Table_1_Assessment of Multicolor Flow Cytometry Panels to Study Leukocyte Subset Alterations in Water Buffalo (Bubalus bubalis) During BVDV Acute Infection.docx

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.

识别能够识别水牛中同源白细胞分化分子（LDM）的交叉反应性单克隆抗体（mAbs），已克服了限制病原体免疫反应研究和疫苗开发的一个主要障碍。据报道，进行了两项试点试验以实现两个目标：（1）证明多参数流式细胞术在水牛身上同样可以良好地进行，无论是通过直接还是间接标记荧光染料的mAbs，均适用于研究；（2）利用牛病毒性腹泻病毒（BVDV）作为模型病原体，利用流式细胞术比较和扩展对水牛经济重要性疾病的 研究。怀孕的水牛母牛在人工授精后81天（试验1）和203天（试验2）感染了BVDV-1，并在感染后0天、3天、4天和14天进行了流式细胞术评估。在试验1中使用了荧光素偶联的mAbs，而在试验2中则使用了荧光素偶联的山羊同型特异性抗小鼠抗体来标记mAbs。流式细胞术分析显示，感染后第1天出现了一过性淋巴细胞减少症，这与报道的牛的淋巴细胞减少症相似。特别是，感染前后T淋巴细胞的绝对值之间存在显著差异（-56%，P < 0.01），CD21+ B淋巴细胞（-65%，P = 0.04）和自然杀伤细胞（-72%，P < 0.001）。单核细胞和中性粒细胞的绝对值，或CD4:CD8比例未见显著差异。动物健康状况在产犊后15天进行了跟踪。在评估期间未观察到感染的临床症状，然而，在试验1中的动物在感染后出现了并发症。一头母牛在感染后57天流产，另一头母牛在分娩后一天发生脱垂并死亡。与怀孕203天感染的水牛相比，这两头动物也表现出更明显的淋巴细胞减少症（例如，3 dpi时T淋巴细胞的减少分别为-77%和-22%）。试点研究已证明，使用多色多参数流式细胞术研究影响水牛健康的病原体引起的免疫反应是可行的。