Evaluating Effects of AIV Infection Status on Ducks Using a Flow Cytometry-Based Differential Blood Count

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.