Spatial mapping of influenza and coronavirus receptors in the respiratory and intestinal tract epithelium of beef cattle using advanced PixF image analysis

Abstract

Influenza A viruses (IAV) and coronaviruses (CoV) pose significant threats to various animal species, including cattle. Reports of SARS-CoV-2 infections and recent outbreaks of highly pathogenic avian influenza (HPAI H5N1) in dairy cattle highlight the need to understand receptor distributions critical for viral entry. This study investigates the spatial distribution of IAV and CoV receptors in bovine tissues using PixF, a novel and newly developed web-based image analysis tool. Respiratory (trachea and lung) and intestinal (small and large intestine) tissues from crossbred Holstein-Angus steers were analyzed. Lectin histochemistry staining with fluorescently labeled Sambucus nigra (SA α2,6-Gal receptors) and Maackia amurensis (SA α2,3-Gal receptors) identified IAV receptors, while coronavirus receptors ACE2, TMPRSS2, APN, DPP4, and CEACAM1 were assessed using indirect immunofluorescence. PixF provides an initial yet tailored image processing framework for quantifying and mapping receptor expression, revealing a predominance of SA α2,3-Gal receptors in epithelial regions, while SA α2,6-Gal receptors were confined to glandular tissues of the respiratory tract. Coronavirus receptors exhibited variable expression across tissues; TMPRSS2, APN, and DPP4 are highly expressed in the respiratory mucosal epithelium; ACE2, TMPRSS2, and DPP4 are highly expressed in the intestinal mucosal epithelium, while CEACAM1 is notably low across tissues. These findings demonstrate the potential utility of PixF, a simple prototypic fluorescence quantification tool customized for the use case detailed in this work, to provide a browser interface that prioritizes ease-of-use, enabling non-specialists to obtain essential quantification and spatial information quickly. PixF was utilized to elucidate receptor co-localization and enhance our understanding of host-pathogen interactions in cattle, offering a reproducible, accessible, and biologically informed analysis pipeline.

Keywords: ACE2; Beef cattle; Coronavirus; Influenza virus; Intestinal tract; Respiratory tract; Sialic acid.

Fig. 1

PixF workflow—color scheme analysis, chromaticity mapping, and image segmentation modules. (A) PixF has been built to learn multiple color schemes used for different biological imaging experiments spanning scales of life. (B) By reasoning over the data, PixF can identify a color palette (albeit without directionality) where the user should indicate which end of the spectrum is high and which is low. (C) A chromaticity analysis enables projecting the travel of a color palette from a given image on the visible spectrum (convex hull). This is important for calculating the definitions of neighborhoods for a given color in the image. For example, Rainbow and Custom Scales have a larger travel and hence have a larger neighborhood for each color in comparison to FuchsiaTones. This means sensitivity towards variance in pixel-level intensity for FuchsiaTones must be stringent during PixF analysis. (D) Overview of the image segmentation, color picker (without a palette), and analyzer modules in PixF.

Fig. 2

Sialic acid distribution in the beef cattle respiratory tract. (A–JJ) Confocal microscopy images of fluorescently labeled lectins Sambucus nigra (SNA, FITC, green) specific for sialic acid (SA) α2,6-Gal, Maackia amurensis II (MAL II, DyLight 650, red) specific for SA α2,3-Galβ (1–3) GlcNAc, and Maackia amurensis I (MAL I, FITC, green) specific for SA α2,3-Galβ (1–4) GlcNAc. Blue represents nuclear staining using DAPI; scale bar = 20 µM. Representative merged images showing SNA/MAL II binding (A–E) and MAL I/MAL II binding (F–J). Multifocal, apical membranous (white arrow) MAL II labeling was observed on trachea epithelium and extensively on basal cells (orange arrow) (A), while goblet cells (arrowhead) showing intense cytoplasmic SNA (Ai) and MAL II (Aii) labeling. Image analysis of tracheal epithelial lining (3D plot of insert in A) showing greater MAL II labeling even in co-localized areas (arrow), and slightly higher SNA in goblet cell (arrowhead) (Aiii). Sub-mucosal areas showing both SNA and MAL II (B), with higher SNA in lamina propria connective tissue (asterisk) and higher MAL II in glands (dashed white outline). The 3D plot of insert in (B) demonstrated varying levels of expression of MAL II (arrow) and SNA (arrowhead) (Biii). Goblet cells and epithelial lining of the bronchus, bronchiole, and alveoli show intense MAL II labeling (arrow) (C, D, E) with SNA labeling in the lamina propria of the bronchus (white asterisk) (C) and the interstitial region of the alveolar wall (asterisk) (E). Quantitative image analysis confirmed that the lining of epithelial cells has higher membranous MAL II labeling (arrow) (Ciii, Diii, Eiii). MAL I demonstrated a labeling distribution analogous to that of MAL II, albeit with attenuated signal intensity (F, H–J), reflected in quantitative image analysis (Fiii, Hiii–Jiii). MAL I is more robust in the submucosal glands of the trachea (dashed white outline) (G, Giii). (K–O) Confocal images of the respiratory tract showing SA N-glycolylneuraminic acid (Neu5Gc, FITC, green) following immunofluorescence staining. Neu5Gc was detected on the apical membrane and cell borders of ciliated pseudostratified epithelia of the trachea (K), multifocally on the epithelial lining of the bronchus (M), bronchiole (N), and the alveoli (O). The sub-epithelial glands in the tracheal region also showed Neu5Gc (L).

Fig. 3

Sialic acid distribution in the beef cattle intestinal tract. (A-H) Confocal microscopy images of fluorescently labeled lectins Sambucus nigra (SNA, FITC, green) specific for sialic acid (SA) α2,6-Gal, Maackia amurensis II (MAL II, DyLight 650, red) specific for SA α2,3-Galβ (1–3) GlcNAc, and Maackia amurensis I (MAL I, FITC, green) specific for SA α2,3-Galβ (1–4) GlcNAc. Blue represents nuclear staining using DAPI; scale bar = 20 µM. Representative merged images showing SNA/MAL II binding (A–D) and MAL I/MAL II binding (E–H). The epithelial lining of the small and large intestine shows MAL II labeling (insets of A, C), which was evident in the quantitative image analysis (arrow) (Aiii, Ciii). Goblet cells located in the crypt and apical region of the intestines show abundant MAL II labeling (A–D). SNA labeling was limited to lamina propria (asterisk) (A, B) and goblet cells in the crypt region of the intestines (arrowhead) (B, D). MAL I and MAL II were labeled largely co-localized (E–H) in both the epithelial lining and goblet cells. Quantitative image analysis indicates MAL II was comparatively higher in the co-localized areas (arrow) (insets of E, G, Eiii, Giii). Uniform labeling of MAL I in goblet cells distributed toward apical or crypt regions of the small intestine (Ei, Fi). Note the gradient decrease in MAL I labeling in the goblet cells distributed toward the apical to crypt regions of the large intestine (Gi, Hi). (I–L) Confocal images of small and large intestines showing SA N-glycolylneuraminic acid (Neu5Gc, FITC, green) following immunofluorescence staining. The SA Neu5Gc was expressed on the epithelial lining and goblet cells of the small intestine (I, J). Neu5Gc expression was more scattered in the large intestine, and there was no evident expression on the epithelial lining (K, L).

Fig. 4

Coronavirus receptors in the respiratory tract and intestinal tracts of beef cattle. Confocal images showing angiotensin-converting enzyme 2 (ACE2, A), transmembrane protease serine 2 (TMPRSS2, B), aminopeptidase N (APN, C), dipeptidyl peptidase 4 (DPP4, D), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, E) expression following immunofluorescence staining. Primary antibodies, ACE2 (4 µg/ml, Cat# sc-390851), TMPRSS2 (0.2 µg/ml; Cat# sc-515727), APN (0.16 µg/ml; Cat# sc-166105), CEACAM1 (4 µg/ml; Cat# sc-166453), and DPP4 (1:50 dilution; Cat# BOV2078, Washington State University Monoclonal Antibody Center, Pullman, WA, USA); Secondary antibody, AffiniPure™ Donkey Anti-Mouse IgG (H + L) conjugated Alexa Fluor^® 488 (15 µg/ml; Cat# 715-545-150). Blue represents nuclear staining using DAPI; scale bar = 20 µM. Representative images of coronavirus receptor expression across tissues are shown. The epithelial lining of the trachea (Ai), alveoli (Aiv), small intestine (Av), and large intestine (Avi) showed strong ACE2 expression compared to the bronchus (Aii) and bronchiole (Aiii). The respiratory epithelium showed extensive TMPRSS2, APN, and DPP4 expression with a multifocal expression on the alveoli (Bi–iv, Ci–iv, Di–iv). TMPRSS2 (Bv–vi) and DPP4 (Dv–vi) were detected uniformly on villi and crypt regions in the small and large intestine epithelia, while APN levels were comparatively low in both intestines (Cv–vi). With the exception of the bronchus (Eii) and bronchiole (Eiii), the levels of CEACAM1 were low to no detection on the epithelia of all the tissues assessed (Ei, Eiv–vi).

Fig. 5

Heat map showing the distribution of influenza and coronavirus receptors in the respiratory and intestinal tracts of beef cattle. Heatmap was generated using data from PixF image analysis showing the average log₁₀ total intensity (sum of intensity in all the pixels with signal) of cropped images (n = 3) by a 350*350-pixel region of interest (ROI) on the epithelium of respiratory and intestinal tract tissues. The color scale of lectin staining (orange, high; light yellow, medium; green, low) and immunofluorescence assay (red, high; white, medium; blue, low; dark blue < 5) indicates the level of log total intensity. ACE2, angiotensin-converting enzyme 2; TMPRSS2, transmembrane protease serine 2; APN, aminopeptidase N; DPP4, dipeptidyl peptidase 4; CEACAM1, carcinoembryonic antigen-related cell adhesion molecule 1. Respiratory and intestinal tissues were analyzed separately. For lectin staining data was analyzed with two-way ANOVA, and multiple comparisons with post hoc Tukey test. The levels of sialic acids within the same tissue (⁺denotes significant higher with P < 0.05) and one SA across different tissues (^#denotes significant higher with P < 0.05). Immunofluorescence assay was analyzed with one-way ANOVA (within the same receptor), and multiple comparison with post hoc Tukey test was conducted to compare between different tissues (^*denotes significant higher with P < 0.05).