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`International application number: PCT/US2007/082096
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`International filing date:
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`22 October 2007 (22.10.2007)
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`Document type:
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`Number:
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`Filing date:
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`United States Patent am! ‘I‘yafimmrk Office
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`Norman James
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`Assays and Devices for Identifying Pathogens
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`SIGNATURE
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`TYPED or PRINTED NAME
`JennIfer A. ZarutskIe
`
`(617) 832-1754
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`October 20, 2006
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`50.558
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`ISA-127.60
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`1. Provisional Application for Patent Cover Sheet (1 original and 1 copy;
`2 pages each);
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`Katelyn Nelson
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`ISA-127.60
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`

`

`We claim:
`
`1.
`
`A method, comprising:
`
`(a)
`
`contacting a support comprising a binding agent capable of binding a
`
`ribosome with a sample comprising ribosomes;
`
`(b)
`
`(c)
`
`washing the support after contact with the sample;
`
`incubating the support in the presence of a solution comprising mRNA
`
`and amino acids under conditions suitable to allow ribosomes bound to the binding agent
`
`to translate the mRNA in the solution into a polypeptide; and
`
`(d)
`
`determining the presence of the polypeptide.
`
`2.
`
`The method of claim 1, wherein the ribosomes are specific to a pathogen and the
`
`method further comprises determining the presence of the pathogen in the sample based
`
`on the presence of the polypeptide.
`
`3.
`
`4.
`
`The method of claim 2, wherein the pathogen is a bacterium.
`
`The method of claim 1, wherein the binding agent is capable of specifically
`
`binding a ribosome.
`
`5.
`
`The method of claim 3, wherein the ribosomes are bacterial ribosomes and the
`
`method further comprises releasing the ribosomes from at least one bacterium.
`
`6.
`
`7.
`
`8.
`
`9.
`
`The method of claim 1, wherein the support is a porous material.
`
`The method of claim 3, wherein the porous material is nitrocellulose.
`
`The method of claim 1, wherein the binding agent is an antibody.
`
`The method of claim 1, wherein the binding agent is an agent capable of binding
`
`ribosomal RNA.
`
`10.
`
`The method of claim 9, wherein the agent capable of binding ribosomal RNA is a
`
`nucleic acid.
`
`11.
`
`The method of claim 1, wherein the solution comprising mRNA and amino acids
`
`further comprises a substrate.
`
`12.
`
`The method of claim 11, wherein the polypeptide is an enzyme.
`
`-17-
`
`

`

`13.
`
`The method of claim 12, wherein determining the polypeptide comprises
`
`determining the presence of a reaction product resulting from reaction of the enzyme with
`
`a substrate for the enzyme.
`
`14.
`
`The method of claim 13, wherein determining the polypeptide comprises optically
`
`detecting the presence of the reaction product.
`
`15.
`
`A method, comprising:
`
`(a)
`
`capturing ribosomes on a support using a binding agent capable of binding
`
`the ribosomes;
`
`(b)
`
`incubating the captured ribosomes with a solution comprising mRNA and
`
`amino acids; and
`
`(c)
`
`detecting the polypeptide produced by the incubated, captured ribosomes.
`
`16.
`
`A device, comprising a support comprising a binding agent capable of binding a
`
`ribosome.
`
`17.
`
`The device of claim 16, wherein the support is a porous material.
`
`18.
`
`19.
`
`The device of claim 17, wherein the porous material is nitrocellulose.
`
`The device of claim 16, wherein the binding agent is an antibody.
`
`20.
`
`The device of claim 16, wherein the binding agent is an agent capable of binding
`
`ribosomal RNA.
`
`21.
`
`The device of claim 20, wherein the agent capable of binding ribosomal RNA is a
`
`nucleic acid.
`
`-18-
`
`

`

`ASSA YS AND DEVICES FOR IDENTIFYING PA THOGENS
`
`ISA-127.60
`
`BACKGROUND OF THE INVENTION
`
`[0001] Detection of the bacteria that have infected a subject, including metabolites,
`
`nucleic acids, and proteins thereof, is a fimdamental component in the diagnosis and
`
`treatment of medical disorders, as well as in research. A number of methodologies are
`
`currently in use for detection. These methodologies can generally be divided into
`
`antibody-based diagnostic assays for proteins, either components of the bacteria or
`
`byproducts of the disease, and diagnostic assays for nucleic acids, such as the genetic
`
`material encoding a component of the bacteria.
`
`[0002] Existing methodologies for nucleic acid detection require a high degree of
`
`technical competence for reliability due to the complexity of the reaction conditions (for
`
`example, PCR requires thermocycling) and may be extremely sensitive to contamination
`
`resulting in false positives; they are difficult to use quantitatively rather than qualitatively
`
`and thus their sensitivity is compromised. Further, they often take hours to complete.
`
`Methodologies for protein detection generally rely on conjugation of an enzyme, usually
`
`to additional components of the assay, to increase signal generation and amplification.
`
`The use of these additional ligands increases the noise of the system, with higher
`
`background and false positives, and necessitates several levels of control reactions.
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`
`SUMMARY OF THE INVENTION
`
`[0003] Provided are methods of using ribosomes from pathogens to identify the
`
`pathogens, e. g., for diagnostic or treatment purposes. Ribosomes are specific to a
`
`particular pathogen, and thus the identity or presence of a pathogen in a sample may be
`
`determined based on determining whether a polypeptide is able to be produced using a
`
`25
`
`nucleic acid template with ribosomes isolated using a binding agent specific for the
`
`pathogen’s ribosomes. The methods may be performed at a single temperature, avoiding
`
`-1-
`
`

`

`complex reaction conditions. Further, the amplification of the template by the ribosomes
`
`is rapid — at least about 20,000 target molecules are produced every 10 seconds - and is
`
`very sensitive. Still further, the amplification reaction itself is generic, with only the
`
`nucleic acid template and binding agent used to immobilize the ribosomes differing from
`
`test to test based on the pathogen to be detected. Thus, manufacturing of test kits and
`
`devices for the practice of the methods could be drastically simplified.
`
`[0004] The methods may be incorporated into any test format or device suitable for the
`
`practice of the methods. Also provided are kits, reagents, etc. for the practice of the
`
`methods.
`
`[0005] Further objectives and advantages of the present invention will become apparent
`
`as the description proceeds. To gain a full appreciation of the scope of the present
`
`invention, it will be further recognized that various aspects of the present invention can
`
`be combined to make desirable embodiments of the invention.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0006] FIGURE 1 depicts a schematic of an exemplary embodiment of the use of
`
`ribosomal amplification to detect Streptococcus A bacteria.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0007] Unless defined otherwise above, all technical and scientific terms used herein
`
`have the same meaning as commonly understood by one of ordinary skill in the art to
`
`which this invention belongs. Where a term is provided in the singular, the inventor also
`
`contemplates the plural of that term. The nomenclature used herein and the procedures
`
`described below are those well known and commonly employed in the art.
`
`[0008] The term “amino acid” is intended to embrace all molecules, whether natural or
`
`synthetic, which include both an amino functionality and an acid fimctionality and
`
`capable of being included in a polymer of naturally-occurring amino acids. Exemplary
`
`amino acids include naturally-occurring amino acids; analogs, derivatives and congeners
`
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`

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`thereof; amino acid analogs having variant side chains; and all stereoisomers of any of
`
`any of the foregoing. The names of the natural amino acids are abbreviated herein in
`
`accordance with the recommendations of IUPAC-IUB.
`
`[0009] The term “antibody” refers to an immunoglobulin, derivatives thereof which
`
`maintain specific binding ability, and proteins having a binding domain which is
`
`homologous or largely homologous to an immunoglobulin binding domain. These
`
`proteins may be derived from natural sources, or partly or wholly synthetically produced. ,
`
`An antibody may be monoclonal or polyclonal. The antibody may be a member of any
`
`immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
`
`In exemplary embodiments, antibodies used with the methods and compositions
`
`described herein are derivatives of the IgG class.
`
`[0010] The term “antibody fragment” refers to any derivative of an antibody which is
`
`less than full—length. In exemplary embodiments, the antibody fragment retains at least a
`
`significant portion of the full-length antibody’s specific binding ability. Examples of
`
`antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFV
`
`diabody, and Fd fragments. The antibody fragment may be produced by any means. For
`
`instance, the antibody fragment may be enzymatically or chemically produced by
`
`fragmentation of an intact antibody, it may be recombinantly produced from a gene
`
`encoding the partial antibody sequence, or it may be wholly or partially synthetically
`
`produced. The antibody fragment may optionally be a single chain antibody fragment.
`
`Alternatively, the fragment may comprise multiple chains which are linked together, for
`
`instance, by disulfide linkages. The fragment may also optionally be a multimolecular
`
`complex. A functional antibody fragment will typically comprise at least about 50 amino
`
`acids and more typically will comprise at least about 200 amino acids.
`
`[0011] The terms “comprise” and “comprising” is used in the inclusive, open sense,
`
`meaning that additional elements may be included.
`
`[0012] The term “including” is used herein to mean “including but not limited to”.
`
`“Including” and “including but not limited to” are used interchangeably.
`
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`

`[0013] The term “mRNA” refers to messenger RNA, or the RNA that serves as a
`
`template for protein synthesis in a cell. The sequence of a strand of mRNA is based on
`
`the sequence of a complementary strand of DNA comprising a sequence coding for the
`
`protein to be synthesized.
`
`[0014] “Nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA),
`
`and, where appropriate, ribonucleic acid (RNA). The term should also be understood to
`
`include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs,
`
`and, as applicable to the embodiment being described, single (sense or antisense) and
`
`double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are
`
`representative examples of molecules that may be referred to as nucleic acids.
`
`[0015] The term “pathogen” refers to any organism which may cause disease in a subject,
`
`such as a bacterium, fungus, parasite, virus, etc.
`
`[0016] “Protein” (if single-chain), “polypeptide” and “peptide” are used interchangeably
`
`herein when referring to a gene product, e. g., as may be encoded by a coding sequence.
`
`When referring to “polypeptide” herein, a person of skill in the art will recognize that a
`
`protein can be used instead, unless the context clearly indicates otherwise. A “protein”
`
`may also refer to an association of one or more polypeptides.
`[0017] The term “sample” refers to any sample potentially containing pathogens
`
`containing ribosomes.
`
`[0018] The term “ribosomal RNA” or “rRN ” refers to the RNA component of ribosome
`
`subunits. Ribosomes and their subunits are described further below.
`
`[0019] Provided in one aspect is a method, comprising:
`
`[0020]
`
`(a)
`
`contacting a support comprising a binding agent capable of binding
`
`a ribosome with a sample comprising ribosomes;
`
`[0021]
`
`[0022]
`
`(b)
`
`(c)
`
`washing the support after contact with the sample;
`
`incubating the support in the presence of a solution comprising
`
`mRNA and amino acids under conditions suitable to allow ribosomes bound to the
`
`binding agent to translate the mRNA in the solution into a polypeptide; and
`
`[0023]
`
`(d)
`
`determining the presence of the polypeptide.
`
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`

`[0024] In certain embodiments, the ribosomes are specific to a pathogen and the method
`
`further comprises determining the presence of the pathogen in the sample based on the
`
`presence of the polypeptide.
`
`[0025] Provided in another aspect is a method, comprising:
`
`[0026] (a)
`
`capturing ribosomes on a support using a binding agent capable of binding
`
`the ribosomes;
`
`[0027] (b)
`
`incubating the captured ribosomes with a solution comprising mRNA and
`
`amino acids; and
`
`[0028] (c)
`
`detecting the polypeptide produced by the incubated, captured ribosomes.
`
`[0029] Ribosomes are ribonucleoproteins which are present in both prokaryotes and
`
`eukaryotes. They comprise about two-thirds RNA and one—third protein. Ribosomes are
`
`the cellular organelles responsible for protein synthesis. During gene expression,
`
`ribosomes translate the genetic information encoded in a messenger RNA into protein
`
`(Garrett et al. (2000) “The Ribosome: Structure, Function, Antibiotics and Cellular
`
`Interactions,” American Society for Microbiology, Washington, D.C.).
`
`[0030] Ribosomes comprise two nonequivalent ribonucleoprotein subunits. The larger
`
`subunit (also known as the “large ribosomal subunit”) is about twice the size of the
`
`smaller subunit (also known as the “small ribosomal subunit”). The small ribosomal
`
`subunit binds messenger RNA (mRNA) and mediates the interactions between mRNA
`
`and transfer RNA (tRNA) anticodons on which the fidelity of translation depends. The
`
`large ribosomal subunit catalyzes peptide bond formation--the peptidyl-transferase
`
`reaction of protein synthesis--and includes (at least) two different tRNA binding sites: the
`
`A—site which accommodates the incoming aminoacyl-tRNA, which is to contribute its
`
`amino acid to the growing peptide chain, and the P—site which accommodates the
`
`peptidyl-tRNA complex, i.e., the tRNA linked to all the amino acids that have so far been
`
`added to the peptide chain. The large ribosomal subunit also includes one or more
`
`binding sites for G-protein factors that assist in the initiation, elongation, and termination
`
`phases of protein synthesis. The large and small ribosomal subunits behave
`
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`

`

`independently during the initiation phase of protein synthesis; however, they assemble
`
`into complete ribosomes when elongation is about to begin.
`
`[0031] Accordingly, as used herein, the term “ribosome” refers to a complex comprising
`
`a large ribosomal subunit and small ribosomal subunit. The large ribosomal subunit and
`
`small ribosomal subunit are 50 S and 30 S subunits respectively in bacteria and 60 S and
`
`40 S subunits respectively in eukaryotes.
`
`[0032] Protocols describing the preparation of samples comprising ribosomes are
`
`available in the literature and can be adapted where needed by those skilled in the art.
`
`For example, the preparation of ribosomes from bacteria can be done essentially as
`
`described by Youmans and Youmans, 1965 and adapted as described by Gregory et al.,
`
`1983. In general, but particularly when using virulent Microbes (pathogenic), is
`
`recommended to kill the cells prior to further use, for example by treatment with formalin
`
`as described by Michalek and McGhee, 1977, and adjust concentrations to 108 bacterial
`
`or fungal cells/m1 or 107 protozoa/ml. The preparation can be established to be sterile
`
`when no multiplication occurs upon inoculation on Sheep blood and Mitis Salivarius
`
`agars (DIFCO) or other adapted rich culture medium. Aliquots are stored at -80.degree.
`
`C. Subsequently they are thawed rapidly at 37.degrees. C., and 1 g of whole cells is re-
`
`suspended with 1 g of micro-glass beads (0.17—0.18 m) in 1 ml of PMB to which 3
`
`.mu.g/m1Dnase (SIGMA ) is added. The cells are disrupted by shaking for three 2-
`
`minute cycles in a Braun homogenizer. Intact cells and debris are removed by two
`
`centrifiigations (27 .000.times.g followed by 47.000.times.g; 10 minutes each).
`
`[0033] Preparation of ribosomes from fungi and protozoa follow essentially the same
`
`procedure but require adaptation of culture conditions and lysis methods. Given that
`
`culture conditions of cultivatable pathogenic microbes are widely available in published
`
`literature, preparation of ribosomes from such microbes is well within the possibilities of
`
`a person skilled in the art.
`
`[0034] Integrity of the ribosomal subunits is important. In particular the stabilization of
`
`enclosed large ribosomal RNA’s by divalent cations such as provided by MgClz,
`
`concentration which may need adaptation depending on the microbe and extraction
`
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`

`protocol methods used. The ribosomes in the supernatant can be harvested by
`
`centrifugation at 180.000 to 250.000.times.g for 2 to 3 hr and then subjected to 5
`
`successive washes in PMB at 180.000 to 250.000.times. g for 2 to 3 hr each. The
`
`ribosomal preparation is then clarified twice by two 20-min. centrifugations at
`
`47.000.times. g and the supernatant is filtered through a sterile 0.45 micron Millipore
`
`filter (Millipore Filter Corp.). Non-dissociated (=intact) ribosomes can be prepared from
`
`gram-negative, Rnase-minus mutant bacteria such as Escherichia coli MRE600 following
`
`the method of Staehilin et. al., 1969, with modifications as described by M. M. Yusupov
`
`and A. S. Spirin. 1988. The preparations can then adjusted to, for example, 20 mg/ml on
`
`the basis of protein content by standard protein quantification methods, using, for
`
`example, bovine serum albumin as a standard, and maintained at -80.degree. C. until
`
`used. Characterization of the ribosomal fraction and purity can be determined by spectral
`
`analysis at 235, 280 and 260 nm in order to determine the contamination of ribosomal
`
`RNA by DNA polyacrylamide gel electrophoresis permits to evaluate the presence of
`
`ribosomal proteins and potential contaminating proteins. The degree of intactness can be
`
`evaluated by loading a sample of the original homogenate onto a 10% to 40% sucrose
`
`gradient, containing an appropriate concentration of Mg C12 and centrifugation. The
`
`elusion profile of the sucrose gradient will show the different fractions: lOOS=dimers of
`
`708 ribosomes, 7OS=intact ribosomes, 6OS=interacting SOS and 308 ribosomal subunits,
`
`SOS=large ribosomal subunit, 303=small ribosomal subunit, material less than
`
`3OS=degradation products and contaminants. In good preparations that target non-
`
`dissociated ribosomes, the 708 peak contains over 80% of all material. Optionally, the
`
`708 peak containing the target non-dissociated ribosomes may constitute at least 50%,
`
`60%, 70% or 90% of all material.
`
`[0035] Pathogens that may be detected using the above methods include any organism
`
`comprising ribosomes. Organisms from which ribosomes may be isolated include, but
`
`are not limited to, the following:
`
`[0036] Ribosomes from bacteria such as: Acinetobacter calcoacetz'cus, A. haemolyticus,
`
`Aeromonas hydrophilia, Bacteroidesfiagilis, B. distasonis, Bacteroides 3452A homology
`
`-7-
`
`10
`
`15
`
`20
`
`25
`
`

`

`group, B. vulgatus, B. ovalus, B. thetaiotaomicron, B. uniformis, B. eggerthii, B.
`
`splanchnicus, Branhamella catarrhalis, Campylobacterfetus, C. jejuni, C. coli,
`
`Citrobacterfreuna'ii, Clostridium diflicile, C. diphtheriae, C. ulcerans, C. accolens, C.
`
`afiermentans. C. amycolatum, C. argentorense, C. auris, C. bovis, C. confusum, C.
`
`coyleae, C. durum, C. falsenii, C. glucuronolyticum, C. imitans, C. jeikeium, C. kutscheri,
`
`C. kroppenstedtii, C. lipophilum, C. macginleyi, C. matruchoti, C. mucifacz’ens, C.
`
`pilosum, C. propinquum, C. renale, C. riegelii, C. sanguinis, C. singulare, C. striatum, C.
`
`sundsvallense, C. thomssenii, C. urealyticum, C. xerosis, Enterobacter cloacae, E.
`
`aerogenes, Enterococcus avium, E. casseliflavus, E. cecorum, E. dispar, E. durans, E.
`
`faecalis, E. faecium, E. flavescens, E. gallinarum, E. hirae, E. malodoratus, E. mundtii,
`
`E. pseudoavium, E. rafi’inosus, E. solitarius, Francisella tularensis, Gardnerella
`
`vaginalis, Helicobacter pylori, Kingella dentrificans, K. kingae, K. oralis, Klebsiella
`
`pneumoniae, K. oxytoca, Moraxella catarrhalis, M atlam‘ae, M lacunata, M
`
`nonliquefacz'ens, M osloensis, M phenylpyruvica, Morganella morganii, Parachlamydia
`
`acanthamoebae, Pasteurella multocida, P. haemolytica, Proteus mirabilis, Proteus
`
`vulgaris, Providencia alcalifaciens, P. rettgeri, P. stuartii, Serratia marcescens,
`
`Simkania negevensis, Streptococcus pneumoniae, S. agalactiae, S. pyogenes, Treponema
`
`pallidum, Vibrio cholerae, and V. parahaemolyticus.
`
`[0037] Ribosomes fi'om facultative intracellular bacteria such as: Bordetella pertussis, B.
`
`parapertussis, B. bronchiseptica, Burkholderia cepacia, Escherichia coli, Haemophilus
`
`actinomycetemcomitans, H. aegyptius, H. aphrophilus, H. ducreyi, H. felis, H.
`
`haemoglobinophilus, H. haemolyticus. H. influenzae, H. paragallinarum, H.
`
`parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. paraphrophilus, H.
`
`parasuis, H. piscium, H. segnis, H. somnus, H. vaginalis, Legionella adelaidensis, L.
`
`anisa, L. beliardensis, L. birminghamensis, L. bozemanii, L. brunensis, L. cherrii, L.
`
`cincinnatiensis, Legionella drozanskii L. dumoflli, L. erythra, L. fairfieldensis, L. fallonii,
`
`L. feeleii, L. geestiana, L. gormam'i, L. gratiana, L. gresilensis, L. hackeliae, L.
`
`israelensis, L. jordanis, L. lansingensis, Legionella londiniensis L. longbeachae,
`
`Legionella lytica L. maceachernii, L. micdadei, L. moravica, L. nautarum, L.
`
`10
`
`15
`
`20
`
`25
`
`

`

`oakridgensis, L. parisiensis, L. pittsburghensis, L. pneumophila, L. quateirensis, L.
`
`. quinlivanii, L. rowbothamii, L. rubrilucens, L. sainthelensi, L. santicrucis, L.
`
`shakespearei, L. spiritensis, L. steigerwaltii, L. taurinensz's, L. tucsonensis, L.
`
`wadsworthii, L. waltersii, L. worslez'ensis, Listeria denitrificans, L. grayi, L. innocua, L.
`
`ivanovii, L. monocytogenes, L. seeligeri, L. welshimeri, Mycobacterium abscessus, M
`
`afiicanum, M agri, M aichiense, M alvei, M asiaticum, M aurum, M austroafricanum,
`
`M avium, M bohemicum, M bovis, M branderi, M brumae, M celatum, M chelonae,
`
`M chitae, M chlorophenolicum, M chubuense, M confluentis, M conspicuum, M
`
`cookii, M diernhoferi, M doricum, M duvalz'i, M elephantis, M fallax, M farcinogenes,
`M flavescens, M fortuitum, M frederikshergense, M gadium, M gastri, M genavense,
`
`M gilvum, M goodii, M gordonae, M haemophilum, M hassiacum, M heckeshornense,
`
`M heidelbergense, M hiberniae, M immunogenum, M intracellulare, M interjectum,
`
`M intermedium, M kansasii, M komossense, M kubicae, M lentiflavum, M leprae, M
`
`lepraemurium, M luteum, M. madagascariense, M mageritense, M malmoense, M
`
`marinum, M microti, M moriokaense, M mucogenicum, M murale, M neoaurum, M
`
`nonchromogem'cum, M novocastrense, M obuense, M parqfortuitum, M
`
`paratuberculosis, M peregrinum, M phage, M phlei, M porcinum, M poriferae, M
`
`pulveris, M rhodesiae, M scrofulaceum, M senegalense, M septicum, M shimoidei, M
`
`simiae, M smegmatis, M sphagm', M szulgai, M terrae, M thermoresistibz'le, M
`
`tokaiense, M triplex, M triviale, M tuberculosis, M tusciae, M ulcerans, M vaccae, M
`
`wolinskyi, M xenopi, Neisseria animalis, N. canis, N. cinerea, N. denitrificans, N.
`
`dentiae, N. elongata, N. flava, N. flavescens, N. gonorrhoeae, N. iguanae, N. lactamica,
`
`N. macacae, N. meningitidis, N. mucosa, N. ovis, N. perflava, N. pharyngis var. flava, N.
`
`polysaccharea, N. sicca, N. subflava, N. weaveri, Pseudomonas aeruginosa, P.
`
`alcaligenes, P. chlororaphis, P. fluorescens, P. luteola, P. mendocina, P. monteilii, P.
`
`oryzihabitans, P. pertocinogena, P. pseudalcaligenes, P. putida, P. stutzeri, Salmonella
`
`bacteriophage, S. bongori, S. choleraesuis, S. enterica, S. enteritidis, S. paratyphi, S.
`
`typhi, S. typhimurium, S. typhz’murium, S. typhimurium, S. typhimurium bacteriophage,
`
`Shigella boydii, S. dysenteriae, S. flexneri, S. sonnei. Staphylococcus arlettae, S. aureus,
`
`10
`
`15
`
`20
`
`25
`
`

`

`5
`
`10
`
`15
`
`20
`
`25
`
`S. auricularis, S. bacteriophage, S. capitis, S. caprae, S. carnosus, S. caseolyticus, S.
`
`chromogenes, S. cohnii, S. delphim', S. epidermidis, S. equorum, S. feliS, S. fleurettii, S.
`
`gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S. lentus, S.
`
`lugdunensis, S. lutrae, S. muscae, S. mutans, S. pasteuri, S. phage, S. pisc1fermentans, S.
`
`pulvereri, S. saccharolyticus, S. saprophyticus, S. schlez'feri, S. sciuri, S. simulans, S.
`
`succinus, S. vitulinus, S. warneri, S. xylosus, Ureaplasma urealyticum, Yersim'a aldovae,
`
`Y. bercovieri, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y.
`
`mollaretii, Y. pestis, Y. philomiragia, Y. pseudotuberculosis, Y. rohdei, and Y. ruckeri.
`
`[0038] Ribosomes from obligate intracellular bacteria, such as: Anaplasma bovis, A.
`
`caudatum, A. centrale, A. marginale A. ovis, A. phagocytophila, A. platys, Bartonella
`
`bacilliform is, B. clarridgeiae, B. elizabethae, B. henselae, B. henselae phage, B.
`
`quintana, B. taylorii, B. vinsonii, Borrelia afzelii, B. andersonii, B. anserina, B. bissettii,
`
`B. burgdorferi, B. crocidurae, B. garinii, B. hermsii, B. japonica, B. miyamotoi, B.
`
`parkeri, B. recurrentis, B. turdi, B. turicatae, B. valaisiana, Brucella abortus, B.
`
`melitensis, Chlamydia pneumoniae, C. psittaci, C. trachomatis, Cowdria ruminantium,
`
`Coxiella burnetii, Ehrlichia canis, E. chafleensis, E. equi, E. ewingii, E. muris, E.
`
`phagocytophila, E. platys, E. risticii, E. ruminantium, E. sennetsu, Haemobartonella
`
`canis, H. felis, H. muris, Mycoplasma arthriditis, M buccale, M faucium, M fermentans,
`
`M genitalium, M hominis, M laidlawiz', M lipophilum, M orale, M penetrans, M
`
`pirum, M. pneumoniae, M salivarium, M spermatophilum, Rickettsia australis, R.
`
`conorii, R. felis, R. helvetica, R. japonica, R. massiliae, R. montanensis, R. peacockii, R.
`
`prowazekz'i, R. rhipicephali, R. rickettsii, R. sibirica, and R. typhi.
`
`[0039] Ribosomes from facultative intracellular fungi, such as: Candida Candida aaseri,
`
`C. acidothermophilum, C. acutus, C. albicans, C. anatomiae, C. apis, C. apis var.
`
`galacta, C. atlantica, C. atmospherica, C. auringiensis. C. bertae, C. berthtae var.
`
`chiloensis, C. berthetii, C. blankii, C. boidim'i, C. boleticola, C. bombi, C. bombicola, C.
`
`buinensis, C. butyri, C. cacaoi, C. cantarellii, C. cariosilignicola, C. castellii, C.
`
`castrensis, C. catenulata, C. chilensis, C. chiropterorum,