Susceptibility of calves fed unpasteurized milk from cows experimentally infected with highly pathogenic avian influenza H5N1

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype B3.13 was confirmed in a dairy cow in Texas on March 25, 2024, by the US Department of Agriculture (USDA) National Veterinary Services Laboratories (NVSL) in response to a multi-state investigation into milk production losses. The amount and duration of virus shed in milk from the inoculated mammary quarters point to milk as a critical source of virus spread within and between dairy herds. Pasteurization has been shown to inactivate the virus in milk, however, domestic cats consuming raw milk from affected cows have developed fatal systemic influenza infection, raising the concern for calves fed unpasteurized milk. Investigations on affected dairy herds demonstrated the presence of viral RNA in milk samples for up to 2 weeks before the appearance of clinical signs and diagnostic confirmation of infection. During this lag, unpasteurized milk may pose a risk to calves. Here, we sought to determine if H5N1 genotype B3.13 strain could be transmitted to calves fed unpasteurized milk from virus positive lactating cows. Nine Holstein calves of approximately 7 to 11 weeks old were fed 0.95 L of unpasteurized milk twice a day via bucket. Milk from two non-inoculated cows was fed to one negative control calf for four days. Infected milk was collected from two lactating Holstein cows following intramammary inoculation, early in infection and prior to seroconversion, and fed to four calves for four consecutive days. In addition, milk was collected from another two lactating Holstein cows, inoculated via the intramammary route, in mid-infection following seroconversion, pooled with milk from cows early in infection, and fed to four calves for four consecutive days. Following the 4 initial days, all nine calves were fed milk from non-inoculated cows. Calves fed infected milk without antibodies showed clinical signs including nasal discharge, mild fever, mild lethargy, loose stool and slightly increased respiratory effort for 5–6 days. Viral RNA was consistently detected in nasal swabs from 4/4 calves from 2 to 4 DPI, with detection persisting after calves were transitioned to milk from non-inoculated cows. Infection was confirmed by viral RNA detected in nasal swabs, antigen and viral RNA detection in lung lesions, lymph nodes and pharyngeal tonsil, and seroconversion. Positive PCR results of the nasal swabs were significantly correlated (-0.55) with lethargy in the calves. Calves fed infected milk from cows shedding neutralizing antibodies were partially protected against disease and did not show clinical signs, only 1/4 calves had viral RNA in nasal swabs, and seroconversion was not observed. These data demonstrate that milk diverted from the human food supply in H5N1 positive dairy herds or from suspect cows should not be fed to calves without pasteurization. As characterization of HPAI in the dairy community continues the determination of routes of transmission is an essential first step to inform subsequent research on intervention and vaccination strategies.