Abstract
The identification of cross-reactive monoclonal antibodies (mAbs) that recognize orthologous leukocyte differentiation molecules (LDM) in buffaloes has overcome a major impediment limiting research on the immune response to pathogens and development of vaccines. As reported, two pilot trials were conducted to accomplish two objectives: (1) demonstrate that multiparameter flow cytometry can be conducted equally well in buffalo with mAbs directly and indirectly labeled with fluorochromes in research and (2) flow cytometry can be used to compare and extend studies on diseases of economic importance to buffalo using bovine viral diarrhea virus (BVDV) as a model pathogen. Pregnant buffalo cows were infected with BVDV-1 at 81 (trial 1) and 203 (trial 2) days post artificial insemination and flow cytometric evaluations were performed at 0, 3, 4, and 14 days after infection (dpi). Fluorochrome conjugated mAbs were used in trial 1, and fluorochrome conjugated goat isotype specific anti-mouse antibodies were used to label mAbs in trial 2. Flow cytometric analysis revealed a transient lymphopenia occurs during the 1st days following infection similar to lymphopenia reported in cattle. In particular, significant differences were observed between pre- and post-infection absolute values of T lymphocytes (−56%, P < 0.01). CD21+ B lymphocytes (−65%, P = 0.04), and Natural Killer cells (−72%, P < 0.001). No significant differences were observed in monocytes and neutrophil absolute values, or the CD4:CD8 ratio. Animal health status was followed until 15 days after calving. No clinical signs of infection were observed during the evaluation period, however, animals in trial 1 developed complications later the infection. One cow aborted at 57 days post-infection, the second cow developed a prolapse a day after calving and died. These two animals also showed a more pronounced lymphopenia in comparison with animals infected at 203 days of pregnancy (e.g., −77 vs. −22% T lymphocytes at 3 dpi, respectively). The pilot studies have demonstrated that it is possible to use multicolour multiparameter flow cytometry to study the immune response to pathogens affecting the health of buffalo.
Highlights
Flow cytometry (FCM) is an important and versatile technology: flexibility, accuracy and multiparametric analysis are some of its characteristics
Between 14 and 27 dpi, and all calves born from these buffaloes had BVDV antibodies and were negative for viral antigen, except the calf born from Buffalo2 which was negative for both
Considerable progress has been made in identifying monoclonal antibodies (mAbs) for use in buffalo, there is still a need to expand the mAb reagents for use in this species
Summary
Flow cytometry (FCM) is an important and versatile technology: flexibility, accuracy and multiparametric analysis are some of its characteristics. Flow cytometry was introduced into veterinary sciences in the 80’s concurrent with pioneering studies to develop monoclonal antibodies (mAbs) against leukocyte differentiation molecules (LDM) in cattle and other livestock species [1,2,3]. During this same timeframe studies were initiated to make use of the first characterized mAbs to determine the frequency of leukocyte subsets in blood and milk [4, 5]. The availability of the large set of crossreactivity mAbs affords opportunity to expand research on the immune response to pathogens and vaccine development in buffalo
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