Five beagles, including three female and two male, 14~16 weeks old were obtained from the Center of Laboratory Animals of the Southern Medical University (China). The dogs were anesthetized using xylazine hydrochloride (Hanhe, China) with 0.3 mL/kg body weight and sacrificed. Samples of tissue were then taken from the trachea, lung, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, liver, spleen, and kidney. All tissues were cut into 4-µm sections using routine methods [15].

Lectins used in this research were biotinylated Maackia (M.) amurensis lectin II (specific for α-2,3-linked sialic acid in avian influenza virus receptors) and Sambucus nigra agglutinin (SNA) (specific for α-2,6-linked sialic acid in human influenza virus receptors) from Vector Laboratories (USA). Histochemical staining with the lectins was performed as described in our previous publication [15]. Briefly, the pretreated tissue sections were incubated by SNA and M. amurensis agglutinin (Vector Laboratories, USA) and then the specific labeling was got using streptavidin-biotin complex kit (Vector Laboratories, USA) and diaminobenzidine kit (Promega, USA). Specificity of the lectin staining was confirmed by pretreatment with neuraminidase (NEB, USA) as previously described [15,25]. Briefly, after the slides were treated by neuraminidase, lectin staining was performed as described above. Negative controls were treated by phosphate buffered saline.

In the trachea, goblet cells of the mucosa were strongly positive for α-2,3-sialic acid-linked receptors while only some of these cells showed weak to intermediate staining for α-2,6-sialic acid-linked influenza virus receptors. Ciliated cells were diffusely positive for α-2,3-sialic acid-linked influenza virus receptors but negative for α-2,6-sialic acid-linked influenza virus receptors. The lamina propria of the mucosa was diffusely positive for α-2,3-sialic acid-linked influenza virus receptors, but foci of α-2,6-sialic acid-linked influenza virus receptors were observed. The submucosa showed slightly diffuse staining for α-2,3-sialic acid-linked influenza virus receptors while foci of α-2,6-sialic acid-linked influenza virus receptors were detected (Fig. 1).

In the bronchus, distribution of both receptor types was similar to that found in the trachea. In the lamina propria of the mucosa and submucosa, diffuse staining for α-2,3-sialic acid-linked influenza virus receptors was observed and foci of α-2,6-sialic acid-linked influenza virus receptors were seen. In the respiratory zone of the lung, staining for α-2,3-sialic acid-linked influenza virus receptors was diffuse in ciliated and non-ciliated cells of the bronchi and bronchioles along with the alveolar cells of the pulmonary alveoli. In contrast, almost no positive staining for α-2,6-sialic acid-linked influenza virus receptors was detected (Fig. 1).

In the stomach, most endothelial cells of the mucosa and glands in lamina propria did not express α-2,3-sialic acid-linked influenza virus receptors. However, a small number of endothelial cells in the mucosa, lamina propria of the mucosa, and gland connective tissues in the lamina propria, submucosa, and adventitia were positive. Staining for α-2,6-sialic acid-linked influenza virus receptors in mucosal endothelial cells was weak while the glands were strongly positive. The lamina propria of the mucosa and connective tissues of the glands were negative.

In the duodenum, epithelial cells of the mucosa were negative for α-2,3-sialic acid-linked influenza virus receptors but epithelial cells of the central lacteal and submucosa layer were positive. Goblet epithelial cells of the mucosa were weakly positive for α-2,6-sialic acid-linked influenza virus receptors as were epithelial cells of the central lacteal, lamina propria of the mucosa, and submucosa layer. In the jejunum, epithelial cells of the mucosa were negative for α-2,3- and α-2,6-sialic acid-linked influenza virus receptors, but positive staining for both receptors was observed in the submucosa layer, lamina propria of the mucosa, and connective tissues between the glands. In the ileum, distribution of α-2,3-and α-2,6-sialic acid-linked influenza virus receptors was similar to that found in the duodenum. In the cecum and colon, the lamina propria of the mucosa was strongly positive for α-2,3-sialic acid-linked influenza virus receptors while endothelial cells of the glands were weakly positive. Endothelial cells of the mucosa were also positive. In the rectum, goblet cells of the mucosa and endothelial cells of the glands were positive for both types of receptors. In contrast, the lamina propria of the mucosa was negative for α-2,3-sialic acid-linked influenza virus receptors but positive for α-2,6-sialic acid-linked influenza virus receptors.

Cerebrum neurons and ground substance of the brain were negative for both types of lectins, except for endothelial cells in blood vessels that were positive for α-2,3- and α-2,6-sialic acid-linked influenza virus receptors. In the pancreas, the endocrine portions were positive for α-2,3- and α-2,6-sialic acid-linked influenza virus receptors as were the duct epithelium and connective tissues. The exocrine portion was negative for both receptors. Kupffer cells, bile duct epithelium, and endothelial cells of blood vessels in the liver were focally positive for both receptors, but the hepatocytes were negative.

Cardiac muscle cells were weakly positive for α-2,3-sialic acid-linked influenza virus receptors but negative for α-2,6-sialic acid-linked influenza virus receptors. Connective tissues between the muscle cells and endothelial cells of blood vessels in the heart were positive for both receptors. In spleen, α-2,3-sialic acid-linked influenza virus receptors were detected primarily in the periarterial lymphatic sheath and marginal zone. Focal staining specific for this receptor was also observed in cells of the lymphoid nodules, although most of these cells were negative. In contrast, α-2,6-sialic-acid-linked influenza virus receptors were widely detected, primarily in lymphoid nodule cells, while most cells of the periarterial lymphatic sheath and marginal zone were negative. In the kidney, glomerulus endothelial cells contained diffuse staining for α-2,3- and α-2,6-sialic acid-linked influenza virus receptors. Epithelial cells of the proximal tubule were diffusely positive for both receptors although the thin segment and distal tubules were negative for both receptors.