More people are being infected than known because surveillance has been so poor.

A team of US researchers has revealed evidence of highly pathogenic H5N1 avian flu infections in two Texas farmworkers not previously confirmed to have the disease, and the investigators also cultured infectious H5N1 virus from milk and cattle samples taken from two Texas dairy farms that previously had H5N1 outbreaks.

The research, led by scientists with the University of Texas Medical Branch (UTMB) at Galveston, was published yesterday on the preprint server medRxiv, meaning that it has not yet been peer-reviewed. It comes as the US Department of Agriculture (USDA) confirmed two more dairy farms affected by H5N1 in cows, as well as newly infected mammalian species.

Two of 14 workers test positive

"Knowing that we had a research proposal to study livestock farms for evidence of novel respiratory viruses," the authors wrote, "we were invited by farm owners to study two dairy farms in Texas as they were recovering from incursions of avian influenza A H5N1 virus in their cattle. The identity and locations of two dairy farms (Farm A and B) are protected through nondisclosure agreements."

The investigators visited Farm A on April 3 and Farm B on April 4. They had previously studied specimens from Farm A, but this was their first visit to the other farm. Farm A housed 7,200 dairy cows and employed 180 workers. Farm B had 8,200 dairy cattle as well as beef cattle, but the two types were housed separately. It employed 45 farmworkers.

We were invited by farm owners to study two dairy farms in Texas as they were recovering from incursions of avian influenza A H5N1 virus.

In the 30 days before the researchers visited the farms, both locales reported sick cattle showing signs of respiratory disease, including coughing, nasal discharge, difficulty breathing, and fever. About 5% of the Farm A herd was sick, compared with 14% on Farm B. Farm B also reported that 15 to 20 of roughly 40 feral cats on the property had died after they exhibited influenza-like symptoms.

The research team enrolled 17 adult farm workers in the study, 10 from Farm A and 7 from Farm B. Twelve were men and 15 were Latino. All agreed to nasopharyngeal swab collection, but 3 workers on Farm B declined blood draws for sera specimens.

All 17 nasopharyngeal swabs collected from the farm workers were negative by molecular assays for influenza A viruses and coronaviruses. Microneutralization assays conducted on the 14 farm workers' sera samples demonstrated that 2 workers, both from Farm A, had antibodies to influenza A H5N1 virus, indicating a previous infection.

These workers, both of whom had recent respiratory symptoms, are not included in the US total of 13 human cases of H5 avian flu confirmed since April. Four of these infections are associated with exposure to sick dairy cows, and 9 are tied to H5N1-infected poultry.

One of the two Farm A workers who tested positive for H5N1 antibodies reported no respiratory illness in the previous year but had a cough and was taking cough medicine at the time of the study. The second employee worked in the Farm A cafeteria and had just recovered from a respiratory illness.

"I am very confident there are more people being infected than we know about," senior author Gregory Gray, MD, MPH, a UTMB infectious disease researcher, told NPR. "Largely, that's because our surveillance has been so poor."

Most milk samples test positive

The researchers tested 14 milk samples, 9 of which (64%) tested positive for H5N1 avian flu via polymerase chain reaction (PCR) and were also grown in culture, indicating the presence of live, infectious virus.

Of 39 deep nasal swabs collected from cattle on both farms, 1 tested positive via PCR and cell culture. It was from a recuperating cow. The investigators also detected SARS-CoV-2, the virus that causes COVID-19, in a nasal swab from a sick cow.

In addition, the team detected H5N1 in a great-tailed grackle found dead in an open-air dairy cattle barn.

For environmental sampling, they obtained 12 bioaerosol samples from each farm, as well, to detect airborne influenza viruses but found none. They added, "A single cattle fecal slurry sample was collected on Farm B and it had molecular evidence of influenza A virus."

They wrote, "Our isolates had multiple mutations associated with increased spillover potential."

The authors conclude, "It now seems especially prudent that we find ways to prospectively and more intensely study dairy farms to better quantify serological evidence of infections in both livestock and dairy workers. Before we can perform such important research, we need to find ways to fully protect the dairy businesses from any economic harm that might arise through such intensive study."

USDA confirms H5N1 in cows in Michigan, Texas

In related news, today the USDA Animal and Plant Health Inspection Service (APHIS) confirmed 2 new H5N1 avian flu outbreaks in dairy cows, raising the national total to 175 in 13 states. The new positive samples are from Michigan, which the state announced earlier this week, plus Texas, which hasn't had an outbreak in several weeks.

In addition, APHIS yesterday added three new species—deer mouse, prairie vole, and desert cottontail—all collected in Weld, County, Colorado—to its list of US mammals in which H5N1 avian flu has been confirmed.

APHIS also noted other mammal detections in Colorado, including 2 domestic cats, bringing the total reported in the United States since March to 36.