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 Highly Pathogenic Avian Influenza H5N1 in lactating dairy cows in March 2024, research has focused on the lactating dairy cows which appear to show the most clinical signs. However, research is lagging in the replacement heifers born around the time of outbreaks in the lactating herd. This study sought to evaluate antibody levels to highly pathogenic avian influenza H5N1 in replacement heifers’ months after the outbreak to see if exposure in utero or post-partum to lactating cows and pasteurized pooled colostrum created any serologic response in non-lactating replacement heifers. The test utilized to evaluate antibodies was the multi-species competitive enzyme-linked immunosorbent assay for H5. The only group of heifers that mounted a lasting seroprevalence was not in contact with clinical cows, their milk, or their colostrum during the dairy herd’s confirmed outbreak.

Materials and methods:

The heifer portion of the study was broken into three different age groups, with 50 head in each group. Group #1 consisted of heifers born in February/March 2024 and moved off site, in one group, beginning of May 2024 to the heifer ranch 25 miles away. Group #1 was 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 cows. They lived at the dairy during the outbreak. Group #2 was sampled a total of two times, at the heifer ranch and dairy’s dry cow lot. Group #3 consisted of heifers born October 2024 from cows that were clinically affected in June 2024 and less than 125 days carrying calf when the dam was clinically ill. Group #3 was sampled only one time, at the heifer ranch.
Samples were taken from tail veins with a red-top vacutainer tube. The needle was changed between each heifer. The test used to evaluate the antibodies in the heifers was ID Screen Influenza H5 Antibody Competition 3.0 Multi-species test (Innovative Diagnostics).. This is a competitive ELISA for the detection of antibodies against the hemagglutinin H5 of the Influenza A virus. This test utilizes whole virus. The known sensitivity is 99.6% and specificity is 97.6% for this test (Hochmanl, Orie, et. al.). Results were given a numerical number of S/N (sample to negative ratio). Any samples reading less than 0.4 were called positive for the H5 antibody. The samples from 0.41-0.59 are labeled suspect positive. 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. Neutralizing antibody endpoint titers were read under a fluorescence microscope and expressed as the reciprocal of the highest dilution of serum capable of a 90% endpoint reduction in fluorescent foci relative to controls. Note that negative and positive control sera were included in the assay.

In conjunction with University of Minnesota Veterinary College the milking cows of the herd were sampled to further develop research in lasting antibodies of H5N1 in affected milking herds S. Wells (personal communication, November 11, 2025). The methods of their study were to collect blood samples from cows with and without history of clinical signs of illness during the 2024 outbreak of illness. Samples were taken from the tail vein with a red-top tube and vacutainer. A new needle was used between each cow. They ran their samples at Minnesota VDL to detect antibody using IDEXX ELISA Influenza A antibody test. A total of 200 samples were taken, 100 being from clinically affected cows and 100 being from clinically unaffected cows. The results showed a seroprevalence of 36% 15 months post H5N1 positive status. Of these results 53% of clinically affected cows were still seropositive and 23% of cows with no clinical signs were positive. In their comparison to the dairy in this study vs all herds tested, results were repeatable. When comparing heifer H5 antibody results with UMN cow antibody results there was no correlation between the dam antibodies status and the offspring’s antibody status.

Results:

Group #1 heifers were born 3-4 months before the clinical outbreak and moved away from the lactating herd to the heifer site 1 month before the outbreak. They had 6.4% of the heifers seropositive and another 6.4% of the heifers suspect seropositive 20 months after the outbreak. These positive and negative replacement heifers were repeatedly tested as they moved back into the herd with the dry cows and lactating cows and the seronegative replacement heifers remained negative. The seropositive heifers remained positive over five months of testing. Repeated measures were conducted on the 22 heifers sampled at every interval (October, November, January, and February) to determine if the change in antibody levels was non-random. 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 is no evidence supporting new infections, 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. The absence of new infections is indicative that the Group #1 heifers had one single viral introduction prior to leaving the outdoor heifer site. Group #2 heifers were born during the outbreak from clinically affected cows, housed with affected clinical cows, and fed pooled pasteurized colostrum from clinical cows. One out of 50 heifers were seropositive 15 months after the outbreak. Group #3 heifers were born to clinical dams 4 months after the outbreak. They were all seronegative 15 months after the outbreak.
The SVN test was run on all heifers that showed a positive, or suspect positive, result on the cELISA test. The SVN results indicated positive serum neutralization when it had a log-transformed titter greater than 1. 92% of the VN-positives were cELISA-positive. 75% of the VN-negatives were cELISA-negative. 73% of the ELISA-positives were VN-positive. 92% of the ELISA-negatives were VN-negative. When graphed to compare the correlation between H5 antibody and ability to neutralize the virus, an upward trend was noted. The Pearson correlation was used to measure the strength and direction of this data set; the r value of the correlation is 0.766.

Significance:

Of the three different cohorts studied, Group #1 was the only group that showed significant serum antibodies to HPAI 15 months after the outbreak. While the time and route of exposure to HPAI cannot be determined, it is noteworthy that this group was not in contact with clinical cows, their milk, or their colostrum during the dairy herd’s confirmed outbreak. This suggests that further studies are needed to understand the transmission route of HPAI to replacement heifers. A trend is observed as the H5 antibody increases there is a correlation between the ability to neutralize, it is likely to assume exposure to HPAI H5N1 leads to protective antibodies. Also, given the lack of serum antibodies in replacement heifers born during and after an outbreak, it is reasonable to consider this population might be at risk of infection if exposure to HPAI would occur in the future. Finally, reintroduction of naive heifers into a previously infected milking herd does not show serologic evidence of circulating virus within the herd 16-20 months after the initial outbreak. With comparison of the heifer antibody levels over time, it is reasonable to conclude H5N1 a has lasting seroprevalence in affected groups.