Validation of a binary ethylenimine (BEI) inactivation procedure for biosafety treatment of foot-and-mouth disease viruses (FMDV), vesicular stomatitis viruses (VSV), and swine vesicular disease virus (SVDV).

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV.

Transport of coliphage in the presence and absence of manure suspension.

Mechanisms of coliphage transport and fate in the presence and absence of manure suspension were studied in saturated column experiments. In the presence of manure suspension, little inactivation of indigenous somatic coliphage occurred and the transport was controlled by deposition. The deposition followed a power law distribution with depth, and the magnitude increased with decreasing sand size. Comparison of the cumulative size distribution of manure components in the suspension initially and after passage through sand, suggested that particles retained by mechanical filtration and/or straining decreased the effective pore size and potentially induced straining of the somatic coliphage. A 2-site kinetic deposition model was used to estimate the magnitudes of attachment and straining in the presence of manure suspension, and provided a good description of the data. Modeling results indicated that straining accounted for 16 to 42% of the deposited somatic coliphage, and that both straining and attachment increased with decreasing sand size due to smaller pores and higher surface area, respectively. In the absence of manure suspension, phiX174 (a representative somatic coliphage) and MS2 (a male-specific RNA coliphage) transport was controlled by inactivation induced by the solid phase. This conclusion was based on comparison of coliphage transport behavior at 5 and 20 degrees C, mass balance information, and numerical modeling. Comparison of somatic coliphage transport data in the presence and absence of manure suspension revealed much higher effluent concentrations in the presence of manure. This difference was attributed to lower inactivation and higher detachment rates. The observed coliphage transport behavior suggests that survival of viruses may be extended in the presence of manure suspensions, and that transport studies conducted in the absence of manure suspension may not accurately characterize the transport potential of viruses in manure-contaminated environments.

Transport of giardia and manure suspensions in saturated porous media.

Experiments were conducted to elucidate the transport behavior of cysts of Giardia and manure suspensions through several aquifer sands. Decreasing the median grain size of the sand resulted in lower peak effluent concentrations and increased deposition of the Giardia and manure particles in the sand near the column inlet. The effluent concentration curves for the manure suspensions also exhibited asymmetric shapes that tended to include larger particle sizes as the manure suspension was continuously added. Simulations of the transport of Giardia and manure particles using a simple and flexible power law model for the solid-water mass exchange term provided a satisfactory description of the effluent and spatial distribution data. The cumulative size distribution (CSD) of manure particles in the suspension initially and after passage through the packed columns was used to identify the mechanisms that were controlling the deposition of manure particles and Giardia. The CSD data indicated that manure particles were completely removed at early times by mechanical filtration and/or straining when the ratio of the particle to the median grain diameter was greater than 0.003 to 0.017. However, the CSD changed with increasing time due to deposition-induced filling of straining sites. The Giardia transport was controlled by straining. For a given sand, higher effluent concentrations of Giardia were observed in the presence than in the absence of manure suspension. The relative increase of Giardia in the effluent concentrations varied from 75 to 172%. Hence, pathogen transport studies conducted in the absence of manure suspension may underestimate transport potential in manure-contaminated environments.