qPCR quantification of contaminated bacteria in down and feather samples. (a) Scanning electron microscopic images of unwashed and washed down and feather. Red arrowheads indicate debris. (b) Colony-forming unit (CFU) of down and feather samples. CFU was calculated using standard agar plate protocol. n = 6, error bars indicate SD. (c) qPCR analysis of bacterial numbers in down and feather samples. n = 6, error bars indicate SD. (d) Bacterial proliferation in the down and feather samples. Red signal indicates mCherry-expressing E. coli.

Zebrafish embryo toxicity test (ZFET) of down and feather samples. (a) Scanning electron microscopic images of glue down. Red arrowheads indicate glue chemicals. (b) Representative images of 72 hours-post-fertilization (hpf) zebrafish in ZFET. (c) Survivals and anomalies in ZFET. ** p < 0.01 vs. control. n = 4–6, error bars indicate SD. (d) Quantification of contaminated bacterial in down and feather samples used in ZFET. n = 4, error bars indicate SD. (e) Representative images of macrophage-EGFP zebrafish used in ZFET. Green indicates EGFP-expressed macrophages. Unwashed and glue down immersion water were diluted 5 times to reduce mortality. (f) Quantification of numbers of macrophages in the tail fin. *** p < 0.001 vs. control. n = 8, error bars indicate SD.