Submitted to: Aerobiologia
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/13/2009
Publication Date: 12/1/2009
Citation: Dungan, R.S., Leytem, A.B. 2009. The Effect of Extraction, Storage, and Analysis Techniques on the Measurement of Airborne Endotoxin From a Large Dairy. Aerobiologia. 25:265-273. Interpretive Summary: Endotoxins, which are a major outer membrane constituent of Gram-negative bacteria, are a potent inducer of inflammatory reactions in the respiratory tract when inhaled. High concentrations of airborne endotoxin generated at concentrated animal feeding operations (CAFOs) can be a concern from both a human and animal health perspective. While there are methods available to measure endotoxin concentrations, these methods have not been validated for use in this situation and have knowledge gaps with respect to the extraction, storage, and analysis of airborne endotoxin concentrations measured from CAFOs. We utilized polycarbonate filters to collect total airborne endotoxins, sonication as the extraction technique, and Tween 20 (polysorbate surfactant) in pyrogen-free water (PFW) as the extraction solution. Endotoxin concentrations were determined via a chromogenic method known as the Limulus amebocyte lysate (LAL) assay. Based on the results from our study, we recommend that airborne endotoxins be extracted from polycarbonate filters with PFW-Tween and sonicated for no longer than 30 min in glass or polypropylene tubes to avoid deactivating the endotoxin. After sonication, immediate vortexing of the extracts for up to 15 min had no effect on the concentration. The extracts can also be subjected to multiple freeze-thaw cycles, with little or no effect on the final endotoxin concentration. In order to avoid overestimation of endotoxin concentrations due to the quick reaction time of the LAL assay, it is also recommended to simultaneously add the LAL reagent using a 96-chanel pipette and immediately place the microplate in a plate reader.
Technical Abstract: The objective of this study was to fill in additional knowledge gaps with respect to the extraction, storage, and analysis of airborne endotoxin, with a specific focus on samples from a dairy production facility. We utilized polycarbonate filters to collect total airborne endotoxins, sonication as the extraction technique, and 0.05% Tween 20 in pyrogen-free water (PFW) as the extraction solution. Endotoxin concentrations were determined via the Limulus amebocyte lysate (LAL) assay. The endotoxin concentrations in extracts after 15 and 30 min of filter sonication were similar, while the concentration in 60 min extracts was about 2-fold lower. Rapidly vortexing samples for up to 15 min after sonication did not increase the endotoxin concentration. However, concentrations were 13% and 26% lower in extracts that were centrifuged at 1,000 and 10,000 x g for up to 15 min, respectively. Field samples and endotoxin standard were also sonicated in glass or polypropylene tubes for up to 120 min. Regardless of the extraction vessel, a decrease in endotoxin concentration occurred when sonicated for >30 min. Samples and endotoxin standard subjected to 12 freeze-thaw cycles at -20C only showed a slight but not significant decrease in endotoxin concentration.