PDF(Pubmed)
Abstract：
Ducks have recently received a lot of attention from the research community due to their importance as natural reservoirs of avian influenza virus (AIV). Still, there is a lack of tools to efficiently determine the immune status of ducks. The purpose of this work was to develop an automated differential blood count for the mallard duck (Anas platyrhynchos), to assess reference values of white blood cell (WBC) counts in this species, and to apply the protocol in an AIV field study. We established a flow cytometry-based duck WBC differential based on a no-lyse no-wash single-step one-tube technique, applying a combination of newly generated monoclonal antibodies with available duck-specific as well as cross-reacting chicken markers. The blood cell count enables quantification of mallard thrombocytes, granulocytes, monocytes, B cells, CD4+ T cells (T helper) and CD8+ cytotoxic T cells. The technique is reproducible, accurate, and much faster than traditional evaluations of blood smears. Stabilization of blood samples enables analysis up to 1 week after sampling, thus allowing for evaluation of blood samples collected in the field. We used the new technique to investigate a possible influence of sex, age, and AIV infection status on WBC counts in wild mallards. We show that age has an effect on the WBC counts in mallards, as does sex in juvenile mallards. Interestingly, males naturally infected with low pathogenic AIV showed a reduction of lymphocytes (lymphocytopenia) and thrombocytes (thrombocytopenia), which are both common in influenza A infection in humans. IMPORTANCE Outbreaks of avian influenza in poultry and humans are a global public health concern. Aquatic birds are the primary natural reservoir of avian influenza viruses (AIVs), and strikingly, AIVs mainly cause asymptomatic or mild infection in these species. Hence, immunological studies in aquatic birds are important for investigating variation in disease outcome of different hosts to AIV and may aid in early recognition and a better understanding of zoonotic events. Unfortunately, immunological studies in these species were so far hampered by the lack of diagnostic tools. Here, we present a technique that enables high-throughput white blood cell (WBC) analysis in the mallard and report changes in WBC counts in wild mallards naturally infected with AIV. Our protocol permits large-scale immune status monitoring in a widespread wild and domesticated duck species and provides a tool to further investigate the immune response in an important reservoir host of zoonotic viruses.
摘要：
由于鸭子作为禽流感病毒（AIV）的天然储库的重要性，最近受到了研究界的广泛关注。尽管如此，缺乏有效确定鸭子免疫状态的工具。这项工作的目的是为野鸭（Anasplatyrhynchos）开发自动分类血细胞计数，为了评估该物种的白细胞（WBC）计数参考值，并将该方案应用于AIV现场研究。我们建立了基于流式细胞术的鸭WBC差异基于无裂解无洗单步单管技术，应用新产生的单克隆抗体与可用的鸭特异性以及交叉反应的鸡标记的组合。血细胞计数能够量化野鸭血小板，粒细胞,单核细胞,B细胞,CD4+T细胞(T辅助细胞)和CD8+细胞毒性T细胞。这项技术是可重复的，准确,比传统的血涂片评估要快得多。稳定血液样本可以在采样后1周内进行分析，从而允许评估在现场收集的血液样本。我们使用新技术来调查性别的可能影响，年龄,野生野鸭白细胞计数上的AIV感染状况。我们表明年龄对野鸭的白细胞计数有影响，幼年野鸭的性行为也是如此。有趣的是,自然感染低致病性AIV的男性显示淋巴细胞（淋巴细胞减少症）和血小板（血小板减少症）减少，这在人类甲型流感感染中都很常见。禽流感在家禽和人类中的爆发是全球公共卫生关注的问题。水生鸟类是禽流感病毒（AIV）的主要天然宿主，惊人的是，AIV主要在这些物种中引起无症状或轻度感染。因此，水鸟的免疫学研究对于研究不同宿主对AIV的疾病结果变化非常重要，可能有助于早期识别和更好地了解人畜共患事件。不幸的是,到目前为止，由于缺乏诊断工具，这些物种的免疫学研究受到了阻碍。这里,我们提出了一种能够对野鸭进行高通量白细胞(WBC)分析的技术,并报告了自然感染AIV的野鸭中WBC计数的变化.我们的方案允许在广泛的野生和驯化鸭物种中进行大规模的免疫状态监测，并提供了一种工具来进一步研究人畜共患病毒重要宿主中的免疫反应。
