Study finds dairy cows vulnerable to flu strains from birds, humans, and pigs

By Vijay Kumar MalesuReviewed by Danielle Ellis, B.Sc.Jul 18 2024

In a recent study published in Emerging Infectious Diseases, a group of researchers compared the distribution of sialic acid (SA) receptors in the respiratory tract and mammary glands of dairy cattle naturally infected with the highly pathogenic avian influenza (HPAI) A(H5N1) virus (highly contagious and deadly avian influenza virus strain H5N1).

Background 

In March 2024, the United States (US) Department of Agriculture reported the first detection of HPAI H5N1 virus in dairy cattle in the US. The presence of specific virus receptors on host cells influences the susceptibility to HPAI H5N1 infection. Still, the distribution of SA receptors in dairy cattle, particularly in mammary glands, remains poorly understood. Further research is needed to clarify the mechanisms of HPAI H5N1 transmission and persistence in dairy cattle and its implications for public health and the dairy industry.

About the study

In March 2024, two adult Holstein dairy cows in Texas, were diagnosed with HPAI H5N1 virus infection at the Iowa State University Veterinary Diagnostic Laboratory. The cows exhibited symptoms including reduced lactation and yellow, thickened milk.

The diagnosis was confirmed through real-time reverse transcription polymerase chain reaction (rRT-PCR) and immunohistochemistry in the mammary glands and lungs. Formalin-fixed and paraffin-embedded tissue sections of trachea, lung, and mammary gland tissues, as well as milk samples, were collected for further analysis.

Tissue sections were verified to be positive for the Influenza A virus (IAV) matrix gene nucleic acid using an RNA In Situ Hybridization Assay (RNAscope), and both macroscopic and microscopic lesions were tested using IAV chromogenic immunohistochemistry and rRT-PCR. 

Milk samples were cytocentrifuged and stained with a modified Wright stain for cytologic evaluation. Differential cell counts were determined under ×1,000 magnification by counting 100 nucleated cells. For receptor characterization, formalin-fixed, paraffin-embedded mammary gland sections were prepared and stained using a dual lectin and immunochemistry staining method.

The staining process involved incubating sections with specific lectins and streptavidin conjugated with Alexa Fluor 647, followed by immunostaining with influenza A nucleoprotein (IAV-Np). Multicolor immunofluorescent staining was performed using antibodies against cytokeratin and Ionized calcium-binding adapter molecule 1 (Iba1) (macrophage marker).

The slides were examined and imaged using an Olympus trinocular microscope equipped with a DP23 camera and CellSyns Dimension software. Negative controls consisted of primary lectin or antibody omission, and positive controls were performed on porcine tissues and known IAV-infected porcine lung tissue. 

Study results 

Two naturally infected lactating Holstein dairy cows with HPAI H5N1 virus infections were selected for retrospective evaluation. The cows exhibited clinicopathologic manifestations, and their infection was confirmed through previously reported detection methods and sequencing data.

Histological examination of the mammary gland revealed acute moderate multifocal mastitis characterized by epithelial attenuation and intraluminal neutrophilic inflammation. Immunohistochemistry for IAV-Np in the mammary gland showed intranuclear and intracytoplasmic immunoreactivity in alveolar and interlobular ductal epithelial cells. Cytologic evaluation of milk from the affected gland found moderate to marked neutrophilic and mild macrophagic inflammation consistent with mastitis.

The study aimed to evaluate the distribution of SA α2-3 and α2-6 receptors in the respiratory and mammary tissues of the infected cows using fluorescent and chromogenic-based lectin histochemistry.

Sambucus nigra agglutinin (SNA) (a lectin that binds specifically to sialic acid linked α2,6 to galactose or N-acetylgalactosamine) labeling was observed in submucosal glands, goblet cells, and intraepithelial and lamina proprial immune cells of the trachea and bronchi. Maackia amurensis lectin I (MAL-I) (a lectin that binds specifically to sialic acid linked α2,3 to galactose) and MAL-II labeling was weak to moderate, multifocal, and observed in the respiratory epithelium of the trachea. In the mammary gland, MAL-II and SNA labeling were expressed in unaffected secretory alveoli, with SNA also observed in unaffected interlobular ducts. No detectable MAL-I or IAV-Np labeling was observed in unaffected mammary gland sections.

Fluorescent labeling provided more definitive sensitivity and localization compared to chromogenic, lectin-based assays. In the infected mammary gland, pancytokeratin (epithelial marker) and Iba1 were used to evaluate intracellular distribution of IAV-Np. Co-labeling of IAV-Np with pancytokeratin was observed in cells lining the interlobular ducts and secretory alveoli, with more intense labeling in the alveolar epithelium. Intraluminal cells within secretory alveoli and ducts exhibited both intranuclear and cytoplasmic IAV-Np expression, commonly labeled with pancytokeratin and rare Iba1-positive cells.

Intranuclear IAV-Np labeling co-localized with MAL-II and SNA in epithelial cells lining the secretory alveoli. Co-labeling of IAV-Np and SNA was also observed in ductal epithelial cells, while MAL-II was not detected in ducts. MAL-I labeling was not found in the secretory ducts or alveoli, precluding its detection with IAV-Np. 

Conclusions

To summarize, the presence of HPAI H5N1 virus in cattle, especially in the mammary gland and milk, highlights the adaptability of IAV to nontraditional species and the need for active surveillance. IAVs utilize specific SA receptors for host infection. Avian IAVs prefer SA α2,3 linkages, while mammalian IAVs, including those from humans and swine, favor SA α2,6 linkages. This study found both types of SA receptors in the respiratory tract and mammary glands of dairy cattle, indicating potential for cross-species infection.