Dairy cows inoculated with highly pathogenic avian influenza virus H5N1

Abstract

Highly pathogenic avian influenza (HPAI) H5N1 haemagglutinin clade 2.3.4.4b was detected in the USA in 2021. These HPAI viruses caused mortality events in poultry, wild birds and wild mammals. On 25 March 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multistate investigation into milk production losses¹. More than 200 positive herds were identified in 14 US states. The case description included reduced feed intake and rumen motility in lactating cows, decreased milk production and thick yellow milk^2,3. The diagnostic investigation revealed viral RNA in milk and alveolar epithelial degeneration and necrosis and positive immunoreactivity of glandular epithelium in mammary tissue. A single transmission event, probably from birds, was followed by limited local transmission and onward horizontal transmission of H5N1 clade 2.3.4.4b genotype B3.13 (ref. ⁴). Here we sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows. Heifers were inoculated by an aerosol respiratory route and cows by an intramammary route. Clinical disease was mild in heifers, but infection was confirmed by virus detection, lesions and seroconversion. Clinical disease in lactating cows included decreased rumen motility, changes to milk appearance and production losses. Infection was confirmed by high levels of viral RNA detected in milk, virus isolation, lesions in mammary tissue and seroconversion. This study provides the foundation to investigate additional routes of infection, pathogenesis, transmission and intervention strategies.

Fig. 1. Clinical signs and viral detection in lactating dairy cows.

a, Representative milk sampling demonstrating thickening, flakes or clots, and change in colour in the upper left and right photographs, and gel formation in positive California mastitis tests in the lower left photograph. In all photos, changes were observed in the inoculated front right and rear left quarters in the upper right and lower left cups of the milk collection paddle in each photo. b, Daily rumination time in minutes for cow 2,112 (blue) and cow 2,129 (orange) measured using an ear-tag accelerometer sensor. Rumination time decreased at 1 DPI and recovered to pre-challenged levels at 7 DPI. c, Each individual milking machine bucket was weighed once daily to monitor production, for cow 2,112 (blue) and cow 2,129 (orange). Milk production steadily declined from 1 to 4 DPI, remained low until 10–12 DPI, and was 71–77% of pre-inoculation production at 23 DPI. d, Milk was stripped from each teat before milking and a sample was taken from the bucket after milking, for cow 2,112 (blue) and cow 2,129 (orange). An RT–qPCR assay detected viral RNA beginning on 1 DPI until study termination at 24 DPI in the inoculated quarters (solid lines), with Ct on the y axis and DPI on the x axis. The positive detections in non-inoculated quarters (dashed lines) were probably cross-contamination due to the non-sterile collection of milk from each teat. The pink shading indicates time points when live virus isolation was attempted from the bucket samples and samples were negative by egg inoculation. One inoculated quarter from cow 2,112 was positive for virus isolation on 12 DPI, but all tested samples beyond 12 DPI were negative for virus isolation.

Fig. 2. Correlation between viral RNA detection and clinical signs.

Significant (P < 0.05, two-sided) Pearson correlation coefficients between Ct values for milk samples from the bucket (n = 34) as well as the inoculated teats (front right (FR) (n = 34) and rear left (RL) (n = 34)) and rumination time (n = 46), milk production (n = 44) and consistency (n = 48) and colour (n = 48) scores of milk from the inoculated teats across all time points. Empty squares in the matrix indicate the correlation was not significant (P < 0.05, two-sided).

Fig. 3. Fibrosis in inoculated mammary gland tissue.

a,c, Representative photomicrographs of the interlobular (arrowheads) and intralobular (arrows) fibrosis and inflammatory infiltrate (chevrons) of the left rear mammary gland in cows 2,112 (a) and 2,129 (c). b,d, A matched Masson’s trichrome stain (fibrosis is blue) demonstrates the extent of fibrosis in cows 2,112 (b) and 2,129 (d). e,f, A representative photomicrograph showing haematoxylin–eosin (e) and Masson’s trichrome (f) staining in a non-inoculated teat (left front) of cow 2,112 for comparison. All photomicrographs are at ×100 magnification and the scale bar (200 μm) applies to all panels.

Fig. 4. IHC in mammary tissue.

a–d, IHC detection of IAV antigen in epithelial cells lining secretory alveoli of the left rear mammary glands (arrowheads in a,b) with higher-magnification images showing staining in the cytoplasm (arrows in c,d) and nucleus (arrowheads in c,d), for cows 2,112 (a,c) and 2,129 (b,d). Photomicrographs at ×100 (a,b) and ×400 (c,d) magnification. Scale bars, 100 μm (a,b) and 20 μm (c,d).

Fig. 5. Histopathologic changes in heifer lung tissue.

a, Peribronchiolar mononuclear cell inflammatory infiltrate (arrowheads) and accumulation of intraluminal inflammatory cells (arrow) in a bronchiole in the cranial part of the left cranial lung lobe of heifer 2,311. b, Replication of HPAI virus in the respiratory epithelium lining the affected bronchiole of heifer 2,311 shown in a, as demonstrated by IHC (arrowheads). c, Peribronchiolar mononuclear cell inflammatory infiltrate (arrowheads) in another bronchiole in the cranial part of the left cranial lung lobe of heifer 2,311. d, Replication of HPAI virus in the respiratory epithelium lining the affected bronchiole of heifer 2,311 shown in c, as demonstrated by IHC (arrowheads). e, Bronchiolitis obliterans in heifer 2,316. The lumen of a bronchiole (asterisk) is partially occluded by a polyp lined by attenuated epithelial cells (arrowheads). The polyp is composed of fibroblasts, fibrin and lymphocytes (arrow). Adjacent alveoli contain increased inflammatory cells (probable alveolar macrophages; brace). An adjacent arteriole is circumferentially surrounded by a mononuclear cell inflammatory infiltrate composed predominately of lymphocytes and fewer plasma cells (chevron). f, Replication of HPAI virus in the respiratory epithelium lining the affected bronchiole of heifer 2,316 as demonstrated by IHC (arrowheads), and in probable type II pneumocytes (arrow) and alveolar macrophage (brace). All photomicrographs are at ×200 magnification and the scale bar (50 μm) applies to all panels.

Extended Data Fig. 1. Evolutionary history of H5N1 clade 2.3.4.4b hemagglutinin genes from dairy cattle in the United States between March and May 2024.

The HA gene and whole genome phylogeny demonstrated a single introduction of the virus from wild birds to dairy cattle with subsequent dissemination across nine US states. The A/dairy cattle/Texas/24-008749-002/2024 study strain, indicated by a red star, was 99.94% identical to a B3.13 whole genome consensus strain (9 nucleotide substitutions across the genome); and at the amino acid level, the TX/24 HA protein was identical to a consensus B3.13 HA sequence, with only 2 synonymous substitutions detected at the nucleotide level.

Extended Data Fig. 2. Macroscopic lung lesions in a heifer.

Minimal multifocal obstructive atelectasis (arrows and insets) consistent with influenza A virus infection in Heifer 2316.

Extended Data Fig. 3. Influenza A virus immunohistochemistry in lymph node and mammary gland.

Influenza A virus detection by immunohistochemistry (arrowheads) in the germinal center light zone of the supramammary lymph node of cow 2129 (A). Immunohistochemistry did not detect Influenza A virus antigen in the uninoculated left front mammary gland of 2112 (B). Photomicrographs at 200X (A) and 100X (B) magnification.

Extended Data Fig. 4. Inoculation of Holstein calves and cows.

A) Schematic of lactating cow study design. B) Photographs of intramammary inoculation in the lactating Holstein cows via teat canula. C) Schematic of heifer calf study design. D) Photographs of aerosol respiratory inoculation in the Holstein heifer calves using nebulizer masks. The graphics in a,c were created with BioRender.com.