Mastitis represents one of the most important infectious diseases in camels with heavy economic losses due to reduced milk quantity and quality. Balanced immune cell composition and function in the mammary gland are essential for effective immune response to mastitis pathogens. The objective of the present study was to characterize the cellular immune response to subclinical mastitis in the mammary gland of dromedary camels. Therefore, immunostaining and flow cytometry were used to compare the cellular composition, leukocyte phenotype, and cell viability in camel milk from healthy she-camels (n = 8) and she-camels with subclinical mastitis (SCM; n = 6). In addition, the ex vivo phagocytic activity of milk phagocytes was compared between healthy and affected animals. The health status of the mammary gland was evaluated based on the California Mastitis Test (CMT) score. SCM (CMT score of ≥3 in the absence of clinical signs of mastitis) was found in six of the 56 sampled quarters (10.7 %) with only one affected quarter per animal. In comparison to milk from healthy camels, milk from SCM animals showed higher somatic cell count (SCC), higher numbers of CD45+ leukocytes with an expanded fraction of CD172a+ myeloid cells. Within the myeloid cell population, there was an increase in the percentage of granulocytes (CD172a+CD14low) with a decreased percentage of macrophages (CD172a+CD14high) in milk from affected animals compared to healthy animals. The decrease in lymphoid cells in SCM milk was mainly due to the decreased fraction of CD4+ helper T cells. Camel SCM was also associated with a stimulated phenotype, increased cell viability, and enhanced phagocytic activity of the milk phagocytes, macrophages and granulocytes. Collectively, the present study identified significant changes in SCC, leukocyte count, phenotype, viability, and function in association with subclinical mastitis in camels. The results of the present study support a better understanding of host-pathogen interaction mechanisms in the camel mammary gland.

Immunophenotyping of separated leukocytes is a common technique used to evaluate the changes in cellular immunity during clinical studies. For fixed cells or blood specimens infected with hazardous pathogens, cell fixation is performed before immunofluorescence. The impact of camel leukocytes fixation before staining on the reactivity of cell surface markers with monoclonal antibodies has not been investigated so far. The aim of the present study was, therefore, to compare cell staining of fixed and unfixed camel leukocytes with monoclonal antibodies to several cell surface antigens. Leukocytes were separated from camel blood and were fixed with paraformaldehyde (PFA) or left without fixation. Cells were labeled with monoclonal antibodies to several leukocyte antigens and the expression pattern of the antigens was compared between fixed and non-fixed cells using flow cytometry. The mean fluorescence intensity of each cell marker was calculated and compared between fixed and unfixed cells. Leukocyte fixation with PFA changed the binding activity of the monoclonal antibodies to CD163 and WC1 markedly, making it unable to stain any cell population. Although the cell staining efficacy of other molecules (such as CD14, CD172a, MHCII, CD11a, CD18, CD44, and CD45) was reduced, they were still able to define the target cells. The fixation-induced changes in the expression density of the analyzed monocytic markers may, however, lead to the misinterpretation of immunophenotyping studies of fixed monocytes or macrophages. Collectively, the obtained results indicated significant changes in the staining efficacy of monoclonal antibodies against several cell surface antigens of camel leukocytes, which should be considered when PFA-fixed cellular targets on camel leukocytes are to be analyzed.