Select Agents and Toxins Exclusions

Attenuated strains of Overlap Select Agents excluded from the requirements of 9 CFR Part 121 and 42 CFR part 73:

  • Bacillus anthracis strains devoid of both plasmids pX01 and pX02. (effective 2-27-2003)
    Bacillus anthracis strains devoid of both virulence plasmids pX01 and pX02 are excluded based on published studies evaluating the attenuation of strains containing different combinations of the two plasmids).

    1. Hambleton P, Carman JA, Melling J. Anthrax: the disease in relation to vaccines. Vaccine. 1984 Jun; 2 (2):125-32.
    2. Shlyakhov EN , Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine. 1994 Jun; 12(8):727-30.
    3. Sterne M. Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind. 1946 Mar; 21:41-3.

  • Bacillus anthracis strains devoid of the plasmid pX02 (e.g., Bacillus anthracis Sterne, pX01+ pX02 -). (effective 2-27-2003)
    Bacillus anthracis strains lacking the virulence plasmid pX02 (e.g., Sterne, pX01+ and pX02 -) indicate that these strains were 105 to 107 fold less virulent than isogenic strains with both plasmids. These strains have been used to vaccinate both humans and animals.

    1. Hambleton P, Camman JA, Melling J. Anthrax: The disease in relation to vaccines. Vaccine. 1984 Jun; 2(2):125-32.
    2. Shlyakhov EN, Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine. 1994 Jun; 12(8):727-30.
    3. Sterne M. Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind. 1946 Mar; 21:41-3.

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  • ∆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 Unpublished data showed that the strain retained sensitivity to ampicillin, doxycycline, gentamicin, kanamycin, rifampicin, chloramphenicol, and streptomycin.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.

    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.

  • Brucella melitensis strain 16M∆vjbR (effective December 22, 2014)
    This vaccine candidate strain contains a deletion of the vjbR locus (BMEII1116) from B. melitensis 16M (ATCC#23456).  Restoration of virulence in the presence of complementing plasmid confirmed that this genetic defect is solely responsible for the reduction in virulence. In contrast to the parental virulent organism, the 16MΔvjbR knockout mutant fails to induce symptoms associated with disease in nonhuman primates, the 16MΔvjbR knockout mutant fails to cause death in immune-deficient mice, and the mutant fails to cause reticulo-endothelial symptoms (hepato- and splenomegaly) observed with both virulent organisms and currently available vaccine strains (data unpublished). The threat to humans associated with animal abortion and/or sustained secretion of the vaccine candidate from the mammary gland of animals appears to be greatly reduced based on attenuated virulence observed in several model systems. Finally, the attenuated phenotype in primates clearly demonstrates enhanced safety of the vaccine candidate reducing the risk of disease despite the potential consequence of transmission.

    1. Arenas-Gamboa AM, Ficht TA, Kahl-McDonagh MM, & Rice-Ficht AC (2008) Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge. Infect. Immun. 76(6):2448-2455
    2. Arenas-Gamboa AM, Ficht TA, Kahl-McDonagh MM, Gomez G, & Rice-Ficht AC (2009) The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle. Infect. Immun. 77(2):877-884
    3. Mense MG, Borschel RH, Wilhelmsen CL, Pitt ML, Hoover DL. Pathologic changes associated with brucellosis experimentally induced by aerosol exposure in rhesus macaques (Macaca mulatta). Am J Vet Res. 2004;65(5):644-52. PubMed PMID:15141886.
    4. Rajashekara G, et al. (2005) Unraveling Brucella genomics and pathogenesis in immunocompromised IRF-1-/-mice. Am. J. Reprod. Immunol. 54(6):358-368.
    5. Rambow-Larsen AA, Rajashekara G, Petersen E, Splitter G. Putative quorum-sensing regulator BlxR of Brucella melitensis regulates virulence factors including the type IV secretion system and flagella. Journal of bacteriology.2008;190(9):3274-82. PubMed Central PMCID: PMC2347389.
    6. Sprynski N, Felix C, O'Callaghan D, Vergunst AC. Restoring virulence to mutants lacking subunits of multiprotein machines: functional complementation of a Brucella virB5 mutant. FEBS open bio. 2012;2:71-5. PubMed Central PMCID: PMC3642115.
    7. Weeks JN, Galindo CL, Drake KL, Adams GL, Garner HR, Ficht TA. Brucella melitensis VjbR and C12-HSL regulons: contributions of the N-dodecanoyl homoserine lactone signaling molecule and LuxR homologue VjbR to gene expression. BMC microbiology. 2010;0:167. PubMed Central PMCID: PMC2898763.
    8. Yingst SL, Huzella LM, Chuvala L, Wolcott M. A rhesus macaque (Macaca mulatta) model of aerosol-exposure brucellosis (Brucella suis): pathology and diagnostic implications. J Med Microbiol. 2010;59(Pt 6):724-30. Epub 2010/03/13. doi: jmm.0.017285-0 [pii] 10.1099/jmm.0.017285-0. PubMed PMID: 20223898.

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