Rebecca Dorn, BS ¹ ; Christopher Chase, DVM, PhD ¹ ; Corale Dorn, DVM ³ ; Scott Wells, DVM, PhD ⁴ ; Steve Lawson, PhD ² ; Eric Nelson, PhD ² ; Guillermo Castillo, DVM, PhD ⁵ ;
¹Professional Program in Veterinary Medicine, South Dakota State University, Brookings SD, 57007
²Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings SD, 57007
³Dells Veterinary Services, Dell Rapids SD, 57022
⁴College of Veterinary Medicine, University of Minnesota, St. Paul MN, 55108
⁵Center for Animal Health and Food Safety, University of Minnesota, St. Paul MN, 55108

Introduction:

Since the diagnosis of H5N1 influenza A virus in lactating dairy cows in March 2024, research has focused on lactating dairy cows which show the obvious clinical signs of infection. However, research is lagging in dairy replacement heifers born around the time of outbreaks in the lactating herd. The objective of this study was to evaluate naturally occurring antibody responses to H5N1 influenza A virus in dairy replacement heifers months after the outbreak to evaluate if exposure in utero or post-partum to lactating cows and pasteurized pooled colostrum created detectable serologic responses in non-lactating replacement heifers. The test utilized to evaluate antibodies was a multi-species competitive enzyme-linked immunosorbent assay for H5-specific antibodies. Study results indicated that the only group of heifers that mounted a lasting serologic response were those in contact with clinical cows, their milk, and their colostrum prior to, but not following the confirmed date of the dairy herd’s clinical outbreak.

Materials and methods:

This study was performed in a 1,200-milking head Holstein dairy in Western Minnesota. For this study, evaluated dairy heifers were selected in three different age groups, with approximately 50 animals in each group. Group 1 consisted of heifers born between November 2023 to March 2024 and were moved off site, by May 4, 2024, to the heifer ranch 25 miles away. Group 1 heifers were sampled a total of four times, at the heifer ranch, the dairy’s dry cow lot, the close-up pen and the milking pens. Group 2 consisted of heifers born in June 2024 to clinically affected dams. These heifers were located at the dairy during the outbreak. Heifers from Group 2 were sampled a total of two times, at the heifer ranch and dairy’s dry cow lot. Group 3 consisted of heifers born in October 2024 from cows that were clinically affected in June 2024 and less than 125 days of gestation when the dam was clinically ill. Heifers from group 3 were sampled only one time, at the heifer ranch. Samples were collected from caudal tail veins of sampled dairy heifers from each of the three groups into a red-top vacutainer tube, using a new needle for each heifer.
The test used to evaluate the antibodies in the heifers was ID Screen Influenza H5 Antibody Competition 3.0 Multi-species test. This is a competitive ELISA for the detection of antibodies against the hemagglutinin H5 of the Influenza A virus. The positive and suspect positive cELISA results were then challenged with a serum virus neutralization assay. A fluorescent-focus serum virus neutralization assay using live virus was previously developed for the quantification of neutralizing antibodies produced in response to HPAI (H5N1) virus infection.

Results:

Group 1 heifers were born 3-7 months before the clinical outbreak and moved away from the lactating herd to the heifer site 30 days before the SCR health system indicated a health problem on June 4. While at the heifer ranch in October 2025, testing indicated that 9% (5/54) of the heifers were seropositive and another 9% (5/54) suspect positive. These heifers that tested either seropositive or suspect positive were born between February 25, 2024, to March 21, 2024. 90% (9/10) of the confirmed positive and suspect positive heifers were born within 10 days of each other, from February 25, 2024, to March 5, 2024. Once the heifers were moved from the heifer ranch and into the dry cow lot, 13% (6/45) of the heifers were seropositive and another 7% (3/45) suspect positive. From the dry cow lot the heifers moved into the close-up pens inside the milking barn, where 3% (1/38) of the heifers were seropositive, with another 5% (2/38) suspect positive. The final sampling took place while the heifers were in the milking herd, intermingled with older lactation cows. Testing indicated that 6% (3/47) of the heifers seropositive and another 6% (3/47) of the heifers suspect seropositive at this point 20 months after the outbreak confirmation. This group of replacement heifers was repeatedly tested as they moved into the milking herd with the dry cows and lactating cows. Overall, 13% (7/53) heifers in this group were cELISA positive at least once.
Repeated measures analysis was performed using the 22 Group 1 heifers sampled at every interval (October, November, January, and February). To rule out a reintroduction of the virus as the heifers are exposed to dry cows and milking cows, the “negative” population was monitored for seroconversion. There was no evidence supporting new infections in this heifer group, 100% of the heifers that tested negative in October 2025, at the heifer ranch, remained negative as they moved throughout the milking herd in February 2026.
One out of 51 heifers in Group 2 were seropositive 15 months after the outbreak. Group 2 heifers were born between May 31, 2024 to July 11, 2024, with the confirmed bulk tank PCR outbreak on June 7th, 2024. The date of birth of the test-positive heifer was on June 2, 2024.
Group 3 heifers were born to clinical dams 4 months after the outbreak. All heifers in this group (0/46) were seronegative 15 months after the outbreak.

Significance:

Of the three different dairy heifer cohorts evaluated, Group 1 was the only group with significant serum antibodies to influenza A virus 15 months after the outbreak was confirmed. While the time and route of exposure to the virus cannot be definitively confirmed, it is noteworthy that 90% (9/10) of the positive heifers were born within 10 days of each other and not in contact with reported clinically ill cows, their milk, or their colostrum following the confirmation date of the dairy herd’s outbreak. Further studies are needed to better understand the transmission route of H5N1 influenza A virus to replacement heifers, including early in the outbreak prior to detection. Further, given the lack of serum antibodies in replacement heifers born during and after an outbreak, it is reasonable to consider this population may be at risk of infection if viral exposure would occur in the future. Finally, reintroduction of test-negative heifers into a previously infected milking herd did not result in serological evidence of circulating virus within the herd 16-20 months after the initial outbreak. Through comparison of heifer and cow antibody levels, it is reasonable to conclude H5N1 influenzas A virus has a lasting seroprevalence in both groups affected.