Federal Select Agent Program SA-Grams

Date of issuance: 9/1/15 Title: Exclusion of Brucella melitensis and Burkholderia mallei strains Text: Based upon consultations with subject matter experts and a review of relevant published studies and information provided by the entities requesting the exclusions, the Federal Select Agent Program has determined that the following attenuated strains are not subject to the requirements of the select agent regulations. • ∆norD ∆znuA Brucella melitensis-lacZ strain (effective July 7, 2015) The ∆norD ∆znuA Brucella melitensis-lacZ strain contains the deletion of two virulence genes. The znuA gene constitutes a high-affinity periplasmic binding protein-dependent and ATP-binding cassette (ABC) transport system for Zn2+. The norD gene is a member of the norEFCBQD operon encoding a nitric oxide reductase. The strain also contains the E.coli lacZ marker gene to differentiate between the ∆norD ∆znuA Brucella melitensis-lacZ strain and wild-type B. melitensis. Unpublished data showed that the strain retained sensitivity to ampicillin, doxycycline, gentamicin, kanamycin, rifampicin, chloramphenicol, and streptomycin. The strain also failed to replicate in RAW 264.7 and bovine macrophages. The ∆norD ∆znuA Brucella melitensis-lacZ strain cleared from mice at a rate similar to the excluded ∆norD ∆znuA Brucella abortus-lacZ strain. Nasal vaccination of BALB/c mice with 1 x109 CFUs of ∆norD ∆znuA Brucella melitensis-lacZ was protective against pulmonary challenge with wild-type B. melitensis 16M. References 1. Clapp B, Skyberg JA, Yang X, Thornburg T, Walters N, Pascual DW. Protective live oral brucellosis vaccines stimulate Th1 and th17 cell responses. Infect Immun. 2011 Oct;79(10):4165-74. doi: 10.1128/IAI.05080-11. Epub 2011 Jul 18 2. Lewis DA, Klesney-Tait J, Lumbley SR, Ward CK, Latimer JL, Ison CA, Hansen EJ. Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi. Infect Immun. 1999 Oct;67(10):5060-8. 3. Beard SJ, Hashim R, Wu G, Binet MR, Hughes MN, Poole RK. Evidence for the transport of zinc(II) ions via the pit inorganic phosphate transport system in Escherichia coli. FEMS Microbiol Lett. 2000 Mar 15;184(2):231-5. 4. Kim S, Watanabe K, Shirahata T, Watarai M. Zinc uptake system (znuA locus) of Brucella abortus is essential for intracellular survival and virulence in mice. J Vet Med Sci. 2004 Sep;66(9):1059-63. 5. Loisel-Meyer S, Jiménez de Bagüés MP, Bassères E, Dornand J, Köhler S, Liautard JP, Jubier-Maurin V. Requirement of norD for Brucella suis virulence in a murine model of in vitro and in vivo infection. Infect Immun. 2006 Mar;74(3):1973-6.

• Burkholderia mallei strain CHL001, a ∆tonB ∆hcp1 mutant of B. mallei strain ATCC 23344 (potential vaccine candidate strain) (effective August 20, 2015) B. mallei strain CHL001 was derived from the B. mallei tonB mutant (TMM001) deficient in iron acquisition. The tonB gene encodes a periplasmic protein required for siderophore-mediated iron uptake. In addition, the strain contains a 162 base pair intragenic in-frame deletion of the hemolysin co- regulated protein-1 (hcp1) gene, which encodes for a protein required for the assembly and functionality of the type six secretion system cluster 1 (T6SS-1). The construction of a double deletion mutant eliminates the possibility of reversion to wild-type virulence. The CHL001 strain was administered to BALB/c mice via a single intranasal dose of 1.5x104 CFU or via three intranasal doses (administered bi- weekly) of either 1.5x104 or 1.5x105 CFU. The mice exhibited 100% survival (unpublished data). In addition, immunocompromised mice (NSG) were challenged with 1.5x104 CFU of CLH001. After 21 days, the immunocompromised mice did not display any overt signs of infection and exhibited 100% survival. The strain could not be recovered from the lungs, liver, or spleen.

References

1. Mott TM, et al., Characterization of the Burkho/deria ma//el tonB Mutant and its potential as a backbone strain for vaccine development. PLoS Neg/ Trop Dis 2015. [In press]. 2. Burtnick MN, DeShazer D, Nair V, Gherardini FC, Brett PJ. Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages. Infect Immun. 2010 Jan;78(1):88-99. 3. Schell MA, Ulrich RL, Ribot WJ, Brueggemann EE, Hines HB, Chen D, Lipscomb L, Kim HS, Mrázek J, Nierman WC, Deshazer D. Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol. 2007 Jun;64(6):1466-85= 4. Lever MS, Nelson M, Ireland PI, Stagg AJ, Beedham RJ, Hall GA, Knight G, Titball RW. Experimental aerogenic Burkholderia mallei (glanders) infection in the BALB/c mouse. J Med Microbiol. 2003 Dec;52(Pt 12):1109-15. 5. Massey S, Johnston K, Mott TM, Judy BM, Kvitko BH, Schweizer HP, Estes DM, Torres AG. In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model.Front Microbiol. 2011 Aug 26;2:174.

Additional information regarding the excluded strains can be found at: http://www.selectagents.gov/exclusions-overlap.html.

If you have any questions regarding this email, please contact your File Manager with the Federal Select Agent Program for APHIS/AgSAS entities, 301-851-3300 option 3 (voice only) and for CDC/DSAT entities, 404-718-2000.