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Notification of Exclusion

The regulations in 7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73 establish a procedure by which the Secretary, Department of Health and Human Services (HHS) and the Secretary, United States Department of Agriculture (USDA) may exclude attenuated strains of select agents or toxins upon a determination by the Secretary that an attenuated strain of a select agent or toxin that does not pose a severe threat to plant or animal health, plant or animal products, or public health and safety may be excluded from the list of select agents and toxins.

To apply for an exclusion of an attenuated strain of a select agent or toxin, an individual or entity must submit a written request and supporting scientific information. Refer to 9 CFR §121.4, 42 CFR §73.3(e) and §73.4(e) for details on applying for an exclusion.

Based upon consultations with subject matter experts and a review of relevant published studies and information provided by the entities requesting the exclusions, HHS Secretary and USDA Secretary have determined that the following attenuated strains are not subject to the requirements of 42 CFR Part 73 and 9 CFR Part 121. The fact that these strains are excluded from the requirements should in no way be interpreted that these agents and toxins should be handled under anything less than appropriate safety conditions. In addition, an excluded attenuated strain will be subject to the regulations if there is any reintroduction of factor(s) associated with virulence or other manipulations that modify the attenuation such that virulence is restored or enhanced.

Please note that research with excluded strains of select agents and toxins that also involve recombinant DNA may be subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines). The NIH Guidelines can be accessed at http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html. If your research is otherwise subject to the NIH Guidelines, the transfer of recombinant DNA to a Risk Group 2, 3, 4, or restricted agent (Variola major and Variola minor) falls under Section III-D of the NIH Guidelines.

Attenuated strains of HHS select agents and toxins excluded:

Attenuated strains of Overlap select agents and toxins excluded:

Compiled background list

Notifications of Exclusions for USDA-only agents are located at:
http://www.aphis.usda.gov/programs/ag_selectagent/index.html.

Compiled Background List

Attenuated strains of HHS select agents and toxins excluded
Attenuated strains of Overlap select agents and toxins excluded

Attenuated strains of HHS select agents and toxins excluded:

Coccidioides posadasii:

  1. The data demonstrates that deletion of the single copy of the class V chitin synthase using a gene replacement strategy results in a stable avirulent phenotype that lacks the ability to form infectious arthroconidia. This mutant is unable to form spherules in vivo as exemplified by its inability to survive or kill mice following intraperitoneal inoculation. Based upon consultations with subject matter experts and information provided by the requestor, HHS has determined that Coccidioides posadasii ∆chs5 mutant strain does not pose a severe threat to public health and safety.
  2. A triple gene knock-out mutant of Coccidioides posadasii ( ∆cts2/ ∆ard1/ ∆cts3) is attenuated through the deletion of two coccidioidal chitinase genes and a D-arabinitol 2-dehydrogenase gene. Unpublished data demonstrate that this mutant strain, unlike the wild-type Coccidioides posadasii, is unable to produce endospores which give rise to the next generation of spherules. These data further demonstrate that the mutant strain is stable, and that there is no possibility of spontaneous reversion to the wild-type gene. Based upon consultations with subject matter experts and information provided by the requestor, HHS has determined that Coccidioides posadasii ( ∆cts2/ ∆ard1/ ∆cts3) mutant strain does not pose a severe threat to public health and safety.

Conotoxins:

The term "conotoxin" is used broadly to comprise a very large number of polypeptides isolated from the venom of fish-hunting marine snails of the Conus genus of gastropod mollusks; many of these molecules are neurologically active in mammals [1-3]. Based upon available experimental evidence, however, the following conotoxins (i.e. conopeptides) do not possess sufficient acute toxicity to pose a significant public health threat, and many are employed as useful research tools or potential human therapeutics. Select Agent conotoxins excluded are: the class of sodium channel antagonist μ-conotoxins, including GIIIA [3]; the class of calcium channel antagonist μ-conotoxins, including GVIA, GVII, MVIIA, MVIIC, and their analogs or synthetic derivatives [3,4]; the class of NMDA-antagonist conantokins, including con-G, con-R, con-T and their analogs or synthetic derivatives [5]; and the putative neurotensin agonist, contulakin-G and its synthetic derivatives [6].

References:

  1. Olivera, B.M., Gray, W.R., Zeikus, R., McIntosh, J.M., Varga, J., Rivier, J., de Santos, V. and Cruz, L.J. (1985). Peptide neurotoxins from fish-hunting cone snails. Science 230:1338-1343.
  2. Olivera, B.M., Rivier, J., Scott, J.K., Hillyard, D.R. and Cruz, L.J. (1991). Conotoxins. J. Biol. Chem. 266:22067-22070.
  3. Olivera, B.M. and Cruz, L.J. (2001). Conotoxins, in retrospect. Toxicon 39:7-14.
  4. Yoshikami, D., Bagabaldo, Z. and Olivera, B.M. (1989). The inhibitory effects of omega-conotoxins on Ca channels and synapses. Ann. N.Y. Acad. Sci. 560:230-248.
  5. Prorok, M. and Castellino, F.J. (2001). Structure-function relationships of the NMDA receptor antagonist conantokin peptides. Curr. Drug Targets 2(3):313-322.
  6. Craig, A.G., Norberg, T., Griffin, D., Hoeger, C., Akhtar, M., Schmidt, K., Low, W., Dykert, J., Richelson, E., Navarro, V., Mazella, J., Watkins, M., Hillyard, D., Imperial, J., Cruz, L.J., and Olivera, B.M. (1999). Contulakin-G, an O-glycosylated invertebrate neurotensin. J. Biol. Chem. 274:13752-13759.

Yersinia pestis:

  1. Strains of Yersinia pestis that lack the 75 kb low-calcium response (Lcr) virulence plasmid are excluded. Strains lacking the Lcr plasmid (Lcr-) are irreversibly attenuated due to the loss of a virulence plasmid. An Lcr- strain of Yersinia pestis (Tjiwidej S) has been extensively used as a live vaccine in humans in Java. Thus, these strains pose no significant threat to public health.

Reference:

  1. Meyer, K.F. et al. (1974). Plague immunization. I. Past and present trends, J. Infect. Dis. 129 (suppl.): S13-S18.
  2. Pgm- mutants of Yersinia pestis occur at a high frequency (ca 10-5) (1) and result in avirulence and Pgm- strains such as the EV 76 strain have been used for years as live human vaccines with no significant plague-associated problems. The mutation in question is due to the excision of about 102-kb of chromosomal DNA via reciprocal recombination between adjacent IS 100 elements (2). The lost DNA sequence encodes the ability to synthesize and utilize the siderophore yersiniabactin, which is necessary for growth in mammalian peripheral tissue, as well as the Hms+ locus, which is necessary for biofilm production in the flea vector (3). However, PCR and/or Southern blot analysis will be required to ensure that "Pgm-" derivatives have undergone this deletion rather than a mutation in the hemin storage genes (hms), which also causes loss of Congo red (CR) binding, which is the most common characteristic used to evaluate the pigmentation phenotype (4).

References:

  1. Brubaker, R. R. 1969. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 98(3):1404-1406.
  2. Fetherston, J.D., P. Scheutze, and R.D. Perry. 1992. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol. Microbiol. 6(18):2693-2704.
  3. Bearden, S.W., and R.D. Perry. 1999. The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol. Microbiol. 32(2):403-414.
  4. Une, T. and R.R. Brubaker. 1984. In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes of Yersiniae. Infect. Immun. 43(3):895-900.

Attenuated strains of Overlap select agents and toxins excluded:

Bacillus anthracis:

  1. Bacillus anthracis strains that are 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.
  2. Bacillus anthracis strains lacking the virulence plasmid pX02 (e.g., Sterne pX01+ and pX02-) are excluded based on information indicating 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 and do not pose a severe threat to the public health and safety.

References:

Brucella abortus:

  1. The Brucella abortus Strain 19 live vaccine, used in the U.S. Department of Agriculture Brucellosis Eradication Program from 1941 to 1996, is effective in the control of clinical brucellosis in cattle.1 For over a decade, B. abortus Strain 19 was also used to immunize more than 8 million people in the USSR.2 While there have been occasional reports of human brucellosis caused by B. abortus Strain 19 as a result of accidental aerosolization or needle sticks, 3, 4 this strain does not pose a severe threat to human or animal health.

References:

  1. Proceedings of the United States Animal Health Association. (1989). 93:640-655.
  2. Joint FAO/WHO Expert Committee on Brucellosis. (1986). No. 740, p. 34-40.
  3. Young, E. (1983). Human brucellosis. Reviews of Infectious Diseases, 5 (5): 821-842
  1. Brucella abortus strain RB51 was conditionally licensed as a vaccine by USDA in 1996 and granted a full license in March 2003. It is used as part of the cooperative State-Federal Brucellosis Eradication Program.1 Brucella abortus strain RB51 is a genetically stable, rough morphology mutant of field strain Brucella. It lacks the polysaccharide O-side chains on the surface of the bacteria. Strain RB51 is less virulent than the Brucella abortus Strain 19 vaccine and field strain2 Brucella abortus. The RB51 strain does not pose a significant threat to human or animal health.

References:

  1. Brucellosis http://www.aphis.usda.gov/vs/nahps/brucellosis/.
  2. Schurig, G.G., Roop, R.M. II, Bagchi, T., Boyle, S., Buhrman, D., and N. Sriranganathan. (1991). Biological properties of RB51: a stable rough strain of Brucella abortus. Vet Microbiol 28(2):171-188.
  3. Stauffer, B., Reppert, J., Van Metre, D., Fingland, R., Kennedy, G., Hansen, G., Pezzino, G., Olsen, S., and D. Ewalt. (1998). Human exposure to Brucella abortus Strain RB51 - Kansas, 1997. MMWR 47(09):172-175.

Coxiella burnetii:

LPS is the only confirmed virulence factor of C. burnetii. Organisms isolated from natural infections or laboratory are in phase I and have a smooth-type LPS. Repeated passage of phase I organisms through embryonated eggs or cultured cells resulted in the conversion to phase II and a change in the LPS to a rough-type. Injection of such laboratory-derived phase II variants into guinea pigs resulted in infection and reversion to phase I. However, plaque-purified (cloned) isolates of the Nine Mile Strain phase II organisms do not undergo phase reversion and are avirulent since inoculation of susceptible animals with phase II cells does not result in infection nor can viable phase II or phase I organisms be recovered from the spleens of these animals. The Nine Mile Strain plaque purified phase II is stable and does not revert to phase I; restriction fragment-length polymorphisms detected after HaeIII digestion of chromosomal DNA and DNA-DNA hybridization, suggests that the Nine Mile Strain plaque purified phase II variant has undergone a deletion. Based upon consultations with subject matter experts and a review of relevant published studies, HHS and USDA have determined that Coxiella burnetii, Phase II, Nine Mile Strain, plaque purified clone 4, does not pose a significant threat to human or animal health.

References:

Francisella tularensis:

  1. The type strain Utah 112 of Francisella tularensis subspecies novicida (also referred to as Francisella novicida) is excluded. The exclusion is only for the type strain, Utah 112. This strain was originally isolated from a water sample taken from Ogden Bay, Utah in 1951. It is experimentally pathogenic for mice, guinea pigs and hamsters, producing lesions similar to those of tularemia; rabbits, white rats and pigeons are resistant. The Utah 112 strain is not known to infect man and thus, is not of public health concern.
  2. Francisella tularensis subspecies holartica LVS (live vaccine strain) is excluded. This and similar strains have been used to vaccinate millions of people including thousands of U.S. military personnel and laboratory workers without major problems.
  3. Francisella tularensis biovar tularensis strain ATCC 6223. This strain has fastidious growth requirements and grows poorly in the laboratory. Mice are used as a model to study the pathogenesis of tularemia (1). The LD50 of virulent strains of F. tularensis biovar tularensis for mice infected via the subcutaneous route is <10 CFU (1). However, mice infected intraperitoneally with 105 CFU or intradermally with 107 CFU of strain ATCC 6223 were not killed. Thus, strain ATCC 6223 does not pose a threat to human or animal health.

References:

  1. Ellis, J., Oyston, P.C.F., Green, M., and R.W. Titball. 2002. Tularemia. Clin. Microbiol. Rev. 15(4):631-646.

Venezuelan Equine Encephalitis (VEE):

  1. Venezuelan Equine Encephalitis (VEE) strain V3526 is an attenuated strain of VEE, which was constructed by site-directed mutagenesis. V3526 contains two mutations relative to the virulent parental clone (1). One of these mutations is a deletion, which renders the virus non-viable; the other mutation restores viability without restoring the pathogenic properties of the parental virus. The stability of the deletion mutation in V3526 fundamentally and significantly decreases the hazard associated with this strain, and makes it unlikely that it can revert to wild type. This strain is considerably less virulent than the excluded vaccine strain TC83. This strain does not pose a significant threat to human or animal health.

Reference:

  1. Davis, N.L., et al. (1995). Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined

 

Page last modified: May 2, 2007