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
  • 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).

    Reference(s):
    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.

    Reference(s):
    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.

    Return to top


BRUCELLA ABORTUS

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 Unpublished data showed that the strain retained sensitivity to ampicillin, doxycycline, gentamicin, kanamycin, rifampicin, chloramphenicol, and streptomycin.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

    Reference(s):
    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.

    Reference(s):
    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.

  • Return to top


BURKHOLDERIA MALLEI

BURKHOLDERIA PSEUDOMALLEI
  • Burkholderia pseudomallei strain PBK001 (ΔtonB Δhcp1) (effective 01-14-2020)
    Burkholderia pseudomallei (Bp)PBK001 strain contains a deletion of the tonBgene, which encodes a periplasmic protein required for siderophore-mediated iron uptake. Iron uptake is important for virulence and establishing an infection in the host. The strain also contains a deletion in the hcp1 gene, which is a component of the type six secretion system utilized by the bacteria for cell-to-cell spread and dissemination. Mice (n=15) were administered a single intranasal dose of 1.5x105 CFU of the Bp PBK001 strain (equivalent to 100 LD50 of wild-type Bp K96243) and the survival was monitored for 28 days. The mice exhibited 100% survival and the bacteria disappeared from the lungs within 2 days of the infection. No bacteria were detected in any of the other target organs (liver and spleen). No symptoms of overt infection (ruffled fur, hunched posture, weight loss, etc.) were observed in these animals.

    Reference(s):

    1. Khakhum, N., et al., Burkholderia pseudomallei ΔtonB Δhcp1 Live Attenuated Vaccine Strain Elicits Full Protective Immunity against Aerosolized Melioidosis Infection. mSphere, 2019. 4: p. pii: e00570-18.
    2. Hatcher CL, Mott TM, Muruato LA, Sbrana E, Torres AG.. Burkholderia mallei CLH001 Attenuated Vaccine Strain Is Immunogenic and Protects against Acute Respiratory Glanders. Infect Immun, 2016. 84: p. 2345-2354.
    3. Khakhum, N., et al., Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis. PLoS Negl Trop Dis, 2019. 13: p. e0007578.
    4. Mott TM, Vijayakumar S, Sbrana E, Endsley JJ, Torres AG. Characterization of the Burkholderia mallei tonB Mutant and its potential as a backbone strain for vaccine development. PLoS Negl Trop Dis 2015. 9: p. e0003863.
    5. Burtnick, M.N., et al., The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei. Infect Immun, 2011. 79: p. 1512-1525.
    6. Wiersinga, W., et al., Melioidosis. Nat Rev Dis Primers, 2018. 4: p. 17107.
    7. Massey, S., et al., Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection. Sci Rep, 2014. 4: p. 4305.




  • Burkholderia pseudomallei Δasd strains (effective 12-13-2017)

    B. pseudomallei Δasd strains
    DL2 Δasd
    DL25 Δasd
    DL28 Δasd
    MSHR503 Δasd
    NAU44A6 Δasd
    MSHR840 Δasd
    MSHR1655 Δasd
    MSHR87 Δasd
    MSHR367b Δasd

    The asd gene, encoding the aspartate semi-aldehyde dehydrogenase gene, is deleted in all B. pseudomallei strains listed in the table above. The asd mutants lack the ability to cross-link their peptidoglycan cell-wall and, in the absence of diaminopimelic acid (DAP), will die due to osmotic stress. DAP is a bacterial specific metabolite that is not made in hosts including mammals; therefore, these mutants are completely attenuated in vivo and are unable to replicate within the host.

    Reference(s):

    1. Norris MH, Propst KL, Kang Y, Dow SW, Schweizer HP, Hoang TT. The Burkholderia pseudomallei Δ asd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice. Infect Immun. 2011 Oct; 79(10):4010-8.
    2. Norris MH, Kang Y, Lu D, Wilcox BA, Hoang TT. Glyphosate resistance as a novel select-agent-compliant, non-antibiotic-selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei. Appl Environ Microbiol. 2009 Oct; 75(19):6062-75.




  • Burkholderia pseudomallei strain 576mn (effective August 18, 2017)
    B. pseudomallei strain 576mn is a ∆purM derivative of the wild-type strain 576a. Studies demonstrated that strain 576mn was auxotrophic for adenine in minimal media, unable to replicate in human cells, significantly attenuated in BALB/C mice studies following high-dose intranasal inoculation (100% animal survival), and significantly attenuated compared to wild-type B. pseudomallei 576a strain.

    Reference(s):

    1. Norris MH, Rahman Khan MS, Schweizer HP, Tuanyok A. An avirulent Burkholderia pseudomallei ∆purM strain with atypical type B LPS: expansion of the toolkit for biosafe studies of melioidosis BMC Microbiol. 2017 Jun 7;17(1):132. doi: 10.1186/s12866-017-1040-4.




  • Burkholderia pseudomalleis capsular polysaccharide mutant strain, JW270 (effective July 2, 2014)
    The JW270 mutant contains a deletion of the capsule biosynthetic cluster (30.8 kb), a virulence determinant characterized in B. pseudomallei. Data from survival analysis and blood culture studies indicate significant attenuation (~4.46 log reduction) in hamster and murine models relative to wild-type B. pseudomallei strain DD503 (data not published).

    Reference(s):
    1. Atkins T, Prior R, Mack K, Russell P, Nelson M, Prior J, Ellis J, Oyston PC, Dougan G, Titball RW. Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J Med Microbiol. 2002 Jul;51(7):539-47.
    2. Burtnick M, Bolton A, Brett P, Watanabe D, Woods D. Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis. Microbiology. 2001 Jan;147(Pt 1):111-20.
    3. Reckseidler SL, DeShazer D, Sokol PA, Woods DE. Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Infect Immun. 2001 Jan;69(1):34-44.
    4. Reckseidler-Zenteno SL, DeVinney R, Woods DE. The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in serum by reducing complement factor C3b deposition. Infect Immun. 2005 Feb;73(2):1106-15.
    5. Reckseidler-Zenteno SL, Viteri DF, Moore R, Wong E, Tuanyok A, Woods DE. Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei. J Med Microbiol. 2010 Dec;59(Pt 12):1403-14. doi: 10.1099/jmm.0.022202-0. Epub 2010 Aug 19.
    6. Sarkar-Tyson M, Thwaite JE, Harding SV, Smither SJ, Oyston PC, Atkins TP, Titball RW. Polysaccharides and virulence of Burkholderia pseudomallei. J Med Microbiol. 2007 Aug;56(Pt 8):1005-10.
    7. Warawa JM, Long D, Rosenke R, Gardner D, Gherardini FC. Role for the Burkholderia pseudomallei capsular polysaccharide encoded by the wcb operon in acute disseminated melioidosis. Infect Immun. 2009 Dec;77(12):5252-61. doi: 10.1128/IAI.00824-09. Epub 2009 Sep 14.
    8. Warawa JM, Long D, Rosenke R, Gardner D, Gherardini FC. Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the burkholderia pseudomallei capsule mutant. Front Microbiol. 2011 Jun 16;2:133. doi: 10.3389/fmicb.2011.00133. eCollection 2011.




  • Burkholderia pseudomalleis strain B0011, a Δasd mutant of B. pseudomallei strain 1026b (effective 12-07-2011)
    This strain contains a deletion in the aspartate-B-semialdehyde dehydrogenase ( asd ) gene, which is auxotrophic for diaminopimelate (DAP). The Δ asd mutant was found to be avirulent in mice and unable to replicate in HeLa or RAW 264.7 cells.

    Reference(s):
    1. Norris MH, Propst KL, Kang Y, Dow SW, Schweizer HP, Hoang TT. The Burkholderia pseudomallei Δ asd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice. Infect Immun. 2011 Oct; 79(10):4010-8.
    2. Norris MH, Kang Y, Lu D, Wilcox BA, Hoang TT. Glyphosate resistance as a novel select-agent-compliant, non-antibiotic-selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei. Appl Environ Microbiol. 2009 Oct; 75(19):6062-75.




  • Burkholderia pseudomallei strain Bp82, a ΔpurM mutant of B. pseudomallei strain 1026b deficient in purine biosynthesis (effective 04-14-2010)
    The B. pseudomallei ΔpurM mutant was shown to be fully attenuated in hyper susceptible animal models, including Syrian hamsters and 129/SvEv mice when infected via the inhalational challenge route. The mutant strain also failed to cause mortality in immune deficient mice. The mutant strain failed to replicate in vivo or disseminate following intranasal challenge. The attenuation of the strain was due to the ΔpurM defect since complementation of the Bp82 ΔpurM allele with wild-type sequence resulted in adenine prototrophy and restored virulence.

    Reference(s):
    1. Propst KL, Mima T, Choi KH , Dow SW, Schweizer HP. A Burkholderia pseudomallei ΔpurM mutant is avirulent in immunocompetent and immunodeficient animals: candidate strain for exclusion from select-agent lists. Infect Immun. 2010 Jul; 78(7):3136-43.

  • Return to top


RIFT VALLEY FEVER VIRUS

VENEZUELAN EQUINE ENCEPHALITIS