Inactivation of VirulentBrucellaSpecies in Culture and Animal Samples

Abstract

In response to several publicized failures of inactivation procedures, the Select Agent program in the United States has recently focused on validation or documentation of inactivation methods appropriate for biological select agents and toxins covered under the regulations. Although some data on inactivation of Brucella spp are available within the historical literature, reports are primarily limited to heat inactivation of milk, and there are discrepancies among studies in time required to eliminate all viable bacteria. In our experiment, complete elimination of viable Brucella bacteria (B abortus, B suis, and B melitensis) within 30 to 60 minutes required temperatures approaching boiling, whereas lower temperatures required much longer heating times (hours). Buffered neutral formalin (10% concentration) was highly effective in inactivating Brucella bacteria by 4 hours from tissue sections that had high levels of colonization. Methods based on methanol or methanol-acetone at 50% to 70% concentrations inactivated viable Brucella from liquid samples within 3 to 5 days. Phenolic disinfectants were also highly effective in quickly inactivating Brucella in solutions. After passage through a 0.22-mm filter, no Brucella were recovered from spiked serum samples or serum samples from experimentally infected animals. Despite demonstration of the effectiveness of these procedures in our laboratory, it is highly recommended that others validate any similar procedures used under their own in vitro conditions. It is also recommended that culture methods be used, as possible, on all inactivated samples to verify the effectiveness of the validated procedure and protect against inadvertent laboratory errors.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory—Bacillus anthracis and Brucella melitensis

“…bacteria within 4 h from tissue sections with high colonization levels. 69 This may be most relevant for conducting laboratory work, that is, prepping and examining tissue sections.…”

“…With viable Brucella bacteria ( B. abortus , B. suis , and B. melitensis ), temperatures approaching boiling allowed complete inactivation to be achieved within 30–60 min; however, lower temperatures require much longer heating times (hours). 69 Autoclaving at 121°C for at least 15 min can decontaminate tools exposed to the B. canis. 71 Autoclave parameters should be validated for different matrices and different Brucellae species.…”