Grandoni Francesco, Martucciello Alessandra, Petrini Stefano, Steri Roberto, Donniacuo Anna, Casciari Cristina, Scatà Maria Carmela, Grassi Carlo, Vecchio Domenico, Feliziani Francesco, De Matteis Giovanna, Davis William C, De Carlo Esterina
CREA-Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Centro di ricerca Zootecnia e Acquacoltura (Research Centre for Animal Production and Aquaculture), Monterotondo, Italy.
Istituto Zooprofilattico Sperimentale del Mezzogiorno, National Reference Centre for Hygiene and Technologies of Water Buffalo Farming and Productions, Salerno, Italy.
Front Vet Sci. 2020 Oct 16;7:574434. doi: 10.3389/fvets.2020.574434. eCollection 2020.
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%, < 0.01). CD21 B lymphocytes (-65%, = 0.04), and Natural Killer cells (-72%, < 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.
识别出能识别水牛直系同源白细胞分化分子（LDM）的交叉反应性单克隆抗体（mAb），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。如报道所述，进行了两项试点试验以实现两个目标：（1）证明在研究中，使用直接和间接用荧光染料标记的单克隆抗体，多参数流式细胞术在水牛中同样可以很好地进行；（2）流式细胞术可用于以牛病毒性腹泻病毒（BVDV）作为模型病原体，比较和扩展对水牛具有经济重要性疾病的研究。在人工授精后第81天（试验1）和第203天（试验2），怀孕的水牛母牛感染了BVDV-1，并在感染后0、3、4和14天进行流式细胞术评估。试验1中使用了荧光染料偶联的单克隆抗体，试验2中使用荧光染料偶联的山羊同型特异性抗小鼠抗体来标记单克隆抗体。流式细胞术分析显示，感染后的头几天会出现短暂的淋巴细胞减少，这与牛中报道的淋巴细胞减少情况相似。特别是，在感染前后，T淋巴细胞的绝对值（-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）存在显著差异。单核细胞和中性粒细胞的绝对值或CD4:CD8比值没有观察到显著差异。跟踪动物健康状况直至产犊后15天。在评估期间未观察到感染的临床症状，然而，试验1中的动物在感染后期出现了并发症。一头母牛在感染后57天流产，第二头母牛在产犊后一天出现脱垂并死亡。与在怀孕203天时感染的动物相比，这两头动物的淋巴细胞减少也更为明显（例如，在感染后3天，T淋巴细胞分别为-77%和-22%）。这些试点研究表明，使用多色多参数流式细胞术来研究影响水牛健康的病原体的免疫反应是可行的。
