Just ten viral particles can infect cows with bird flu

Just 10 viral particles of the H5N1 bird flu that caused hundreds of influenza outbreaks in U.S. dairy cattle can cause infection in cows, a new study shows.

The research also hints at why the outbreaks have confounded scientists, farmers and livestock handlers hoping to contain and prevent the disease – an effort likely complicated by the fact that the virus has an affinity for cow mammary glands rather than airways.

Tests for transmission among cows through milk machinery or through feeding of calves, and between birds and cattle through shared indoor air, didn't show results of disease spread.

Researchers are continuing to pursue more answers, but for now the transmission mystery endures, meaning scientists can't yet provide evidence-based recommendations for practices that would stop the spread.

"How it spreads from cow to cow becomes a very important question. We need to understand if there's a way to change milking practices or farming practices, whatever it is, to limit cow-to-cow transmission because we think spillover is going to happen again. It's just a matter of time," said senior author Andrew Bowman, professor of veterinary preventive medicine at The Ohio State University.

"And right now we don't have a great way to prevent either that spillover or cow-to-cow transmission once it happens."

The study was published recently in Nature Communications.

Highly pathogenic H5N1 avian influenza A viruses are associated with wild birds and poultry, but since 2021 a group of these viruses of clade 2.3.4.4b have circulated globally in mammals and were first reported in U.S. dairy cattle in March 2024.

To date, 1,053 outbreaks of the originally detected virus genotype (B3.13) have been confirmed in dairy herds in 17 states, which have been brought under control through a national milk testing strategy that halted movement of herds producing milk in which the virus was detected. Current federal data suggests small numbers of infections in cows are confirmed in Idaho, Utah and Texas.

This work has all been done in response to the unprecedented spillover of avian influenza into dairy cattle. Initially, we had no idea that cows could even be infected with influenza, let alone that the mammary gland was involved. That in and of itself was a major paradigm shift: It's not respiratory."

Andrew Bowman, professor of veterinary preventive medicine, The Ohio State University

Bowman and colleagues have been following the disease since its emergence on dairy farms, reporting early this year on detection of the virus in retail milk supplies – at that time, 36.3% of samples tested were positive for H5N1 particles.

"Pasteurization is inactivating it. But once a cow's infected, they produce high-viral-titer milk for a week-plus," Bowman said.

Those high virus titers, or concentrations, in milk were among the results of the current work, which involved testing the effects of varied levels of viral particle inoculations into individual cow teats, where mammary glands are located.

Results showed that the smallest dose of 10 particles resulted in productive infection but fewer clinical signs compared to higher doses, as well as shedding of milk containing high concentrations of viral particles.

"They are four separate mammary glands that have a common blood supply, but in these small numbers of animals, we're not seeing a lot of viral movement between those glands," Bowman said. "I think that may become important to how sick a cow does or doesn't get because if one of four glands is infected, that's different from a severe infection in all four."

Several experiments aimed to identify potential disease transmission routes.

Researchers transferred contaminated milking equipment directly from an infected cow to a healthy cow twice daily for 14 days. Even though infected cows had clinical signs and their milk was high in viral particles, the healthy cows exposed to contaminated equipment remained unaffected and their milk was virus-free.

Bottle-feeding calves high-viral-titer milk collected from infected cows led to minimal virus detection and inflammatory signs in the calves, suggesting transmission through feeding doesn't spread enough material to establish an infection.

Lactating cows given an intranasal dose of the H5N1 virus did not get sick and the presence of viral RNA was minimal in nasal swabs and absent in milk, but some of their respiratory tissues showed signs of an immune response compared to uninfected cows. These cows were also co-housed with chickens that remained healthy after 17 days. The combined findings suggest airborne transmission may be minimal if it's happening at all.

Experiments were conducted in one of only six Biosafety Level 3 facilities in the country that can accommodate large animals. There is a chance, Bowman said, that the biocontainment conditions were too sterile and airflow-controlled to resemble daily farm life, especially when it comes to airborne transmission.

And performing a limited number of tests on actual cows, rather than surrogate models, could be a "numbers game," he said. "We see about 20% of cows on farms become clinically ill. Two transmission experiments just may not be enough.

"I think the milking equipment is something we still have to consider. The virus replicates in the mammary gland to a really high titer. It comes out in milk at a really high titer. We have common contact equipment that goes from cow to cow with high-risk material to the high-risk tissue. I think the straight line between two points would say the milking equipment still seems to be the likely route of transmission between cows," Bowman said.

"There is still the bigger question of spillover from wild birds into cows. In waterfowl, it's a pathogen replicating in their gut. How in the world does it go from a duck's intestine into a cow's mammary gland? That's a head scratcher."

This work was supported by the Centers of Excellence for Influenza Research and Response funded by the National Institute of Allergy and Infectious Diseases.

Cody Warren and Scott Kenney of Ohio State were co-lead authors. Additional co-authors were Carolyn Lee, Natalie Tarbuck, Hannah Cochran, Bryant Foreman, Patricia Boley, Saroj Khatiwada, Alok Dhakal, Khadijat Adefaye, Jennifer Schrock, Mohammad Jawad Jahid, Thamonpan Laocharoensuk, Raksha Suresh, Olaitan Shekoni, Erika Stevens, Sara Dolatyabi, Christina Sanders, Elizabeth Ohl, Devra Huey, Juliette Hanson, Kara Corps and Renukaradhya Gourapura, all of Ohio State, and Richard Webby of St. Jude Children's Research Hospital.

The Plant and Animal Agrosecurity Research Facility is located at Ohio State's College of Food, Agricultural, and Environmental Sciences Wooster Campus.