Inactivation of virulent Brucella species in culture and animal samples

Authors: Olsen, Steven; Boggiatto, Paola; Vrentas, Catherine

Submitted to: Applied Biosafety
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2017
Publication Date: 11/1/2017
Citation: Olsen, S.C., Boggiatto, P.M., Vrentas, C.E. 2017. Inactivation of virulent Brucella species in culture and animal samples. Applied Biosafety. 22(4):145-151. https://doi.org/10.1177/1535676017734202.
DOI: https://doi.org/10.1177/1535676017734202

Interpretive Summary: Bacteria in the genus Brucella causes reproductive losses in animals and clinical disease in humans. Their characteristics have led them to be considered as potential bioweapons and therefore they are regulated under the Select Agent Act. Recently, Select Agent regulation has emphasized the need to validate inactivation procedures for Select Agents to ensure that live agents are not being improperly removed from appropriate containment areas. In the current study, we characterized the ability of various inactivation procedures to kill high concentrations of Brucella bacteria. The most variability was found between temperatures when using heat inactivation. This work will be of interest to regulatory personnel, research personnel, and diagnostic laboratories that have interests in work involving virulent species of Brucella.

Technical 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. is available within the historical literature, reports are primarily limited to heat inactivation of milk, and there are discrepancies between 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 from tissue sections that had high levels of colonization by 4 hours. Methods using methanol or methanol:acetone at 50 to 70% concentrations inactivated viable Brucella from liquid samples within 3 to 5 days . After passage through a 0.22 um 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.