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Abstract

Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus was identified in 4 cull dairy cows condemned by the U.S. Department of Agriculture because of pneumonia with accompanying systemic changes. Histologic findings were bronchopneumonia in 3 cows and embolic pneumonia and nephritis in 1 cow. In addition to detection of HPAI A(H5N1) virus by reverse-transcription real-time PCR in various formalin-fixed, paraffin-embedded tissues, influenza A virus was detected by immunohistochemistry and in situ hybridization in the pulmonary respiratory epithelium of 2 of the cows with bronchopneumonia and in renal medullary tubules of the cow with nephritis. A PCR panel screening for common bovine respiratory pathogens in the cows with bronchopneumonia revealed variable coinfections with Histophilus somni, Pasteurella multocida, Mannheimia haemolytica, Mycoplasmopsis bovis, and bovine coronavirus. We describe the distribution of HPAI A(H5N1) virus in naturally infected cows while highlighting the need for research on the roles of coinfection and immune response in influenza viral replication.

Keywords

dairy cows highly pathogenic avian influenza HPAI A(H5N1)pneumonia

Since the first report in March 2024 of highly pathogenic avian influenza (HPAI) A(H5N1) virus in milk and mammary glands of lactating dairy cattle, the virus has been found in numerous U.S. states and has spread horizontally through the interstate transportation of cattle.² Transmission to cattle has occurred through unknown mechanisms, with some cattle infected on farms without recent purchases.¹³ As of 2025 Aug 21, outbreaks of HPAI A(H5N1) virus infections in dairy cattle have occurred in 17 states, affecting >1,078 herds.¹⁴ In addition to cattle, HPAI A(H5N1) virus has been detected in poultry and wild birds, with spillover infections occurring in numerous terrestrial and marine mammal species in North America and Europe.^2–5,12,15

Sialic acids (SAs) are the primary receptors for virus attachment and cell entry. In cattle, the respiratory epithelium is rich in primarily SA α2,3-gal-β receptors, compared with mammary gland tissue, which has both SA α2,3-gal-β and α2,6-linked receptors.¹⁰ Genomic analysis of the circulating bovine genotype B3.13 contains mutations associated with a potential increase in binding affinity to the human SA α2,6-linked receptor.⁷ This analysis may help to explain why cases of mastitis caused by HPAI A(H5N1) virus are overrepresented in clinically affected dairy cows, in contrast to fewer cases of cattle developing respiratory disease. However, viral pathogenesis and its relation to receptor binding affinity remains to be fully elucidated.

We focused on 4 cows brought into federally inspected slaughter establishments with pneumonia accompanied by systemic changes and identified as naturally infected with HPAI A(H5N1) virus via PCR testing. All 4 cows in our study were condemned on postmortem evaluation and thus did not enter the food supply. Of the PCR-positive cows, replication of the influenza virus within respiratory epithelium of 2 cows and within renal tubules of a third cow was demonstrated via in situ hybridization (ISH). Interestingly, concurrent infections with other microorganisms were confirmed via PCR testing and histochemical staining within the affected lungs and kidney, respectively. This raises questions regarding interactions between comorbidities and the host immune response on the ability of HPAI A(H5N1) virus to replicate in bovine tissues.

In April 2024, 3 cull dairy cows originating from a known HPAI A(H5N1) virus-positive herd were condemned by the USDA’s Food Safety and Inspection Service (FSIS) by in-plant personnel because of severe pneumonia and lesions consistent with infectious systemic disease. FSIS, the Animal and Plant Health Inspection Service’s National Veterinary Services Laboratories (NVSL; Ames, IA, USA), and the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL; Ames, IA, USA) performed ancillary testing to investigate infection with the HPAI A(H5N1) virus. Preliminary HPAI A(H5N1) virus PCR results from these 3 cows led to the implementation of an exploratory study of the distribution of HPAI A(H5N1) virus in tissues of cull dairy cows condemned during inspection. This exploratory study targeted dairy cows with an unknown HPAI A(H5N1) virus herd status that were condemned because of pneumonia, septicemia, or both by Public Health Veterinarians (PHVs) during routine inspection at U.S. slaughter facilities. From 2024 Apr 3 to 2024 May 13, PHVs sampled at least 10 condemned dairy cows at each of 11 targeted high-throughput cull cow slaughter facilities, operating in 8 states (California [2], Idaho, Michigan, Minnesota, Pennsylvania [2], South Dakota, Texas [2], Wisconsin) for a total of 111 cows. Tissue samples from these cows and the 3 preliminary cows mentioned above were submitted in 10% neutral-buffered formalin to the USDA FSIS Eastern Laboratory Pathology Branch. Tissues collected by PHVs included lung, mediastinal lymph node, kidney, liver, spleen, heart, and diaphragm. Samples were processed to generate formalin-fixed, paraffin-embedded (FFPE) tissue blocks for histologic examination at FSIS, PCR testing at NVSL, and immunohistochemistry and ISH at ISU-VDL.

Mammary gland tissue was excluded from our study because the udders were removed upstream in the slaughter process before inspection, as udders are not considered suitable for human consumption and to prevent contamination of the carcass. Each tissue type from the exploratory study was processed in separate cassettes; tissues collected from the initial 3 cull cows were comingled in tissue cassettes ( Table 1 ; cases 1–3). Scrolls (10-µm thick) of FFPE tissues from each animal were submitted to the NVSL for reverse-transcription real-time PCR (RT-rtPCR) for influenza A virus (IAV), as well as primer-probe sets specific for H5, N1, and H5 clade 2.3.4.4b subtyping by RT-rtPCR. All reactions consisted of 40 cycles, with the IAV RT-rtPCR being semiquantitative and utilizing an exogenous internal control to ensure that the PCR performed within an accepted level of efficiency. The other PCR reactions (H5, N1, H5 clade 2.3.4.4b) were run in parallel or serially on the same extraction, and the qualitative results were used to characterize the virus. Of the FFPE tissue from 114 cows (initial 3 cull cows from the Idaho slaughter plant and 111 cows from the follow-up exploratory study) tested by RT-rtPCR for HPAI A(H5N1) virus, nucleic acid was detected in 4 cows for each of the primer-probe sets specific for IAV, H5, N1, and H5 clade 2.3.4.4b (Table 1). HPAI A(H5N1) virus nucleic acid was detected in each of the lung samples. Other tissues tested from the 4 infected cows tended to have higher Ct values compared with those in the lung. One exception was case 2 in which the kidney Ct value for IAV was lower than that of the lung.

Table 1.

RT-rtPCR results (Ct) for influenza A(H5N1) virus (IAV, EA H5 HPAI, IAV N1, H5 2.3.4.4) in formalin-fixed, paraffin-embedded (FFPE) tissue samples from cull dairy cows.

Case (U.S. state, ID)	FFPE tissue(s)	IAV	EA H5 HPAI	IAV N1	H5 2.3.4.4
1 (ID, B64460)	Liver, kidney, spleen	34.4	33.1	35.2	35.3
	Lung	26.9	25.6	28.8	27.9
	Lymph node	32.2	30.5	32.7	33.3
2 (ID, B64497)	Kidney	32.4	29.2	32.8	30.6
	Lung, liver	35.9	33.3	36.1	35.1
	Lymph node, spleen	38.4	37.2	38.0	37.4
3 (ID, B64464)	Kidney, lymph node	39.3	ND	ND	ND
	Lung	36.8	35.1	37.8	37.4
	Lung, spleen	ND	ND	ND	39.2
4 (MI, B64731)	Kidney	34.9	31.8	35.4	33.1
	Liver	32.5	29.5	32.7	30.4
	Lung	22.3	16.9	21.7	20.2
	Spleen	32.8	29.3	32.8	30.1
	Heart	31.3	27.3	30.8	28.5
	Diaphragm	29.9	23.9	29.3	27.8
	Lymph node	31.4	28.7	31.4	29.1

EA H5 HPAI = cleavage site of Eurasian H5 HPAI; IAV = influenza A virus; H5 2.3.4.4 = goose/Guangdong H5 clade 2.3.4.4b; IAV N1 = influenza A N1 subtype; ND = not detected.

Unstained slides from 4 HPAI A(H5N1) virus PCR-positive cows were submitted to the ISU-VDL for IAV nucleoprotein immunohistochemistry (IHC), as described previously.² ISH targeting the RNA sequence encoding IAV matrix protein was also performed to co-localize genomic material in respiratory tissues. The RNAscope procedure was performed (Formalin-fixed paraffin-embedded (FFPE) sample preparation and pretreatment for the RNAscope 2.5 HD assay, RNAscope 2.5 HD detection reagent–red; Advanced Cell Diagnostics [ACD]) following the manufacturer’s recommendations. ACD probes used included V-influenza-H1N1-H5N1-M (catalog 436211), RNAscope negative control probe–DapB (310043), RNAscope positive control probe–Bt-PPIB (319451). Positive control tissue for IAV IHC and ISH consisted of IAV-positive porcine lung and bovine mammary gland ( Suppl. Fig. 1 ). Each slide was evaluated by at least 5 veterinary anatomic pathologists, and distribution and intensity were subjectively agreed upon by comparison of the tissues in our study. Intensity and distribution of labeling within the evaluated tissues were scored: weak-rare = minimal immunoreactivity confined to the cytoplasm of a few scattered cells; moderate-localized = prominent intracytoplasmic and intranuclear immunoreactivity confined to cells within one region of the tissue where the rest of the tissue was negative; and strong-widespread = marked intracytoplasmic and intranuclear immunoreactivity in many cells throughout the tissue ( Table 2 ).

Table 2.

Influenza A virus detection by immunohistochemistry (IHC) and in situ hybridization (ISH) in tissue samples from cull dairy cows.

Case(U.S. state, ID)	IHC results (ISH results)
Case(U.S. state, ID)	Lung	Liver	Kidney	Spleen	Diaphragm	Heart	Lymph node
1 (ID, B64460)	+++ (+++)	– (–)	– (–)	– (–)	NC	NC	– (–)
2 (ID, B64497)	– (–)	– (–)	++ (+)	– (–)	NC	NC	– (–)
3 (ID, B64464)	– (–)	NC	– (–)	– (–)	NC	NC	– (–)
4 (MI, B64731)	+++ (+++)	– (–)	– (–)	– (–)	+ (–)	– (–)	+ (–)

– = negative; + = weak-rare detection; ++ = moderate-localized detection; +++ = strong-widespread detection; NC = not collected.

Histologically, severe suppurative bronchopneumonia was evident in 3 of the 4 positive cows (Fig. 1A, 1D). Two of these cows were positive for IAV on IHC and ISH in the lung (Tables 1, 2; cases 1, 4). Strong intracytoplasmic and intranuclear immunoreactivity was widespread within the epithelium lining bronchi and bronchioles by IHC ( Fig. 1B, 1E ). Additionally, moderate intracytoplasmic and intranuclear immunoreactivity was observed within sloughed, degenerate epithelium within airways and surrounding alveolar spaces. ISH labeling for viral RNA had a similar distribution, indicating replicating virus ( Fig. 1C, 1F ). One cow (Table 1; case 2), with the lowest RT-rtPCR Ct value (32.4) in the kidney, had histologic evidence of embolic bacterial nephritis ( Fig. 2A, 2B ) and pneumonia. IHC and ISH in this case repeatedly confirmed IAV nucleoprotein antigen and viral RNA within cells and intraluminal material in a few tubules of the renal medulla ( Fig. 2C, 2D ). This cow was negative on IHC and ISH in the lung. In case 4 (Table 2), weak-positive labeling on IHC was detected in the cytoplasm of a few cells within the diaphragm. These cells were within the interstitium surrounding myofibers and identified as possible macrophages or satellite cells. No IAV nucleoprotein antigen or viral RNA was detected in diaphragm myocytes, and ISH was negative on the diaphragm. Similarly, a few macrophages within one lymph node section from the same cow had weak immunoreactivity on IHC. No nuclear IHC labeling was detected. ISH also was negative on the lymph node. Despite HPAI (A)H5N1 virus PCR detections, no IAV nucleoprotein antigen or viral RNA was detected in the lung of case 3 or in the liver, spleen, or heart by IHC or ISH in any cow (Table 2).

Figure 1.

Detection of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in the lung of cull dairy cows with bronchopneumonia. A. Case 1; D. Case 4. Regionalized, suppurative bronchopneumonia. H&E. B. Case 1; E. Case 4. Strong-widespread intracytoplasmic and intranuclear immunoreactivity for IAV nucleoprotein within respiratory epithelium lining bronchioles. Insets: high magnification of IAV nucleoprotein immunoreactive cells. Immunohistochemistry. C. Case 1; F. Case 4. Strong-widespread intracytoplasmic and intranuclear labeling for IAV matrix nucleic acid within respiratory epithelium lining bronchioles Insets: high magnification of IAV labeled cells. In situ hybridization.

Figure 2.

Detection of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in the kidney of a cull dairy cow (case 2) with embolic nephritis. A. Extensive coagulative necrosis and suppurative inflammation of the renal cortex. H&E. B. Colonies of gram-positive bacteria within glomeruli and free within areas of necrosis. Inset: high magnification of colonies. Gram stain. C. Moderate-localized immunoreactivity for IAV nucleoprotein in renal medullary tubules. Immunohistochemistry. D. Weak-rare labeling for IAV matrix nucleic acid in renal medullary tubules. In situ hybridization.

Additionally, FFPE lung tissue from these same 4 dairy cows was evaluated via a bovine respiratory panel real-time PCR (rtPCR), which included bovine alphaherpesvirus 1 (BoAHV1), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), Mycoplasmopsis bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. FFPE tissue was extracted (MagMAX FFPE DNA/RNA Ultra kit; ThermoFisher). The bovine respiratory panel rtPCR panel was conducted per the ISU-VDL protocol as a multiplex panel with primers and probes (available on request) targeting BCoV, BRSV, BVDV, BoAHV1, M. haemolytica, P. multocida, H. somni, and M. bovis, using a 25 µL reaction, as described previously.¹¹

In cases 1, 3, and 4 with bronchopneumonia, H. somni and P. multocida were detected by rtPCR testing ( Table 3 ). M. haemolytica nucleic acid was detected in cases 1 and 3. BCoV nucleic acid was detected in case 4 and M. bovis in case 3. Case 2 with embolic nephritis and pneumonia, but lacking histologic evidence of bronchopneumonia, was negative for all viral and bacterial agents in the bovine respiratory panel.

Table 3.

Bovine respiratory panel rtPCR Ct results for formalin-fixed, paraffin-embedded lung samples from cull dairy cows.

Case(U.S. state, ID)	Lung lesion	BoAHV1	BCoV	BVDV	BRSV	Pm	Mh	Hs	Mb
1 (ID, B64460)	Bronchopneumonia	>35	>35	>35	>35	32.0	34.4	21.6	>35
2 (ID, B64497)	Embolic pneumonia	>35	>35	>35	>35	>35	>35	>35	>35
3 (ID, B64464)	Bronchopneumonia	>35	>35	>35	>35	23.7	27.2	22.0	25.4
4 (MI, B64731)	Bronchopneumonia	>35	31.8	>35	>35	32.5	>35	21.5	>35

BoAHV1 = bovine alphaherpesvirus 1; BCoV = bovine coronavirus; BVDV = bovine viral diarrhea virus; BRSV = bovine respiratory syncytial virus; Hs = Histophilus somni; Mb = Mycoplasmopsis bovis; Mh = Mannheimia haemolytica; Pm = Pasteurella multocida. Ct >35 = negative.

Our findings demonstrate viral replication within respiratory epithelium of the lung. The results further reveal co-infection of HPAI A(H5N1) virus with common bovine respiratory pathogens in cows with bronchopneumonia. Interestingly, the kidney of one cow with embolic bacterial nephritis had possible HPAI A(H5N1) viral replication within a few tubules of the renal medulla. Initial pathogenesis studies suggest that HPAI A(H5N1) virus is not a primary cause of respiratory disease in dairy cows.^1,6 Unfortunately, we cannot confirm whether these animals had viral mastitis, because no mammary gland tissue or milk was sampled at slaughter. Furthermore, it is unclear if a bacterial bronchopneumonia developed secondary to an underlying HPAI A(H5N1) viral infection, or if damage to the lung tissue by bacterial pathogens created conditions that facilitated viral replication.

Although HPAI A(H5N1) virus was detected by PCR testing of the diaphragm and heart of case 4, virus was not detected in myofibers by IHC or ISH. The few cells within the diaphragm and lymph node with IHC cytoplasmic immunoreactivity may have been resident macrophages or satellite cells (muscle). The lack of nuclear immunoreactivity may indicate that virus was phagocytosed by these cells without nuclear invasion or viral replication. Due to the close proximity of the diaphragm to the lungs, it is also possible that these cells could have been circulating inflammatory cells from the lungs.

Our study is unique in that natural infection and replication of HPAI A(H5N1) virus were identified in the lungs and kidney of dairy cows brought to slaughter. Pneumonia in cows at postmortem inspection is common and often multifactorial in etiology (e.g., shipping fever). However, until recently, IAV was regarded as an unlikely contributing pathogen. Collectively, our findings raise questions about the circumstances surrounding HPAI A(H5N1) virus replication within the respiratory epithelium of the bovine lung. Can the virus cause primary pneumonia or does the virus take advantage of already diseased tissue? It also raises questions about the immune response and degree of influenza viral replication, given that most cows with shipping fever have at least some degree of immunosuppression. Finally, if replication of HPAI A(H5N1) virus is occurring at high rates in bronchial and bronchiolar epithelial cells, is horizontal transmission via respiratory droplets possible in these herds? Although the detection of IAV by ISH and IHC in the kidney was potentially an outlier, IAV RNA has been detected in urine from a cow on an affected farm.⁸ Further studies on interactions between HPAI A(H5N1) virus and a host immune system that is under the stress of coinfection are warranted.

USDA FSIS Disclaimer: All HPAI A(H5N1) virus-infected cows were condemned at slaughter and did not enter the food supply. Cooking meat to the USDA FSIS recommended minimum internal temperature (71.1ºC for ground beef) has been shown to result in a reduction of avian IAV particles to nondetectable levels.⁹

Supplemental Material

sj-pdf-1-vdi-10.1177_10406387261417354 – Supplemental material for Detection of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cull dairy cows with underlying respiratory and systemic disease

Supplemental material, sj-pdf-1-vdi-10.1177_10406387261417354 for Detection of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cull dairy cows with underlying respiratory and systemic disease by Daniel J. Righter, Erin B. Howey, Chris L. Siepker, Eric R. Burrough, Drew R. Magstadt, Marta Mainenti, Asha Fears, Aaron D. Lehmkuhl, Gleeson Murphy, Kimberly Lehman, Mia Kim Torchetti, Suelee Robbe-Austerman and Carrie E. Schmidt in Journal of Veterinary Diagnostic Investigation

Footnotes

Acknowledgements

We thank Trish Archer, Karen Stockwell, and Shayla Belton at the USDA FSIS Eastern Laboratory Pathology Branch for their collaborative efforts, and PHVs Betina Proof, Leslie Luscher, and William Boyd for critical efforts in identifying and collecting samples from infected dairy cows. We further thank Jennifer Groeltz-Thrush at the Iowa State Veterinary Diagnostic Laboratory for performing IHC and ISH on tissues evaluated in this manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Daniel J. Righter

Erin B. Howey

Chris L. Siepker

Eric R. Burrough

Marta Mainenti

Kimberly Lehman

Suelee Robbe-Austerman

Carrie E. Schmidt

Supplemental material

Supplemental material for this article is available online.

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