Performing Department
(N/A)
Non Technical Summary
This project aims to understand how the stage of reproduction in dairy cows affects their vulnerability to avian influenza H5N1 (bird flu).While bird flu is typically associated with poultry and wild birds, recent field reports and experimental studies have shown that it can infect the udders of lactating dairy cows. However, it is not yet known whether the mammary glands of other groups on the dairy farm--such as pregnant heifers, non-pregnant heifers, or pregnant lactating cows--are also susceptible. In this study, we are focusing on the milk-producing epithelial cells to determine whether their susceptibility to infection changes depending on the cow's reproductive status: pregnant, lactating, both, or neither. In addition, we will study whether glycans (also known as sugars) naturally present in milk or colostrum (the first milk after giving birth) can help protect these cells from infection. To do this, we will grow mammary gland cells in the lab, infect them with the virus, and test how the virus grows in the presence or absence of milk sugars. By studying how milk and pregnancy-related changes in the dairy cow influence infection risk, this research will help us better understand how viruses may spread in dairy herds. These findings will be shared with veterinarians, dairy farmers, and scientists to support efforts in animal health, food safety, and disease prevention.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The primary objective of this project is to investigate the susceptibility of bovine mammary epithelial cellsto H5N1 clade 2.3.4.4b influenza virus across different reproductive life stages and to evaluate the potential protective effects of mammary gland secretions against infection.
Project Methods
Methods for Specific Aim 1: Assess BMEC Susceptibility to H5N1 Across Reproductive Life StagesTo assess the susceptibility of bovine mammary epithelial cells (BMECs) to H5N1 clade 2.3.4.4b, we will collect mammary gland biopsies from four groups of animals (n=6 per group): pregnant heifers, nonpregnant heifers, pregnant lactating cows, and nonpregnant lactating cows. Each biopsy will be divided for both cell culture and histological analysis. For histology, tissues will be fixed in formalin, stored in ethanol, embedded in paraffin, and sectioned for staining. For cell culture, tissues will be enzymatically digested using collagenase and DNase I, filtered, and cryopreserved.Primary BMECs will be thawed and cultured in growth medium at 37°C with 5% CO2. Once cultures reach 70-80% confluence, they will be infected with H5N1 clade 2.3.4.4b at multiplicities of infection (MOIs) of 0.05, 0.1, and 0.5. Samples will be collected at 3, 24, 48, 72, and 96 hours post-infection for downstream analysis.Viral replication will be assessed through multiple approaches. Quantitative PCR (qPCR) will be used to measure viral RNA levels using clade-specific primers and probes. Infectious virus titers will be determined using the tissue culture infectious dose 50% (TCID50)assay in MDCK cells, with cytopathic effect (CPE) used as the readout. Immunohistochemistry (IHC) and immunofluorescence will be used to detect viral antigens and sialic acid receptor expression in tissues and cells.Sialic acid receptor profiling will be performed using lectin histochemistry. Fluorescently-labeled plant-derived lectins--SNA (for α2,6-linked sialic acids), MAL-I, and MAL-II (for α2,3 linkages)--will be used to stain sections and cultured cells. Confocal microscopy will be used for imaging, and signal intensity will be quantified using ImageJ software.To evaluate host gene expression, infected and uninfected BMECs will be lysed for RNA extraction. Total RNA will be used for library preparation and bulk RNA sequencing. Differential gene expression analysis will focus on genes involved in viral susceptibility, including sialidases.Methods for Specific Aim 2: Evaluate the Protective Effects of Mammary SecretionsTo investigate whether mammary secretions protect against H5N1 infection, milk or colostrum will be collected from pregnant heifers, pregnant lactating cows, and nonpregnant lactating cows (n=10 per group). After disinfecting teats, samples will be manually expressed from all four udder quarters and collected into sterile containers.Sialic acid content in the secretions will be analyzed using high-performance liquid chromatography (HPLC). Samples will be centrifuged to remove fat and proteins, and the clarified supernatant will be injected into the HPLC system. Fluorescence detection will be used to quantify individual sialic acid forms.BMEC cultures will be pretreated with mammary secretions for 24 hours prior to infection with H5N1 at the MOI determined in Aim 1. After infection, viral replication will be assessed at multiple time points using qPCR, TCID50assays, and observation of CPE. Protective efficacy will be determined by comparing infection metrics in treated versus untreated cultures.Data Analysis and Statistical MethodsAll in vitro experiments will include three technical replicates per condition. Viral RNA levels, infectious titers (TCID50), and sialic acid concentrations will be analyzed using one-way ANOVA with Tukey's post-hoc tests to compare differences between groups. Categorical outcomes such as CPE scoring will be analyzed using chi-square or Fisher's exact tests. Bulk RNA-seq data will be analyzed using the DESeq2 package, with false discovery rate (FDR) correction applied to control for multiple testing. All statistical analyses will be conducted using Python, and results will be visualized using GraphPad PRISM.