Forty-eight midlactation Holstein cows were used to evaluate dietary treatments arranged as a 4 x 2 x 2 factorial: .26, .38, .48, or .60% Mg, .24 or .62% Na, and 1.14 or 1.59% K. Supplemental Mg, K, and Na were supplied by feed-grade magnesium phosphate, potassium bicarbonate, or sodium chloride. All dietary treatments were equal in Ca and P. There were no effects of dietary Na or K on feed intake or milk production. Feed intakes were equal with .26, .38, and .48% Mg but declined 4.9% with .60% Mg. Milk yields responded curvilinearly to dietary Mg. Similarly, 4% FCM yields responded curvilinearly, increasing 7% with .48% Mg compared with .26% Mg then declining with .60% Mg. Milk fat percentages were unaffected by dietary Mg concentration, but milk fat yields responded curvilinearly. Milk protein percentages declined linearly as dietary Mg increased. Plasma Mg concentrations increased linearly from 2.52 to 2.68 mg/dl as dietary Mg increased. Renal fractional excretions of Ca responded curvilinearly as dietary Mg increased and decreased as dietary K increased. Results of this experiment suggested that current recommendations for dietary Mg do not maximize lactational performance. A companion laboratory experiment showed that feed-grade magnesium phosphate had less alkalizing capacity than two MgO sources.

OBJECTIVES/GOALS: In vitro models that mimic the human respiratory system are needed to assess the toxicity of inhaled contaminants. Therefore, our goal is to establish a Dosimetric Aerosol in-Vitro Inhalation Device (DAVID) that delivers aerosols in different patterns to human lung cells cultured at an air-liquid interface (ALI). METHODS/STUDY POPULATION: The collection unit of DAVID was modified in this study to accommodate different deposition patterns (spots, annular ring, rectangle & circle). CuO aerosols of varying concentrations were generated using a 6-jet Collison nebulizer for varying time periods to achieve different doses. To quantify the doses that were delivered to cells, the samples were digested with nitric acid & analyzed by Inductively Coupled Plasma-Optical Emission Spectrometry. Following the exposure of A549 cells to CuO aerosols, cytotoxicity and mRNA expression (i.e., HMOX1 & IL-8) will be assessed via LDH and RT-qPCR to determine the effect of regional (mass deposited/area of the pattern) and global (mass deposited/area of the cell culture insert) doses in cells. RESULTS/ANTICIPATED RESULTS: The deposition areas covered by rectangular, spot, annular ring, and circular patterns are estimated to be 6, 17, 27 and 85% of the insert’s surface area, onto which cells are cultured. Results for the patterns tested (spots and annular ring) show that both the regional and global doses were greater for spots than annular ring. Also, the regional doses were higher than global doses. Irrespective of the patterns, the global doses were the same for nebulizer suspensions of 0.1-1 mg/mL. Statistical analysis by ANOVA revealed there was no significant difference in doses between replicate inserts used in the same trial. We anticipate that regional doses with aerosol deposition to a larger surface area of the cell culture insert will correspond with higher cytotoxicity and mRNA expression of HMOX1 and IL-8 in cells. DISCUSSION/SIGNIFICANCE: There are limited in vitro exposure systems that can efficiently deliver aerosols to lung cells, while also mimicking inhalation by humans. In addition to addressing this knowledge gap, we will show the role of regional & global doses in studying cellular response & the ability of DAVID to deliver aerosols in different deposition patterns.

Wastewater-based epidemiology (WBE) has been utilized for outbreak monitoring and response efforts in university settings during the coronavirus disease 2019 (COVID-19) pandemic. However, few studies examined the impact of university policies on the effectiveness of WBE to identify cases and mitigate transmission. The objective of this study was to retrospectively assess relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wastewater outcomes and COVID-19 cases in residential buildings of a large university campus across two academic semesters (August 2020–May 2021) under different COVID-19 mitigation policies. Clinical case surveillance data of student residents were obtained from the university COVID-19 response program. We collected and processed building-level wastewater for detection and quantification of SARS-CoV-2 RNA by RT-qPCR. The odds of obtaining a positive wastewater sample increased with COVID-19 clinical cases in the fall semester (OR = 1.50, P value = 0.02), with higher odds in the spring semester (OR = 2.63, P value < 0.0001). We observed linear associations between SARS-CoV-2 wastewater concentrations and COVID-19 clinical cases (parameter estimate = 1.2, P value = 0.006). Our study demonstrated the effectiveness of WBE in the university setting, though it may be limited under different COVID-19 mitigation policies. As a complementary surveillance tool, WBE should be accompanied by robust administrative and clinical testing efforts for the COVID-19 pandemic response.