquired. Flow cytometric data were analyzed with the software FlowJo version 10 (FLOWJO LLC).
2.5 Generation of reactive oxygen species (ROS)
ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) as described earlier  with modifications. Separated camel leukocytes (1 x 106 / well) in RPMI medium were incubated with heat killed staphylococcus aureus (50 bacteria/cell) for 20 min (37°C, 5% CO2). For the detection of ROS, dihydrorhodamine (DHR) 123 (Mobitec, Goettingen, Germany) was added to the cells (150 ng / ml final). To identify monocyte subsets, cells were labeled with monoclonal antibodies to CD14 and MHCII (see above). After washing, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, California, USA). The relative amount of generated ROS was determined by the mean green fluorescence intensity of gated monocyte subsets (based on CD14 and MHCII expression) after acquisition of 100 000 events (n = 15 animals).
2.6 Phagocytosis assay
Heat killed staphylococcus aureus (S. aureus) bacteria (Pansorbin, Calbiochem, Merck, Nottingham, UK) were labeled with fluoresceinisothiocyanate (FITC, Sigma-Aldrich, St. Louis, Missouri, USA). FITC-conjugated and heat killed S. aureus bacteria were suspended in RPMI medium and adjusted to 2x108 bacteria / ml. Separated camel leukocytes were plated in 96 well plates (1x106/well) and incubated (37°C, 5% CO2) with labeled bacteria (50 bacteria / cell) for 40 minutes (37°C, 5% CO2). To identify monocyte subsets, cells were labeled with monoclonal antibodies to CD14 and MHCII (see above). After washing, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, California, USA). After washing, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, California, USA). For each monocyte subset (based on their CD14 and MHCII expression), phagocytosis-positive cells were defined as the percentage of green fluorescing cells among total cells (n = 15 animals). Phagocytosis capacity (as an indicator for the number of bacteria ingested by each monocyte) was defined as the mean green fluorescence intensity of gated phagocytosis-positive monocyte subset.