214 Immuno-detection and stability of SARS-CoV-2 in milk from white-tailed deer

Abstract SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has expanded to species beyond humans, such as domestic and wild animals, underscoring zoonotic transmission and the urgency of epidemiological preparedness in light of spillovers. In humans, the humoral immune response to SARS-CoV-2 is initially dominated by immunoglobulin (Ig) M and later by IgG and IgA isotypes, with IgA playing a role in mucosal protection. These neutralizing antibodies have been detected in a variety of human fluids, including saliva, respiratory secretions, serum, and breast milk. It is speculated that these antibodies serve to reduce the infectivity of human secretions and viral transmission, while also acting as a form of passive immunity. Thus, we asked whether this immune-protective mechanism is present in a common animal that is both wild and farmed, the white-tailed deer (WTD). We present previously unreported evidence of the existence of neutralizing antibodies against SARS-CoV-2 in serum and milk samples from six WTD harvested during the 2022 and 2023 hunting seasons in central Virginia, USA. Identification of antibodies against SARS-CoV-2 was conducted using species-independent qualitative lateral flow latex immunochromatography from three different commercial sources, as well as enzyme-linked immunosorbent assay (ELISA). Three out of six WTD showed positive antibody signals for both serum and milk using all analytical methods. A fourth sample was positive only for serum, while another sample provided a positive result by ELISA but not lateral flow. The last sample tested negative. Breast milk from pre- and post-SARS-Cov-2-infected human volunteers was used to validate the studies. In addition, RT-qPCR targeting SARS-CoV-2 N, E, and S genes yield negative results in milk samples, further supporting previous studies in human milk that established the absence of viral particles in the fluid. Immunohistochemical analysis of WTD mammary tissue revealed a rare histiocyte positive for SARS-CoV-2, a result that more likely reflects a migration event stimulated by inflammation rather than actual infection. Lastly, in vitro stability studies were carried out in deer and human milk spiked with B.1.1, B.1.1.7, or BA.5 variants. Samples were maintained at room temperature, 37 °C, or 63 °C (pasteurization temperature) and collected at various times. The results showed that while B.1.1 and B.1.1.7 remained largely stable in human samples, they rapidly degraded in deer milk. All virus variants were eliminated from human and deer milk at 63 °C, albeit with different kinetics. These findings underscore the potential role of WTD as reservoirs for SARS-CoV-2 neutralizing antibodies, emphasizing the importance of comprehending zoonotic transmission dynamics and viral variant stability. Such insights may have significant implications for informing public health interventions and surveillance strategies.