Patent Grant
7867704
The material on the accompanying compact disc is hereby incorporated by reference into this application. The accompanying compact disc contains three files, Table10.txt, Table8.txt, and Table9.txt, which were created on Sep. 3, 2004. The file named Table10.txt is 1,651 KB, the file named Table8.txt is 4,907 KB, and the file named Table9.txt is 611 MB. The files can be accessed using Microsoft Word on a computer that uses Windows OS.
The Sequence Listing for this application is provided on CD-ROM. The CD-ROM containing the Sequence Listing is hereby incorporated by reference into this application. The CD-ROM containing the Sequence Listing contains one file, 60256371.TXT, which was created on Jun. 7, 2005. The file named 60256371.TXT is 17,837 MB. The file can be accessed using Microsoft Word on a computer that uses Windows OS.
This invention relates to detection of bacteria, and more particularly to detection of Mycobacterium avium subsp. paratuberculosis.
The disorder known as Johne's disease was first described in 1895. Today, Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, is widely distributed both nationally and internationally in domestic ruminants such as cattle, sheep, goats, as well as wildlife such as rabbits, deer, antelopes, and bison. In 1996, the National Animal Health Monitoring System conducted a survey of dairy farms using serological analysis to determine the prevalence of Johne's disease in the U.S. The results of that study showed an estimated 20-40% of surveyed herds have some level of M. paratuberculosis. Furthermore, it is estimated that annual losses in the U.S. from M. paratuberculosis in cattle herds may exceed $220 million.
The pathogenesis of M. paratuberculosis has been recently reviewed by Harris and Barletta (2001, Clin. Microbiol. Rev., 14:489-512). Cattle become infected with M. paratuberculosis as calves but often do not develop clinical signs until 2 to 5 years of age. The primary route of infection is through ingestion of fecal material, milk or colostrum containing M. paratuberculosis microorganisms. Epithelial M cells likely serve as the port of entry for M. paratuberculosis into the lymphatic system similar to other intracellular pathogens such as salmonella. M. paratuberculosis survive and may even replicate within macrophages in the wall of the intestine and in regional lymph nodes. After an incubation period of several years, extensive granulomatous inflammation occurs in the terminal small intestine, which leads to malabsorption and protein-losing enteropathy. Cattle shed minimal amounts of M. paratuberculosis in their feces during the subclinical phase of infection, and yet over time, this shedding can lead to significant contamination of the environment and an insidious spread of infection throughout the herd before the animal is diagnosed. During the clinical phase of infection, fecal shedding of the pathogen is high and can exceed 1010 organisms/g of feces. The terminal clinical stage of disease is characterized by chronic diarrhea, rapid weight loss, diffuse edema, decreased milk production, and infertility. Although transmission of M. paratuberculosis occurs primarily through the fecal-oral route, it has also been isolated from reproductive organs of infected males and females.
The present invention provides nucleic acid molecules unique to M. paratuberculosis. The invention also provides polypeptides encoded by the M. paratuberculosis-specific nucleic acid molecules of the invention, and antibodies having specific binding affinity for the polypeptides encoded by the M. paratuberculosis-specific nucleic acid molecules. The invention further provides for methods of detecting M. paratuberculosis in a sample using nucleic acid molecules, polypeptides, or antibodies of the invention. The invention additionally provides for methods of preventing a M. paratuberculosis infection in an animal.
In one aspect, the invention provides an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 969 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:1 or to the complement of SEQ ID NO:1, wherein any such molecule that is 10 to 35 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Homo sapiens, Pseudomonas aeruginosa, Streptomyces viridochromogenes, Mus musculus, Felis catus, and Xanthomonas campestris using an appropriate third nucleic acid molecule.
For example, a nucleic acid of the invention can have the sequence shown in SEQ ID NO:1. A nucleic acid of the invention can have at least 75% sequence identity to SEQ ID NO:1 (e.g., SEQ ID NO:102). A nucleic acid of the invention can have at least 80% sequence identity to SEQ ID NO:1 (e.g., SEQ ID NO:103). A nucleic acid of the invention can have at least 85% sequence identity to SEQ ID NO:1 (e.g., SEQ ID NO:104). A nucleic acid of the invention can have at least 90% sequence identity to SEQ ID NO:1 (e.g., SEQ ID NO:105). A nucleic acid of the invention can have at least 95% sequence identity to SEQ ID NO:1 (e.g., SEQ ID NO:106). A nucleic acid of the invention can have at least 99% sequence identity to SEQ ID NO:1 (e.g., SEQ ID NO:107).
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 576 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:2 or to the complement of SEQ ID NO:2, wherein any such molecule that is 10 to 35 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Thermotoga sp., Homo sapiens, Pseudomonas aeruginosa, Deinococcus radiodurans, Streptomyces coelicolor, Oryza sativa, Rhizobium leguminosarum, Frankia alni, and Mesorhizobium loti using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 522 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:3 or to the complement of SEQ ID NO:3, wherein any such molecule that is 10 to 35 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Halobacterium NRC-1, Oryza sativa, Glycine max, Streptomyces coelicolor, and Mus musculus using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 582 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:4 or to the complement of SEQ ID NO:4, wherein any such molecule that is 10 to 47 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Oryza sativa, Caenorhabditis elegans, Leishmania mexicana, Drosophila melangaster, Homo sapiens, Zea mays, Halobacterium sp. NRC-1, Pseudomonas aeruginosa, Ralstonia solanacearum, and Streptomyces coelicolor using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 311 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:5 or to the complement of SEQ ID NO:5, wherein any such molecule that is 10 to 36 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Homo sapiens, Streptomyces coelicolor, Ictalurid herpesvirus, Mesorhizobium loti, and Oryza sativa using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 576 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:6 or to the complement of SEQ ID NO:6, wherein any such molecule that is 10 to 41 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Pseudomonas aeruginosa, Ralstonia solanacearum, Arabidopsis thaliana, Pseudomonas fluorescens, Homo sapiens, and Mesorhizobium loti using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 474 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:7 or to the complement of SEQ ID NO:7, wherein any such molecule that is 10 to 35 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Homo sapiens, Pantoea agglomerans, Rattus norvegicus, Erwinia uredovora, Erwinia ananas, and Pantoea ananatis using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 558 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:8 or to the complement of SEQ ID NO:8, wherein any such molecule that is 10 to 36 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Neisseria meningitidis, Homo sapiens, Streptomyces coelicolor, Arabidopsis thaliana, Escherichia coli, Pseudomonas aeruginosa, Streptomyces hygroscopicus var. ascomyceticus, Ralstonia solanacearum, Deinococcus radiodurans, Rhizobium meliloti, Rickettsia typhi, Streptomyces sp., and Mus musculus using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 321 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:9 or to the complement of SEQ ID NO:9, wherein any such molecule that is 10 to 36 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Mycobacterium tuberculosis, Homo sapiens, Streptomyces coelicolor, Drosophila melanogaster, Ralstonia solanacearum, Mesorhizobium loti, and Pseudomonas cruciviae using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 2508 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:10 or to the complement of SEQ ID NO:10, wherein any such molecule that is 10 to 44 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Sinorhizobium meliloti, Xanthomonas albilineans, Halobacterium sp. NRC-1, Ralstonia solanacearum, Deinococcus radiodurans, Halobacterium salinarium, Micromonospora griseorubida, Pseudomonas paucimobilis, and Streptomyces lividans using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 264 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:11 or to the complement of SEQ ID NO:11, wherein any such molecule that is 10 to 36 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Caulobacter crescentus, Brucella melitensis, Pyrobaculum aerophilum, Mycobacterium tuberculosis, Sinorhizobium meliloti, and Mycobacterium leprae using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 1110 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:12 or to the complement of SEQ ID NO:12, wherein any such molecule that is 10 to 39 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Mesorhizobium loti, Bacillus halodurans, Ralstonia solanacearum, Homo sapiens, Drosophila melanogaster, and Rhizobium meliloti using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 672 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:13 or to the complement of SEQ ID NO:13, wherein any such molecule that is 10 to 30 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of M. avium subsp. avium using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 372 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:14 or to the complement of SEQ ID NO:14, wherein any such molecule that is 10 to 30 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Brucella melitensis, Streptomyces coelicolor, Drosophila melanogaster, Mycobacterium tuberculosis, Trypanosoma rangeli, Trypanosoma minasense, Trypanosoma leeuwenhoeki, and Brassica napus using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 600 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:15 or to the complement of SEQ ID NO:15, wherein any such molecule that is 10 to 35 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Ralstonia solanacearum, Sinorhizobium meliloti, Homo sapiens, Mesorhizobium loti, Oryza sativa, Drosophila melanogaster, Rhizobium leguminosarum, Xylella fastidiosa, Deinococcus radiodurans, Achromobacter cycloclastes, and Candida cylindracea using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 540 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:16 or to the complement of SEQ ID NO:16, wherein any such molecule that is 10 to 45 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Streptomyces lavendulae, Xylella fastidiosa, Streptococcus pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Ralstonia solanacearum, Sinorhizobium meliloti, Sus scrofa, Mycobacterium leprae, and Streptomyces coelicolor using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 291 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:17 or to the complement of SEQ ID NO:17, wherein any such molecule that is 10 to 37 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Pseudomonas sp., Homo sapiens, Pseudomonas aeruginosa, Thauera aromatica, Oryza sativa, Ralstonia solanacearum, Rhizobium leguminosarum, Streptomyces coelicolor, Brucella melitensis, Drosophila melanogaster, Deinococcus radiodurans, Streptomyces noursei, Rhizobium meliloti, Synechococcus elongates, Sinorhizobium meliloti, and Mesorhizobium loti using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 225 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:18 or to the complement of SEQ ID NO:18, wherein any such molecule that is 10 to 37 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Rhodobacter capsulatus, Agrobacterium tumefaciens, Mycobacterium smegmatis, Pseudomonas aeruginosa, Ralstonia solanacearum, and Drosophila virilis using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 441 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:19 or to the complement of SEQ ID NO:19, wherein any such molecule that is 10 to 30 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Homo sapiens, Mus musculus, Leishmania major, Pseudomonas aeruginosa, and Botrytis cinerea using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 726 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:20 or to the complement of SEQ ID NO:20, wherein any such molecule that is 10 to 41 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Oryza sativa, Caulobacter crescentus, Rhodobacter sphaeroides, Streptomyces coelicolor, Spermatozopsis similis, Homo sapiens, Sus scrofa, and Giardia intestinalis using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 426 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:21 or to the complement of SEQ ID NO:21, wherein any such molecule that is 10 to 44 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Streptomyces coelicolor, Homo sapiens, Triticum aestivum, Oryza sativa, Brucella melitensis, Caulobacter crescentus, Pseudomonas aeruginosa, Ralstonia solanacearum, Bovine herpesvirus, and Mesorhizobium loti using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 279 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:22 or to the complement of SEQ ID NO:22, wherein any such molecule that is 10 to 45 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Pseudomonas aeruginosa, Oryza sativa, Streptomyces sp., Streptomyces peucetius, Rhizobium sp., Mycobacterium tuberculosis, Caulobacter crescentus, Ralstonia solanacearum, Haloferax volcanii, Mycobacterium leprae, and Streptomyces coelicolor using an appropriate third nucleic acid molecule.
In another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of 10 nucleotides to 4415 nucleotides, the molecule having at least 75% sequence identity to SEQ ID NO:23 or to the complement of SEQ ID NO:23, wherein any such molecule that is 10 to 50 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Ralstonia solanacearum, Sinorhizobium meliloti, Homo sapiens, Mesorhizobium loti, Oryza sativa, Drosophila melanogaster, Rhizobium leguminosarum, Xylella fastidiosa, Deinococcus radiodurans, Achromobacter cycloclastes, Candida cylindracea, Streptomyces lavendulae, Streptococcus pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Sus scrofa, Mycobacterium leprae, Streptomyces coelicolor, Pseudomonas sp., Thauera aromatica, Brucella melitensis, Streptomyces noursei, Rhizobium meliloti, Synechococcus elongates, Rhodobacter capsulatus, Agrobacterium tumefaciens, Mycobacterium smegmatis, Drosophila virilis, Mus musculus, Leishmania major, Botrytis cinerea, Caulobacter crescentus, Rhodobacter sphaeroides, Spermatozopsis similes, Giardia intestinalis, Triticum aestivum, Bovine herpesvirus, Streptomyces sp., Streptomyces peucetius, Rhizobium sp., and Haloferax volcanii using an appropriate third nucleic acid molecule.
Generally, the invention provides an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule of at least 10 nucleotides, the molecule having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NOs:1-23 or to the complement of a sequence selected from the group consisting of SEQ ID NOs:1-23, wherein any such molecule that is 10 to 30 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms selected from the group consisting of Ralstonia solanacearum, Sinorhizobium meliloti, Homo sapiens, Mesorhizobium loti, Oryza sativa, Drosophila melanogaster, Rhizobium leguminosarum, Xylella fastidiosa, Deinococcus radiodurans, Achromobacter cycloclastes, Candida cylindracea, Streptomyces lavendulae, Streptococcus pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Sus scrofa, Mycobacterium leprae, Streptomyces coelicolor, Pseudomonas sp., Thauera aromatica, Brucella melitensis, Streptomyces noursei, Rhizobium meliloti, Synechococcus elongates, Rhodobacter capsulatus, Agrobacterium tumefaciens, Mycobacterium smegmatis, Drosophila virilis, Mus musculus, Leishmania major, Botrytis cinerea, Caulobacter crescentus, Rhodobacter sphaeroides, Spermatozopsis similes, Giardia intestinalis, Triticum aestivum, Bovine herpesvirus, Streptomyces sp., Streptomyces peucetius, Rhizobium sp., Haloferax volcanii, Streptomyces viridochromogenes, Felis catus, Xanthomonas campestris, Thermotoga maritime, Thermotoga neapolitana, Frankia alni, Halobacterium NRC-1, Glycine max, Leishmania tarentolae, Neisseria meningitides, Escherichia coli, Caenorhabditis elegans, Leishmania mexicana, Zea mays, Ictalurid herpesvirus, Rattus norvegicus, Arabidopsis thaliana, Pseudomonasfluorescens, Pantoea agglomerans, Erwinia uredovora, Pantoea ananatis, Streptomyces hygroscopicus, Rickettsia typhi, Pseudomonas cruciviae, Xanthomonas albilineans, Halobacterium salinarium, Micromonospora griseorubida, Pseudomonas paucimobilis, Streptomyces lividans, Pyrobaculum aerophilum, Sinorhizobium meliloti, Mesorhizobium loti, Bacillus halodurans, Trypanosoma rangeli, Trypanosoma minasense, Trypanosoma leeuwenhoeki, and Brassica napus using an appropriate third nucleic acid molecule.
The invention provides for an isolated nucleic acid comprising a nucleic acid molecule of 10 nucleotides to 200 nucleotides having at least 75% sequence identity to one of the sequences set out in SEQ ID NOs:110-1342 or to the complement of that sequence. Using Table 1 as a reference, any such molecule that is 10 to N nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of any of the organisms shown in Table 1 corresponding to the respective SEQ ID NO, using an appropriate third nucleic acid molecule. For example, for SEQ ID NO:110 (segment 26), any such molecule that is 10 to 33 nucleotides in length, under standard amplification conditions, generates an amplification product from M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule, but does not generate an amplification product from nucleic acid of Mus musculus and Rhodobacter sphaeroides, using an appropriate third nucleic acid molecule.
Mus musculus; Rhodobacter sphaeroides
Novosphingobium aromaticivorans; Corynebacterium
efficiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Brucella melitensis; Brucella suis
Mycobacterium avium subsp. avium
Galleria mellonella
Oryctolagus cuniculus; Novosphingobium aromaticivorans
Mycobacterium avium subsp. avium
Oryza sativa; Burkholderia fungorum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum; Zea mays; Oryza
sativa; Streptomyces coelicolor
Neurospora crassa
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Halobacterium sp. NRC-1; Oryza sativa
Mus musculus; Arabidopsis thaliana
Mycobacterium avium subsp. avium
Ralstonia metallidurans; Magnetospirillum
magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Ralstonia metallidurans
Leishmania donovani; Oryza sativa
Oryza sativa
Mycobacterium avium subsp. avium
Mus musculus; Archaeoglobus fulgidus
Mycobacterium avium subsp. avium
Corynebacterium efficiens
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Rhodospirillum rubrum; Ralstonia eutropha
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Actinosynnema pretiosum subsp. auranticum maytansino
Streptomyces coelicolor; Homo sapiens;
Rhodopseudomonas palustris; Triticum aestivum
Mycobacterium avium subsp. avium
Oryza sativa; Spermatozopsis similis
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Pseudomonas sp.; Chlorobium tepidum; Pseudomonas
aeruginosa
Homo sapiens; Lentinus edodes; Geobacter
metallireducens; Thauera aromatica; Oryza sativa
Mycobacterium avium subsp. avium
Chlamydomonas geitleri
Streptomyces lavendulae
Mycobacterium avium subsp. avium
Xanthomonas campestris; Xanthomonas axonopodis
Homo sapiens
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Mus musculus; Mycobacterium avium; Homo sapiens;
Pseudomonas pseudoalcaligenes
Mycobacterium avium subsp. avium
Oryctolagus cuniculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Corynebacterium efficiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Shrimp white spot syndrome
melanogaster; Eimeria tenella; Chlamydomonas
reinhardtii; Bos taurus; Saccharomyces cerevisiae; Homo
sapiens; Gallus gallus
Mycobacterium tuberculosis; Oryza sativa; Azotobacter
vinelandii; Pseudorabies virus; Homo sapiens; Mus
musculus; Desulfitobacterium hafniense; Triticum
aestivum; Streptomyces coelicolor
Mycobacterium tuberculosis; Oryza sativa; Rhodobacter
sphaeroides; Burkholderia fungorum; Desulfitobacterium
hafniense
Mycobacterium tuberculosis
Mycobacterium tuberculosis; Rhodococcus fascians;
Magnetospirillum magnetotacticum; Mycobacterium
leprae; Deinococcus radiodurans; Xanthomonas
campestris; Homo Sapiens; Rhodospirillum rubrum; Oryza
sativa; Streptomyces coelicolor; Penaeus vannamei; Mus
musculus; Caulobacter crescentus
Mycobacterium tuberculosis; Mycobacterium leprae;
Rhodobacter sphaeroides; Rhodospirillum rubrum;
Burkholderia fungorum; Xanthomonas oryzae
Mycobacterium tuberculosis; Ralstonia metallidurans;
Homo sapiens; Azotobacter vinelandii
Mesorhizobium loti
Sinorhizobium meliloti; Xanthomonas campestris;
Rhodopseudomonas palustris; Actinomyces naeslundii;
Streptomyces coelicolor
Pseudomonas fluorescens; Mesorhizobium loti;
Azotobacter vinelandii; Streptomyces coelicolor; Oryza
sativa; Macaca mulatto rhadinovirus; Pseudomonas
aeruginosa
Rhodobacter sphaeroides; Halobacterium sp. NRC-1;
Mycobacterium leprae
Magnetospirillum magnetotacticum; Rhodopseudomonas
palustris; Homo sapiens; Amycolatopsis mediterranei;
Streptomyces coelicolor
Oryza sativa; Homo sapiens; Mus musculus
Mycobacterium tuberculosis; Hordeum vulgare;
Streptomyces coelicolor; Oryza sativa; Desulfitobacterium
hafniense; Haloferax mediterranei; Pseudomonas
aeruginosa; Mus musculus; Haloferax volcanii; Homo
sapiens; Pseudomonas fluorescens; Azotobacter
vinelandii; Sorghum vulgare; Zea mays
Ralstonia metallidurans; Frankia sp.; Homo sapiens;
Streptomyces coelicolor; Oryza sativa
Mycobacterium tuberculosis; Mycobacterium phage Ms6;
Ralstonia solanacearum; Oryza sativa; Rhodobacter
sphaeroides; Rhodopseudomonas palustris; Caulobacter
crescentus; Actinosynnema pretiosum; Homo sapiens;
Thermobifida fusca; Streptomyces noursei; Mesorhizobium
loti; Bifidobacterium longum; Streptomyces coelicolor
Mycobacterium tuberculosis; Pseudomonas fluorescens;
Rhodospirillum rubrum; Ralstonia solanacearum;
Amycolatopsis orientalis; Pseudomonas syringae;
Desulfitobacterium hafniense; Deinococcus radiodurans;
Myxococcus xanthus
Mycobacterium tuberculosis; Streptomyces coelicolor
Ralstonia metallidurans; Azorhizobium caulinodans;
Pseudomonas aeruginosa; Canis familiaris; Sus scrofa;
Neurospora crassa; Rhodobacter sphaeroides;
Rhodospirillum rubrum; Oryza sativa; Streptomyces
coelicolor; Caulobacter crescentus; Papio anubis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Rhizobium meliloti; Sinorhizobium meliloti
Nocardioides sp.; Thermobifida fusca
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Desulfovibrio desulfuricans; Drosophila melanogaster
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Drosophila melanogaster
Cordyceps pseudomilitaris; Oryza sativa
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Oryza sativa;
Magnetospirillum magnetotacticum; Sinorhizobium
meliloti; Burkholderia fungorum; Caulobacter crescentus;
Ralstonia metallidurans; Rhodobacter capsulatus;
Corynebacterium efficiens; Homo sapiens; Mus musculus
Mycobacterium leprae; Mycobacterium tuberculosis;
Oryza sativa; Xanthomonas axonopodis
Mycobacterium avium subsp. avium
Homo Sapiens; Escherichia coli
Mycobacterium tuberculosis; Streptomyces coelicolor;
Oryza Sativa; Pseudomonas aeruginosa; Micromonospora
chersina; Halobacterium sp. NRC-1; Homo sapiens
Deinococcus radiodurans; Zea mays
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Burkholderia fungorum
Magnetospirillum magnetotacticum; Mesorhizobium loti;
Propionibacterium freudenreichii; Streptomyces
coelicolor; Xanthomonas campestris
Mycobacterium tuberculosis; Streptomyces coelicolor;
Oryza sativa; Homo sapiens; Spodoptera frugiperda;
Rhodospirillum rubrum; Thermobifida fusca; Streptomyces
lividans; Corynebacterium efficiens
Mycobacterium kansasii; Burkholderia fungorum;
Ralstonia solanacearum; Halobacterium sp.;
Sinorhizobium meliloti
Gallus gallus; Ralstonia solanacearum; Mycobacterium
avium; Streptomyces avermitilis; Streptomyces sp.
Thermobifida fusca
Mycobacterium tuberculosis; Streptomyces coelicolor;
Micromonospora megalomicea subsp. nigra megalomicin
Mycobacterium leprae; Mycobacterium tuberculosis;
Amycolatopsis sp.; Oryza sativa; Ralstonia solanacearum;
Streptomyces coelicolor
Mycobacterium leprae; Corynebacterium efficiens; Oryza
sativa
Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mesorhizobium loti; Ralstonia solanacearum
Mesorhizobium loti
Bacillus halodurans
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Mycobacterium leprae;
Oryza sativa; Desulfitobacterium hafniense; Xanthomonas
campestris; Bovine herpesvirus; Rhodopseudomonas
palustris; Magnetospirillum magnetotacticum
Homo sapiens; Klebsiella aerogenes; Enterobacter
aerogenes; Pseudomonas sp.; Ralstonia metallidurans;
Magnetospirillum magnetotacticum; Streptomyces
coelicolor; Pseudomonas putida; Bos taurus; Rhodobacter
sphaeroides; Mus musculus
Neurospora crassa; Streptomyces avermitilis; Rubrivivax
gelatinosus; Pseudomonas putida; Hordeum vulgare;
Pseuodmonas stutzeri; Halobacterium sp. NCR-1;
Thermus thermophilus; Caenorhabditis elegans
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Streptomyces coelicolor; Caulobacter crescentus
Mycobacterium avium subsp. avium
Rhodobacter sphaeroides
Mycobacterium tuberculosis
Streptomyces coelicolor; Homo sapiens; Novosphingobium
aromaticivorans; Pan troglodytes; Mus musculus;
Apteronotus albifrons; Ralstonia solanacearum; Sus scofa;
Gallus gallus; Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mesorhizobium loti; Rhodopseudomonas palustris;
Ralstonia solanacearum
Mesorhizobium loti; Rhodobacter sphaeroides
Mycobacterium avium subsp. avium
Bacillus halodurans
Mycobacterium avium subsp. avium
Streptomyces coelicolor; Mycobacterium tuberculosis;
Mycobacterium leprae; Deinococcus radiodurans;
Agrobacterium tumefaciens; Caulobacter crescentus
Mycobacterium tuberculosis; Homo sapiens;
Desulfitobacterium hafniense;
Mycobacterium tuberculosis; Novosphingobium
aromaticivorans; Homo sapiens; Cricetulus griseus; Suid
Mus musculus; Caenorhabditis elegans
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Gallus gallus; Oryza sativa;
Caulobacter crescentus
Mycobacterium tuberculosis; Oryza sativa;
Mycobacterium bovis; Homo sapiens; Novosphingobiuum
aromaticivorans; Mus musculus; Streptomyces coelicolor;
Deinococcus radiodurans; Chlamydomonas reinhardtii
Escherichia coli
Rhodococcus sp.; Mesorhizobium loti
Mycobacterium avium subsp. avium
Ralstonia solanacearum; Pseudomonas aeruginosa; Homo
sapiens; Escherichia coli; Mesorhizobium loti;
Deinococcus radiodurans; Rhodopseudomonas palusiris;
Oryza sativa; Mycobacterium tuberculosis
Mycobacterium tuberculosis; Mycobacterium leprae;
Oryza sativa; Home sapiens; Drosophila melanogaster;
Rhodobacter capsulatus; Alpha proteobacterium; Arabis
Streptomyces coelicoler; Streptomyces galbus
Mycobacterium tuberculosis; Streptomyces coelicolor;
Homo sapiens; Rhodopseudomonas palustris;
Desulfitobacterium hafniense; Mus musculus
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas putida; Magnetospirillum magnetotacticum;
Mycobacterium tuberculosis; Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Mesorhizobium loti; Mycobacterium tuberculosis
Mycobacterium tuberculosis; Xanthomonas axonopodis;
Mycobacterium leprae; Xanthomonas campestris
Home sapiens
Home sapiens; Caulobacter crescentus
Mycobacterium avium subsp. avium
Mycobacterium marinum; Mycobacterium ulcerans;
Leishmania infantum; Desulfitobacterium hafniense;
Rhizobium meliloti; Oryza sativa
Homo sapiens; Bovine herpesvirus; Oryza sativa; Mus
musculus; Burkholderia pseudomallei; Zea mays
Anopheles gambiae; Ralstonia solanacearum; Drosophila
melanogaster; Poncirus trifoliata
Rhodobacter sphaeroides
Pseudomonas syringae
Streptomyces coelicolor; Magnetospirillum
magnetotacticum
Homo sapiens
Desulfitobacterium hafniense; Rhizobium meliloti
Drosophila melanogasler; Ralstonia solanacearum
Bifidobacterium longum; Azotobacter vinelandii;
Rhodospirillum rubrum; Ralstonia solanacearum;
Sinorhizobium meliloti; Streptomyces lincolnensis; Oryza
sativa; Xanthomonas axonopodis; Caulobacter crescentus;
Pseudomonas putida; Mycobacterium phage DS6A;
Pseudomonas fluorescens; Burkholderia fungorum;
Pseudomonas syringae; Mycobacterium tuberculosis;
Streptomyces coelicolor; Pseudomonas aeruginosa
Ralstonia solanacearum
Azotobacter vinelandii; Pseudomonas putida
Corynebacterium efficiens
Pseudomonas aeruginosa; Synechococcus sp.
Corynebacterium glutamicum
Mycobacterium avium subsp. avium
Nitrosomonas europaea
Mycobacterium avium subsp. avium
Pan troglodytes; Homo sapiens
Mycobacterium avium subsp. avium
Gluconacetobacter xylinus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Rhodopseudomonas palustris; Homo sapiens;
Streptomyces noursei; Streptomyces coelicolor; Oryza
sativa
Streptomyces coelicolor; Streptomyces lividans
Mycobacterium avium subsp. avium
Rhodopseudomonas palustris
Synechococcus sp.
Mycobacterium avium subsp. avium
Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo Sapiens; Comamonas sp.
Mycobacterium avium subsp. avium
Corynebacterium glutamicum; Burkholderia fungorum
Mus musculus
Streptomyces maritimus; Streptomyces clavuligerus;
Streptomyces lavendulae; Streptomyces roseofulvus
Homo sapiens
Mycobacterium avium subsp. avium
Streptomyces clavuligerus
Ralstonia eutropha
Rhodobacter sphaeroides; Thermobifida fusca
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Oryza sativa; Zea mays
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Haloferax volcanii; Burkholderia fungorum
Streptomyces coelicolor; Ralstonia solanacearum
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Felis catus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Burkholderia fungorum
Mycobacterium avium subsp. avium
Desulfitobacterium hafniense; Streptomyces coelicolor;
Pseudomonas syringae pv. syringae; Streptomyces
verticillus; Streptomyces avermitilis; Lysobacter sp.
solanacearum; Streptomyces chrysomallus; Stigmatella
aurantiaca; Streptomyces lavendulae; Streptomcyes
toyocaensis; Mesorhizobium loti; Saccharothrix mutabilis
Synechococcus sp.
Mycobacterium avium subsp. avium
Halo ferax mediterranei
Ralstonia solanacearum; Streptomyces verticillus;
Streptomyces coelicolor; Desulfitobacterium hafniense;
Streptomyces noursei
Mycobacterium avium subsp. avium
Streptomyces verticillus; Streptomyces chrysomallus;
Streptomyces avermitilis; Pseudomonas syringae pv.
syringae; Streptomyces coelicolor; Streptomyces
lavendulae; Azotobacter vinelandii; Pseudomonas
fluorescens; Magnetospirillum magnetotacticum;
Thermobifida fusca; Desulfitobacterium hafniense;
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Leishmania major
Burkholderia fungorum; Nitrosomonas europaea;
Pseudomonas sp.
Pseudomonas putida; Sinorhizobium meliloti
Rhodobacter capsulatus; Rat cytomegalovirus
Pseudomonas fluorescens; Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Rhodobacter sphaeroides
Rhodobacter sphaeroides; Oryza sativa; Homo sapiens
Bifidobacterium longum; Caulobacter maris;
Xanthomonas axonopodis pv. citri
Burkholderia fungorum; Streptomyces avermitilis
Streptomyces coelicolor
Rhodobacter sphaeroides; Mycobacterium tuberculosis;
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Xanthomonas campestris pv. Campestris (ATCC 3391)
Mesorhizobium loti; Azotobacter vinelandii; Salmonella
enterica serovar typhi
Mycobacterium avium subsp. avium
Zea mays
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Leishmania major
Homo sapiens; Ralstonia solanacearum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Caulobacter crescentus pv. citri
Xanthomonas axonopodis
Sinorhizobium meliloti
Pseudomonas syringae pv. syringae; Xanthomonas
axonopodis pv. citri; Shigella flexneri
Leishmania major
Pseudomonas fluorescens; Pseudomonas aeruginosa;
Sorangium cellulosum; Drosophila melanogaster
Yersinia enterocolitica; Yersinia pestis; Azotobacter
vinelandii; Escherichia coli; Ralstonia solanacearum;
Streptomyces avermitilis; Pseudomonas fluorescens;
Mycobacterium smegmatis; Streptomyces verticillus;
Streptomyces atroolivaceus
Mesorhizobium loti; Oryza sativa; Novosphingobium
aromaticivorans; Halobacterium sp. NRC-1; Torrubiella
arachnophilus
Agrobacterium tumefaciens
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Oryza sativa
Xanthomonas campestris pv. campestris (ATCC 3391)
Corynebacterium efficiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mus musculus; Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas fluorescens
Rhodospirillum rubrum; Xanthomonas axonopodis pv.
citri
Plasmodium vivax
Pseudomonas putida
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Anopheles stephensi; Drosophila melanogaster
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mus musculus
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Azotobacter vinelandii
Schizosaccharomyces pombe
Streptomyces fradiae
Mycobacterium avium; Desulfitobacterium hafniense
Streptomyces fradiae; Mycobacterium avium subsp.
avium; Homo sapiens; Synechococcus sp.
Mycobacterium avium
Mycobacterium avium; Sinorhizobium meliloti
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Triglochin maritima
Mycobacterium avium subsp. avium
Pseudomonas syringae pv. syringae
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium tuberculosis; Mycobacterium smegmatis;
Mycobacterium avium (strain 2151)
Mycobacterium tuberculosis
Mycobacterium tuberculosis; Mycobacterium leprae,
Mycobacterium avium (strain 2151)
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium avium (strain 2151); Mycobacterium
tuberculosis; Plasmodium falciparum; Thermobifida
fusca; Mus musculus; Xanthomonas campestris pv.
Campestris (ATCC 3391); Mycobacterium smegmatis;
Medicago truncatula
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Mycobacterium bovis
Mycobacterium avium subsp. avium
Rattus norvegicus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Bacteroides thetaiotaomicron
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Homo sapiens
Salmonella enterica serovar typhi; Salmonella
typhimurium
Caenorhabditis elegans
Mycobacterium avium subsp. avium
Streptomyces lavendulae; Mycobacterium tuberculosis;
Homo sapiens; Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Burkholderia fungorum
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mus musculus
Danio rerio
Mycobacterium avium subsp. avium
Salmonella enteritidis
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Mycobacterium avium;
Mycobacterium leprae; Nocardia brasiliensis;
Streptomyces antibioticus; Streptomyces galilaeus;
Streptomyces avermitilis; Agrobacterium tumefaciens;
Streptomyces narbonensis
Mycobacterium tuberculosis; Mycobacterium leprae;
Xanthomonas axonopodis
Mycobacterium avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Nostoc sp.
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Xanthomonas axonopodis
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Burkholderia fungorum
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis; Mycobacterium bovis
Mycobacterium tuberculosis; Mycobacterium leprae;
Mycobacterium bovis
Mycobacterium tuberculosis; Homo sapiens;
Pseudomonas fluorescens; Oryza sativa
Rhodospirillum rubrum; Mycobacterium bovis;
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium bovis; Mycobacterium tuberculosis; Mus
musculus
Sorangium cellulosum
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Myxococcus xanthus
Azotobacter vinelandii; Mycobacterium tuberculosis
Mycobacterium leprae; Mycobacterium bovis;
Mycobacterium tuberculosis; Azotobacter vinelandii;
Streptomyces sp. ; Mus musculus
Mycobacterium tuberculosis; Mycobacterium bovis;
Stigmatella aurantiaca; Micromonospora megalomicea;
Streptomyces hygroscopicus
Mycobacterium tuberculosis; Mycobacterium bovis;
Mycobacterium tuberculosis; Mycobacterium leprae;
Streptomyces avermitilis
Mycobacterium tuberculosis
Saccharopolyspora erythraea; Mycobacterium leprae;
Mycobacterium tuberculosis; Homo sapiens; Caulobacter
crescentus; Mus musculus; Streptomyces nodosus
Mesorhizobium loti; Mycobacterium tuberculosis
Mycobacterium bovis; Mycobacterium tuberculosis;
Streptomyces sp.
Mycobacterium tuberculosis
Sinorhizobium meliloti
Mycobacterium tuberculosis; Amycolatopsis orientalis
Mycobacterium tuberculosis
Gallus gallus
Desulfitobacterium hafniense; Ralstonia solanacearum;
Streptomyces coelicolor; Oryza sativa; Zea mays
Mycobacterium tuberculosis; Desulfitobacterium
hafniense; Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium
Myxococcus xanthus; Ralstonia metallidurans
Mycobacterium avium subsp. avium
Corynebacterium efficiens; Novosphingobium
aromaticivorans; Escherichia coli
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Amycolatopsis orientalis
Magnetospirillum magnetotacticum; Ralstonia
solanacearum; Caulobacter crescentus
Enterococcus saccharolyticus
Mycobacterium avium subsp. avium
Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Bifidobacterium longum;
Neisseria meningitides; Bifidobacterium longum;
Streptomyces avermitilis; Mesorhizobium loti;
Chloroflexus aurantiacus; Xylella fastidiosa;
Pseudomonas syringae pv. syringae; Corynebacterium
efficiens; Agrobacterium tumefaciens; Nitrosomonas
europaea; Rhodobacter capsulatus; Ralstonia
solanacearum; Sinorhizobium meliloti; Halobacterium sp.
Rhodospirillum rubrum; Pseudomonas resinovorans
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa; Ralstonia metallidurans;
Xanthomonas campestris; Xanthomonas axonopodis
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Mycobacterium leprae; Mycobacterium tuberculosis;
Homo sapiens; Streptomyces coelicolor; Oryza sativa
Oryza sativa; Triticum aestivum; Ralstonia solanacearum;
Agrobacterium tumefaciens; Drosophila melanogaster;
Hordeum vulgare
Homo sapiens; Festuca arundinacea; Oryza sativa;
Rhodospirillum rubrum; Magnetospirillum
magnetotacticum; Lolium perenne; Mesorhizobium loti;
Xanthomonas axonopodis; Caulobacter crescentus;
Chlamydomonas reinhardtii
Homo sapiens; Rhodospirillum rubrum; Ralstonia
solanacearum; Mus musculus; Mycobacterium
tuberculosis; Oryza sativa; Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium leprae; Pseudomonas aeruginosa
Mycobacterium leprae
Mycobacterium leprae; Streptomyces nodosus;
Rhodospirillum rubrum; Magnetospirillum
magnetotacticum; Novosphingobium aromaticivorans
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas putida
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Saccharomyces cerevisiae
Mycobacterium avium subsp. avium
Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Brucella melitensis; Brucella suis
Desulfitobacterium hafniense
Mycobacterium avium subsp. avium
Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Mycobacterium leprae; Mycobacterium tuberculosis;
Thermobifida fusca; Pseudomonas sp.; Streptomyces
coelicolor; Mycobacterium bovis; Pseudomonas
fluorescens; Bifidobacterium longum; Corynebacterium
efficiens; Rhodospirillum rubrum; Corynebacterium
glutamicum; Agrobacterium tumefaciens; Shewanella
oneidensis; Rhodobacter capsulatus; Methanosarcina
barkeri; Methanosarcina acetivorans
Pseudomonas denitrificans; Mus musculus;
Mycobacterium tuberculosis; Tupaia herpesvirus
Streptomyces coelicolor; Merxmuellera davyi; Gallus
gallus; Magnetospirillum magnetotacticum; Rhodobacter
capsulatus; Deinococcus radiodurans; Mus musculus
Ralstonia solanacearum; Homo sapiens; Sus scrofa; Oryza
sativa; Mus musculus
Homo sapiens; Frankia sp.; Phleum pretense; Oryza
sativa; Streptomyces plicatus; Mus musculus; Canis
familiaris; Streptomyces violaceoruber; Streptomyces
coelicolor
Mycobacterium tuberculosis; Xanthomonas axonopodis;
Leishmania major
Trypanosoma cruzi; Crematogaster smithi; Salmonella
typhimurium; Leishmania major; Xanthomonas
campestris; Mus musculus; Nitrosomonas europaea;
Drosophila melanogaster; Homo sapiens
Streptomyces coelicolor; Streptomyces lividans;
Xanthomonas campestris
Acetobacter xylinus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Chlamydomonas reinhardtii
Mycobacterium tuberculosis; Burkholderia fungorum;
Corynebacterium efficiens
Mycobacterium tuberculosis
Mycobacterium tuberculosis; Mycobacterium bovis;
Mycobacterium leprae; Polycentropus flavomaculatus;
Caulobacter crescentus; Homo sapiens; Burkholderia
fungorum; Oryza sativa; Cowdria ruminantium;
Pseudomonas aeruginosa
Rhodopseudomonas palustris; Homo sapiens; Oryza
sativa; Leishmania major; Mycobacterium tuberculosis;
Mesorhizobium loti; Ralstonia solanacearum;
Streptomyces pristinaespiralis; Frankia sp.
Desulfitobacterium hafniense; Mycobacterium
tuberculosis
Mus musculus; Burkholderia fungorum; Drosophila
melanogaster
Ralstonia solanacearum; Homo sapiens; Caulobacter
crescentus; Gluconacetobacter xylinus; Rhodobacter
sphaeroides; Rhodospirillum rubrum; Mycobacterium
leprae; Oryza sativa; Pseudomonas aeruginosa;
Pseudomonas putida
Homo sapiens; Rattus norvegicus; Mus musculus
Homo sapiens; Mus musculus; Streptomyces coelicolor;
Hormoconis resinae; Drosophila melanogaster; Oryza
sativa; Gallus gallus
Homo sapiens; Papio anubis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mesorhizobium loti; Mus musculus; Rhodopseudomonas
palustris
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Burkholderia fungorum
Caenorhabditis elegans
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum
Oryza sativa; Rhodobacter sphaeroides; Halobacterium
Klebsiella pneumoniae; Burkholderia fungorum;
Streptomyces coelicolor; Oryza sativa; Ralstonia
metallidurans; Halobacterium sp. NRC-1
Mycobacterium tuberculosis; Ralstonia solanacearum;
Rhodobacter sphaeroides; Ralstonia metallidurans;
Novosphingobium aromaticivorans; Desulfitobacterium
hafniense; Bovine herpesvirus type 1
Mesorhizobium loti; Streptomyces coelicolor;
Burkholderia fungorum; Desulfitobacterium hafniense;
Oryza sativa; Methylovorus sp.
Pseudopleyronectes americanus; Mycobacterium
tuberculosis; Mus musculus; Urochloa panicoides; Oryza
sativa; Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa
Ralstonia solanacearum
Streptomyces coelicolor
Zantedeschia aethiopica; Streptomyces coelicolor
Paucimonas lemoignei
Mycobacterium tuberculosis; Mus musculus; Homo
sapiens; Desulfitobacterium hafniense; Ralstonia
solanacearum; Salmonella typhimurium
Mycobacterium tuberculosis; Streptomyces atroolivaceus;
Ralstonia solanacearum; Lysobacter enzymogenes;
Leishmania major; Cupiennius salei; Oryza sativa;
Streptomyces coelicolor; Pseudomonas aeruginosa;
Magnetospirillum magnetotacticum; Oryza sativa;
Mycobacterium tuberculosis; Desulfitobacterium
hafniense; Rhodopseudomonas palustris; Pseudomonas
aeruginosa; Homo sapiens
Mycobacterium tuberculosis; Streptomyces coelicolor;
Desulfitobacterium hafniense; Oryza sativa; Streptomyces
avermitilis
Desulfitobacterium hafniense; Oryza sativa;
Mycobacterium tuberculosis; Salmonella typhimurium;
Pseudomonas fluorescens; Azotobacter vinelandii;
Spermatozopsis similis
Oryza sativa; Homo sapiens
Mycobacterium tuberculosis; Mycobacterium leprae;
Magnetospirillum magnetotacticum; Oryza sativa
Xanthomonas campestris; Mycobacterium tuberculosis;
Deinococcus radiodurans
Chlamydomonas reinhardtii
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Xanthomonas campestris; Streptomyces nigrifaciens;
Streptomyces phaeochromogenes; Streptomyces noursei
Mycobacterium avium subsp. avium
Desulfitobacterium hafniense
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Arabidopsis thaliana
Xanthomonas axonopodis
Mycobacterium avium subsp. avium
Pseudomonas putida; Mycobacterium smegmatis
Oryza sativa; Homo sapiens
Oryza sativa; Homo sapiens; Rhodospirillum rubrum;
Rhizobium meliloti (Sinorhizobium meliloti)
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens; Pseudomonas fluorescens
Drosophila melanogaster
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Ralstonia metallidurans; Pseudomonas syringae pv.
syringae; Homo sapiens
Mycobacterium avium subsp. avium
Rhodospirillum rubrum; Caenorhabditis elegans
Novosphingobium aromaticivorans; Oryza sativa;
Rhodopseudomonas palustris; Magnetospirillum
magnetotacticum; Corynebacterium glutamicum; Listeria
innocua; Streptomyces coelicolor
Mycobacterium tuberculosis; Streptomyces coelicolor;
Halobacterium sp. NRC-1; Mycobacterium leprae;
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Mycobacterium avium (strain 2151); Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Triticum aestivum
Streptomyces coelicolor; Ralstonia solanacearum;
Microbulbifer degradans; Streptomyces seoulensis;
Caulobacter crescentus; Pseudomonas aeruginosa;
Burkholderia fungorum; Corynebacterium efficiens
Mycobacterium smegmatis; Mycobacterium leprae;
Saccharopolyspora erythraea
Mycobacterium avium (ATCC 35712); Mycobacterium
intracellulare
Mycobacterium avium subsp. avium
Corynebacterium efficiens; Deinococcus radiodurans
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces avermitilis; Magnetospirillum
magnetotacticum; Caulobacter crescentus;
Corynebacterium striatum
Homo sapiens; Halobacterium sp. NRC-1
Caenorhabditis elegans; Pseudomonas fluorescens;
Streptomyces coelicolor; Deinococcus radiodurans
Mycobacterium avium subsp. avium
Desulfitobacterium hafniense; Ralstonia solanacearum
Mycobacterium avium subsp. avium
Agrobacterium tumefaciens; Oryza sativa
Mycobacterium tuberculosis
Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Novosphingobium
aromaticivorans
Alcaligenes eutrophus
Mycobacterium avium subsp. avium
Ictalurid herpesvirus 1 (channel catfish virus)
Rhodobacter sphaeroides
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Drosophila melanogaster
Mycobacterium avium subsp. avium
Glycine max
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Zea mays
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Synechococcus sp.
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Pseudomonas fluorescens; Drosophila melanogaster;
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium; Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Drosophila melanogaster
Homo sapiens
Mycobacterium avium subsp. avium
Halobacterium salinarium
Magnetospirillum magnetotacticum; Ralstonia
solanacearum; Sinorhizobium meliloti
Xanthomonas albilineans
Micromonospora griseorubida
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas paucimobilis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum
Burkholderia fungorum
Mycobacterium avium subsp. avium
Homo sapiens; Mycobacterium bovis; Mycobacterium
tuberculosis
Oryza sativa; Triticum aestivum; Homo sapiens; Mus
musculus; Magnetospirillum magnetotacticum; Hordeum
vulgare; Streptomyces fradiae
Sus scrofa; Desulfitobacterium hafniense; Mycobacterium
tuberculosis; Mus musculus; Homo sapiens; Oryza sativa;
Sinorhizobium meliloti
Deinococcus radiodurans; Burkholderia fungorum;
Desulfovibrio desulfuricans; Mycobacterium tuberculosis
Mycobacterium tuberculosis; Mycobacterium leprae
Mycobacterium tuberculosis; Mus musculus;
Rhodopseudomonas palustris; Streptomyces coelicolor;
Mycobacterium leprae; Rattus norvegicus; Alcaligenes
faecalis; Homo sapiens; Leishmania major; Burkholderia
fungorum; Azotobacter vinelandii; Oryza sativa;
Xanthomonas axonopodis
Streptomyces castaneoglobisporus; Mus musculus; Homo
sapiens; Ralstonia metallidurans; Oryza sativa;
Chlamydomonas reinhardtii; Mycobacterium tuberculosis;
Deinococcus radiodurans
Acidithiobacillus ferroxidans; Rhodobacter sphaeroides;
Mycobacterium leprae; Rhodopseudomonas palustris;
Oryza sativa
Mycobacterium avium subsp. avium
Homo sapiens; Magnetospirillum magnetotacticum; Oryza
sativa; Caulobacter crescentus; Pan troglodytes
Mycobacterium tuberculosis
Pseudomonas aeruginosa
Streptomyces noursei; Burkholderia fungorum; Oryza
sativa; Azospirillum brasilense
Burkholderia mallei
Mycobacterium leprae; Bifidobacterium longum
Mycobacterium avium subsp. avium
Sphingobacterium multivorum; Rhodopseudomonas
palustris; Synechococcus sp.
Pseudomonas aeruginosa
Rhizobium leguminosarum pv. viciae; Oryza sativa
Streptomyces cinnamonensis; Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas putida
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Magnetospirillum magnetotacticum; Geobacter
metallireducens
Mycobacterium avium subsp. avium
Azotobacter vinelandii; Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum; Oryza sativa; Homo
sapiens; Zea mays; Mesorhizobium loti; Azotobacter
vinelandii; Ralstonia solanacearum
Desulfovibrio desulfuricans; Rubrivivax gelatinosus;
Streptomyces coelicolor; Pseudomonas putida; Hepatitis
Azotobacter vinelandii
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas fluorescens; Bifidobacterium longum;
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa
Nostoc punctiforme; Novosphingobium aromaticivorans
Mycobacterium avium subsp. avium
Polyporaceae sp.
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium leprae; Oryza sativa; Mycobacterium
tuberculosis; Rattus norvegicus
Burkholderia cepacia; Halobacterium sp. NRC-1; Homo
sapiens
Rhodobacter sphaeroides; Homo sapiens;
Desulfitobacterium hafniense; Streptomyces coelicolor;
Deinococcus radiodurans; Oryza sativa; Streptomyces
avermitilis; Mesorhizobium loti; Rhodopseudomonas
palustris; Ralstonia metallidurans; Burkholderia
fungorum; Mycobacterium avium (strain GIRO); Ralstonia
solanacearum
Oryza sativa; Homo sapiens; Mesorhizobium loti; Bovine
Mycobacterium avium subsp. avium
Sus scrofa; Mycobacterium smegmatis; Homo sapiens;
Frankia sp.; Streptomyces atroolivaceus
Streptomyces coelicolor; Oryza sativa; Streptomyces
atroolivaceus; Rhodobacter sphaeroides; Rhodospirillum
rubrum; Halobacterium sp. NRC-1
Rhodopseudomonas palustris; Burkholderia fungorum
Mycobacterium avium subsp. avium
Thermobifida fusca
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Pseudomonas aeruginosa;
Magnetospirillum magnetotacticum; Mus musculus
Mus musculus
Streptomyces griseus; Oryza sativa; Halobacterium sp.
Mesorhizobium loti; Pseudomonas fluorescens;
Burkholderia fungorum; Desulfitobacterium hafniense;
Azotobacter vinelandii; Streptomyces coelicolor;
Rhodopseudomonas palustris; Mesorhizobium loti
Mycobacterium tuberculosis; Burkholderia fungorum;
Drosophila melanogaster; Azotobacter vinelandii;
Ralstonia solanacearum; Sinorhizobium meliloti; Oryza
sativa; Pseudomonas fluorescens; Magnetospirillum
magnetotacticum; Zea mays; Cervid herpesvirus
Mycobacterium avium (strain 2151)
Mycobacterium avium subsp. avium
Mycobacterium avium (strain 2151); Streptomyces
coelicolor; Homo sapiens; Magnetospirillum
magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium (strain 2151)
Mycobacterium avium subsp. avium
Thermobifida fusca
Papio anubis; Streptomyces coelicolor
Mycobacterium avium subsp. avium
Rhodopseudomonas palustris; Burkholderia fungorum;
Pseudomonas putida
Mycobacterium avium subsp. avium
Mycobacterium smegmatis
Desulfitobacterium hafniense
Mycobacterium avium subsp. avium
Pseudomonas fluorescens
Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Myxococcus xanthus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mesorhizobium loti
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. aviutn
S. cerevisiae
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Mycobacterium bovis
Mycobacterium tuberculosis; Mycobacterium leprae;
Ralstonia solanacearum; Streptomyces coelicolor;
Caulobacter crescentus; Homo sapiens; Pseudomonas
fluorescens; Streptomyces caelestis; Amycolatopsis
mediterranei; Zea mays
Mycobacterium tuberculosis; Mycobacterium leprae;
Thermobifida fusca; Chlorobium tepidum; Treponema
medium; Spirochete; Treponema denticola; Chloroflexus
aurantiacus; Clostridium thermocellum; Micrococcus
luteus; Deinococcus radiodurans; Mycobacterium phage
Chlorobium tepidum; Mesorhizobium loti; Sinorhizobium
meliloti
Mycobacterium leprae; Mycobacterium tuberculosis;
Streptomyces coelicolor; Bovine herpesvirus type 1;
Lymantria dispar;
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens; Azotobacter vinelandii
Oryza sativa; Streptomyces coelicolor
Mesorhizobium loti
Escherichia coli; Klebsiella pneumoniae; Streptomyces
coelicolor; Enterobacter aerogenes
Streptomyces noursei
Methylobacterium extorquens; Homo sapiens;
Deinococcus radiodurans
Burkholderia fungorum; Desulfitobacterium hafniense;
Brucella abortus; Brucella melitensis; Brucella suis
Mycobacterium avium subsp. avium
Pseudomonas fluorescens;
Streptomyces coelicolor
Rhodopseudomonas palustris; Oryza sativa
Thermosynechococcus elongatus
Rhodospirillum rubrum; Ralstonia solanacearum;
Amycolatopsis mediterranei
Mycobacterium avium subsp. avium
Desulfitobacterium hafniense; Azotobacter vinelandii;
Magnetospirillum magnetotacticum; Caulobacter
crescentus; Pseudomonas aeruginosa
Homo sapiens
Rhodopseudomonas palustris; Microbulbifer degradans;
Deinococcus radiodurans
Homo sapiens; Ralstonia metallidurans
Chromobacterium violaceum; Thermobifida fusca;
Sinorhizobium meliloti; Rhodospirillum rubrum;
Pseudomonas syringae pv. syringae; Rhizobium meliloti
Ralstonia solanacearum; Xanthomonas axonopodis;
Novosphingobium aromaticivorans
Saccharomyces cerevisiae; Ralstonia solanacearum
Mycobacterium tuberculosis; Amycolatopsis lactamdurans
Mycobacterium avium subsp. avium
Pseudomonas aeruginosa; Sinorhizobium meliloti;
Streptomyces coelicolor; Bovine herpesvirus type 1;
Deinococcus radiodurans
Rhodobacter sphaeroides; Rhodopseudomonas palustris;
Rhodospirillum rubrum; Azotobacter vinelandii;
Micromonospora megalomicea subsp. nigra megalomicin;
Agrobacterium tumefaciens; Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Rubrivivax gelatinosus; Ralstonia eutropha
Mycobacterium avium subsp. avium
Homo sapiens
Homo sapiens
Pseudomonas fluorescens; Pseudomonas syringae;
Pseudomonas aeruginosa
Mycobacterium avium subsp. avium
Streptomyces sp. ; Streptomyces coelicolor; Chloroflexus
aurantiacus; Deinococcus radiodurans; Pseudomonas
putida
Rhodopseudomonas palustris; Desulfitobacterium
hafniense
Oryctolagus cuniculus
Streptomyces hygroscopicus
Magnetospirillum magnetotacticum; Streptomyces
coelicolor; Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Oryza sativa; Mesorhizobium loti; Magnetospirillum
magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces coelicolor; Amycolatopsis mediterranei;
Rhodopseudomonas palustris; Rhodospirillum rubrum;
Homo sapiens; Brucella melitensis; Papio anubis; Sus
scrofa; Brucella suis
Pseudomonas aeruginosa
Mycobacterium tuberculosis; Homo sapiens; Bordetella
bronchiseptica
Oryza sativa; Homo sapiens; Streptomyces coelicolor;
Triticum aestivum; Hordeum vulgare; Bos taurus; Mus
musculus
Desulfitobacterium hafniense; Oryza sativa; Homo
sapiens; M. bovis; Streptomyces griseus; Streptomyces
coelicolor; Chlamydomonas reinhardtii; Pseudomonas
putida
Mycobacterium avium subsp. avium
Ralstonia metallidurans
Mycobacterium avium subsp. avium
Deinococcus radiodurans; Streptomyces spheroides;
Ralstonia solanacearum; Streptomyces coelicolor
Homo sapiens; Gallus gallus; Mycobacterium
tuberculosis; Rhodospirillum rubrum; Magnetospirillum
magnetotacticum; Geobacter metallireducens;
Desulfitobacterium hafniense; Streptomyces coelicolor
Mycobacterium tuberculosis; Homo sapiens; Erwinia
chrysanthemi; Streptomyces coelicolor; Xanthomonas
nopodis; Streptomyces avermitilis; Alcaligenes eutrophus;
Desulfitobacterium hafniense; Oryza sativa; Salmonella
enterica; Struthio camelus; Salmonella typhimurium
Mycobacterium avium subsp. avium
Home sapiens; Escherichia coli
Oryza sativa; Chlamydomonas reinhardtii; Homo sapiens;
Mesorhizobium loti; Magnetospirillum magnetotacticum;
Mycobacterium xenopi; Neisseria meningitidis;
Sinorhizobium meliloti; Streptomyces coelicolor;
Myxococcus xanthus;
Oryza sativa; Streptomyces coelicolor; Corynebacterium
efficiens; Myxococcus xanthus; Pyrobaculum aerophilum
Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mesorhizobium loti; Rhodobacter sphaeroides
Mycobacterium avium subsp. avium
Bacillus halodurans
Mycobacterium tuberculosis; Caenorhabditis briggsae
Oryza sativa; Home sapiens
Oryza sativa; Streptomyces coelicolor; Caenorhabditis
briggsae
Mycobacterium tuberculosis; Xanthomonas campestris
Mycobacterium avium subsp. avium
Leishmania major
Mycobacterium tuberculosis; Homo sapiens
Mus musculus; Homo sapiens
Mycobacterium tuberculosis; Mycobacterium leprae;
Chlorobium tepidum; Halobacterium sp.; Rhodospirillum
rubrum; Pseudomonas syringae; Streptomyces coelicolor;
Pseudomonas putida; Leishmania major; Brucella
melitensis; Brucella suis; Rhodobacter sphaeroides;
Sinorhizobium meliloti; Deinococcus radiodurans;
Neurospora crassa
Mycobacterium tuberculosis; Oryza sativa; Molluscum
contagiosum; Rhodopseudomonas palustris;
Mycobacterium bovis; Mus musculus; Magnetospirillum
magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mesorhizobium loti; Rhodopseudomonas palustris;
Ralstonia solanacearum
Mesorhizobium loti
Bacillus halodurans
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Magnetospirillum
magnetotacticum; Azotobacter vinelandii; Ralstonia
solanacearum; Pseudomonas fluorescens; Ralstonia
metallidurans; Magnetococcus sp.; Desulfovibrio
desulfuricans; Brucella melitensis; Brucella suis
Azotobacter vinelandii; Mycobacterium tuberculosis
Oryza sativa; Desulfitobacterium hafniense; Streptomyces
collinus; Aphis gossypii
Chlamydomonas reinhardtii; Homo sapiens;
Mycobacterium tuberculosis; Mycobacterium leprae; Mus
musculus; Magnetospirillum magnetotacticum;
Sinorhizobium meliloti; Tetraodon nigroviridis
Mycobacterium tuberculosis; Streptomyces sp.; Oryza
sativa; Streptomyces griseus; Halobacterium sp.; Homo
sapiens; Pseudomonas fluorescens; Novosphingobium
aromaticivorans; Synechococcus sp.; Drosophila
melanogaster; Streptomyces coelicolor
Mycobacterium avium subsp. avium
Bacillus halodurans
Mycobacterium avium subsp. avium
Mesorhizobium loti; Rhodopseudomonas palustris;
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Sinorhizobium meliloti; Halobacterium sp.
Rhodobacter sphaeroides; Ralstonia solanacearum;
Streptomyces coelicolor
Mycobacterium tuberculosis; Mycobacterium leprae;
Xanthomonas campestris
Oryza sativa; Homo sapiens; Spermatozopsis similis;
Triticum aestivum
Streptomyces verticillus; Frankia sp.; Streptomyces
atroolivaceus; Ralstonia solanacearum; Mycobacterium
tuberculosis; Oryza sativa; Desulfitobacterium hafniense;
Streptomyces coelicolor; Halobacterium sp.; Xanthomonas
campestris; Homo sapiens; Caulobacter crescentus;
Rhodospirillum rubrum; Magnetospirillum
magnetotacticum; Rattus norvegicus; Oryza sativa;
Ralstonia solanacearum; Bovine adenovirus
Mycobacterium tuberculosis; Oryza sativa; Streptomyces
coelicolor; Streptomyces avermitilis; Xanthomonas
campestris; Drosophila melanogaster; Homo sapiens;
Thermobifida fusca
Mycobacterium avium subsp. avium
Oryza sativa; Ralstonia metallidurans; Pseudomonas
putida
Rhodobacter sphaeroides; Ralstonia metallidurans; Zea
mays
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Halobacterium sp.; Corynebacterium efficiens;
Magnetospirillum magnetotacticum; Oryza sativa
Mycobacterium avium subsp. avium
Streptomyces avermitilis
Mycobacterium tuberculosis
Mycobacterium tuberculosis; Oryza sativa
Ralstonia solanacearum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Ralstonia solanacearum
Oryza sativa
Ralstonia metallidurans; Deinococcus radiodurans
Drosophila melanogaster
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Arabidopsis thaliana; Xanthomonas campestris
Mycobacterium avium subsp. avium
Azotobacter vinelandii; Xanthomonas campestris
Mycobacterium avium subsp. avium
Triticum aestivum; Mus musculus
Methylobacterium extorquens
Oryza sativa
Mesorhizobium loti
Streptomycescoelicolor; Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Rhodobacter capsulatus
Mycobacterium avium subsp. avium
Burkholderia fungorum
Streptomyces coelicolor; Drosophila melanogaster
Burkholderia fungorum; S. erythraea; Xanthomonas
axonopodis
Mycobacterium avium subsp. avium
Homo sapiens
Oryza sativa
Methanopyrus kandleri
Thermobifida fusca
Rhodopseudomonas palustris; Desulfonatronum lacustre
Mycobacterium avium subsp. avium
Rhodobacter sphaeroides; Homo sapiens; Sphingomonas
paucimobilis; Caulobacter crescentus;
Mycobacterium avium subsp. avium
Oryza sativa; Novosphingobium aromaticivorans;
Pseudomonas putida
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Streptomyces coelicolor; Pseudomonas aeruginosa;
Ralstonia metallidurans; Magnetospirillum
magnetotacticum; Azoarcus evansii; Rhodobacter
sphaeroides; Halobacterium sp.; Streptomyces collinus;
Caulobacter crescentus
Rhodopseudomonas palustris; Mycobacterium leprae;
Pseudomonas fluorescens; Sinorhizobium meliloti;
Streptomyces coelicolor; Xanthomonas campestris;
Micromonospora echinospora; H. salinarium;
Mesorhizobium loti; Novosphingobium aromaticivorans;
Mycobacterium tuberculosis; Agrobacterium tumefociens
Ralstonia solanacearum
Shigella flexneri; Escherichia coli; Prochlorococcus
marinus; Magnetospirillum magnetotacticum;
Rhodopseudomonas palustris; Burkholderia fungorum;
Thermobifida fusca; Streptomyces coelicolor
Rhodopseudomonas palustris; Pseudomonas aeruginosa;
Burkholderia fungorum; Novosphingobium
aromaticivorans; Streptomyces sp.; Amycolatopsis
mediterranei; Streptomyces coelicolor; Halobacterium sp.;
Pseudomonas putida; Oryza sativa; Micromonospora
megalomicea (subsp. nigra megalomicin); Zea mays
Novosphingobium aromaticivorans; Magnetospirillum
magnetotacticum; Rhodopseudomonas palustris;
Thermobifida fusca; Thauera aromatica; Caulobacter
crescentus
Deinococcus radiodurans
Mycobacterium avium subsp. avium
Mesorhizobium loti; Burkholderia fungorum
Ralstonia solanacearum; Hordeum vulgare; Ralstonia
metallidurans; Xanthomonas axonopodis; Caulobacter
crescentus; Burkholderia fungorum; Streptomyces
antibioticus; Corynebacterium efficiens; Xanthomonas
campestris; Zea mays
Mesorhizobium loti; Ralstonia metallidurans;
Desulfitobacterium hafniense; Agrobacterium tumefaciens
Mycobacterium avium subsp. avium
Chlorobium tepidum; Pseudomonas fluorescens; Ralstonia
metallidurans; Burkholderia fungorum; Mycobacterium
tuberculosis; Streptomyces lavendulae; Novosphingobium
aromaticivorans; Ralstonia solanacearum; Streptomyces
coelicolor; Oryza sativa; Xanthomonas axonopodis;
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces griseus
Ralstonia metallidurans
Mesorhizobium loti; Azotobacter vinelandii; Streptomyces
coelicolor; Homo sapiens
Brucella melitensis; Brucella suis
Burkholderia fungorum; Ralstonia solanacearum
Thermobifida fusca; Oryza sativa
Novosphingobium aromaticivorans; Streptomyces
coelicolor; Mesorhizobium loti; Sinorhizobium meliloti;
Homo sapiens
Mycobacterium avium subsp. avium
Caulobacter crescentus; Magnetospirillum
magnetotacticum; Streptomyces coelicolor
Sinorhizobium meliloti
Saccharopolyspora erythraea
Leishmania major
Mycobacterium avium subsp. avium
Streptomyces coelicolor; Burkholderia fungorum
Ralstonia solanacearum; Thermobifida fusca
Oryza sativa
Sinorhizobium meliloti; Rhizobium sp.
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces coelicolor; Lactococcus lactis subsp. Lactis
Mycobacterium tuberculosis; Desulfitobacterium
hafniense; Ralstonia solanacearum; Streptomyces
coelicolor; Mesorhizobium loti; Homo sapiens;
Rhodospirillum rubrum; Ralstonia metallidurans; Xylella
fastidiosa; Azotobacter vinelandii; Prochlorococcus
marinus; Oryza sativa; Sinorhizobium meliloti
Ralstonia solanacearum; Streptomyces coelicolor;
Magnetospirillum magnetotacticum; Azotobacter
vinelandii; Homo sapiens; Ralstonia solanacearum;
Streptomyces coelicolor; Oryza sativa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Azotobacter vinelandii
Corynebacterium efficiens; Bifidobacterium longum;
Mycobacterium avium (strain 2151)
Mycobacterium avium subsp. avium
Streptomyces lividans; Mycobacterium tuberculosis;
Burkholderia fungorum; Zea mays
Renibacterium salmoninarum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Pseudomonas syringae; Desulfitobacterium hafniense
Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Thermus equiperdum
Mycobacterium avium subsp. avium
Desulfovibrio desulfuricans
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Neurospora crassa
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Escherichia coli
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum
Mycobacterium avium subsp. avium
Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Mastigamoeba balamuthi
Mycobacterium avium subsp. avium
Homo sapiens; Neurospora crassa; Bifidobacterium
longum; Sinorhizobium meliloti
Mycobacterium avium subsp. avium
Halobacterium salinarium; Magnetospirillum
magnetotacticum; Halobacterium sp.; Rhodopseudomonas
palustris; Rhodospirillum rubrum; Streptomyces fradiae;
Zea mays
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis
Agaricus bisporus; Yersinia pestis
Oryza sativa
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Burkholderia fungorum;
Drosophila melanogaster; Thermobifida fusca;
Streptomyces clavuligerus
Drosophila melanogaster; Mycobacterium tuberculosis;
Streptomyces coelicolor
Oryza sativa; Deinococcus radiodurans; Ralstonia
solanacearum; Streptomyces coelicolor
Mycobacterium avium (strain 2151)
Mycobacterium avium (strain 2151); Streptomyces
coelicolor; Magnetospirillum magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium (strain 2151)
Mycobacterium avium; Pseudomonas fluorescens; Oryza
sativa; Pseudomonas putida
Mycobacterium avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Neurospora crassa
Drosophila melanogaster; Halobacterium sp.
Homo sapiens
Mycobacterium avium subsp. avium
Leishmania major; Oryza sativa; Pseudomonas
fluorescens; Homo sapiens; Rhodobacter sphaeroides;
Brucella melitensis; Brucella suis; Zea mays
Neisseria meningitidis; Streptomyces viridochromogenes
Mycobacterium tuberculosis; Mus musculus; Pseudorabies
Mycobacterium tuberculosis; Mycobacterium bovis
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Streptomyces coelicolor;
Micromonospora megalomicea subsp. nigra megalomicin
Mycobacterium leprae; Rhodopseudomonas palustris
Mycobacterium tuberculosis; Mycobacterium leprae;
Oryza sativa; Rhodospirillum rubrum; Magnetospirillum
magnetotacticum; Zea mays; Streptomyces griseus; Homo
sapiens
Mus musculus
Homo sapiens; Magnetospirillum magnetotacticum; Rattus
norvegicus; Oryctolagus cuniculus; Mycobacterium
leprae; Oryza sativa; Mus musculus
Magnetospirillum magnetotacticum; Mus musculus; Homo
sapiens; Rhodopseudomonas palustris; Caulobacter
crescentus
Mycobacterium avium subsp. avium
Acetobacter xylinus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Chlamydomonas reinhardtii;
Burkholderia fungorum; Corynebacterium efficiens
Ralstonia sp.; Ralstonia metallidurans; Bifidobacterium
longum
Sus scrofa; Saimiri sciureus; Bos taurus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium tuberculosis; Burkholderia fungorum;
Acetobacter vinelondii; Pseudomonas aeroginosa;
Salmonella enterica; Salmonella typhi
Oryza sativa; Trichomonas vaginalis
Rhodospirillum rubrum
Streptomyces coelicolor
Magnetospirillum magnetotacticum; Homo sapiens;
Rhodospirillum rubrum; Chloroflexus aurantiacus
Mesorhizobium loti; Pseudomonas syringae
Streptomyces virginiae
Mus musculus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Microbispora bispora
Mycobacterium avium subsp. avium
Burkholderia fungorum; Oryza sativa
Mycobacterium avium subsp. avium
Ralstonia solanacearum; Rhodobacter sphaeroides;
Xanthomonas campestris; Streptomyces avermitilis;
Sphingomonas paucimobilis; Streptomyces coelicolor
Homo sapiens
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Triticum aestivum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Rhodobacter sphaeroides
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum
Drosophila melanogaster
Desulfitobacterium hafniense; Azotobacter vinelandii;
Pseudomonas fluorescens; Leishmania major
Neisseria meningitides
Desulfitobacterium hafniense; Burkholderia fungorum
Mycobacterium avium subsp. avium
Drosophila melanogaster
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Brucella abortus; Brucella melitensis; Brucella suis;
Caenorhabditis elegans
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Salmonella enterica; Salmonella typhimurium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Novosphingobium aromaticivorans
Rhodobacter sphaeroides; Drosophila melanogaster
Amycolatopsis orientalis
Homo sapien
Rhodopseudomonas palustris
Mycobacterium avium subsp. avium
Desulfitobacterium hafniense; Streptomyces coelicolor;
Hordeum vulgare; Thauera aromatica; Mesorhizobium
loti; Ralstonia solanacearum; Xanthomonas campestris;
Pseudomonas aeruginosa
Mesorhizobium loti; Rhodobacter sphaeroides
Mycobacterium avium subsp. avium
Desulfitobacterium hafniense
Caenorhabditis elegans
Halothiobacillus hydrothermalis
Azotobacter vinelandii; Streptomyces coelicolor;
Magnetospirillum magnetotacticum; Pseudomonas
aeruginosa; Streptomyces atroolivaceus
Homo sapiens; Streptomyces coelicolor; Oryza sativa; Zea
mays; Aegilops tauschii; Gallus gallus; Mus musculus;
Rhodobacter sphaeroides; Rhodospirillum rubrum
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Streptomyces coelicolor
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Chloroflexus aurantiacus
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Homo sapiens
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Azotobacter vinelandii; Coffea arabica
Homo sapiens
Agrobacterium tumefaciens
Mycobacterium avium subsp. avium
Mus musculus
Mycobacterium avium subsp. avium
Magnetospirillum magnetotacticum; Xanthomonas
axonopodis;
Streptomyces coelicolor
Mycobacterium leprae
Mycobacterium avium subsp. avium
Mycobacterium avium subsp. avium
Serratia marcescens
Mycobacterium tuberculosis; Mycobacterium leprae;
Deinococcus radiodurans; Xanthomonas campestris
Mus musculus; Streptomyces coelicolor
Rhodococcus opacus; Oryza sativa
Homo sapiens; Ralstonia metallidurans; Desulfovibrio
desulfuricans; Azotobacter vinelandii; Desulfitobacterium
hafniense; Streptomyces coelicolor
Mycobacterium tuberculosis; Pseudomonas syringae;
Burkholderia fungorum; Lysobacter sp.
Pseudomonas aeruginosa; Mycobacterium tuberculosis;
Leishmania major; Burkholderia fungorum; Amycolatopsis
mediterranei; Streptomyces nodosus
Mycobacterium avium subsp. avium
Rhodospirillum rubrum; Thermobifida fusca; Ralstonia
solanacearum
Mycobacterium avium subsp. avium
Thermobifida fusca
Mycobacterium leprae
Bifidobacterium longum
Pseudomonas putida; Desulfitobacterium hafniense
Homo sapiens
Mycobacterium avium subsp. avium
Rhodospirillum rubrum
Mycobacterium avium subsp. avium
Pseudomonas fluorescens
Rhodobacter sphaeroides; Bacillus stearothermophilus
Mycobacterium avium subsp. avium
Mycobacterium kansasii; Desulfitobacterium hafniense;
Rhizobium meliloti; Gallus gallus
Mycobacterium tuberculosis; Azotobacter vinelandii
Mycobacterium avium (strain 2151)
Mycobacterium avium (strain 2151); Streptomyces
coelicolor; Homo sapiens; Magnetospirillum
magnetotacticum
Mycobacterium avium subsp. avium
Mycobacterium avium (strain 2151)
Hordeum vulgare; Amycolatopsis mediterranei; Lymantria
dispar nuclear polyhedrosis virus; Oryza sativa; Bovine
Rhodospirillum rubrum; Triticum aestivum
Mesorhizobium loti; Ralstonia solanacearum;
Thermobifida fusca; Mycobacterium tuberculosis;
Caulobacter crescentus; Homo sapiens
Streptomyces coelicolor; Homo sapiens; Streptomyces
avermitilis; Rhodobacter sphaeroides; Rhodospirillum
rubrum; Desulfitobacterium hafniense; Oryza sativa
Burkholderia fungorum; Homo sapiens; Cavia porcellus;
Azotobacter vinelandii
Mycobacterium tuberculosis; Mycobacterium leprae;
Ralstonia solanacearum; Ralstonia metallidurans; Xylella
fastidiosa; Streptomyces clavuligerus; Pseudomonas
aeruginosa; Mesorhizobium loti; Myxococcus xanthus;
Zea mays; Homo sapiens; Desulfovibrio desulfuricans
Xanthomonas axonopodis; Actinosynnema pretiosum
Azotobacter vinelandii; Ralstonia eutropha;
Bifidobacterium longum; Pseudomonas stutzeri;
Xanthomonas campestris;
Mycobacterium avium subsp. avium
Mesorhizobium loti; Magnetospirillum magnetotacticum;
Mycobacterium tuberculosis; Bifidobacterium longum;
Geobacter metallireducens; Klebsiella pneumoniae;
Pseudomonas aeruginosa
Mycobacterium tuberculosis; Pseudomonas alcaligenes;
Homo sapiens
Actinosynnema pretiosum subsp. auranticum maytansino
Homo sapiens; Sinorhizobium meliloti
Caulobacter crescentus; Ralstonia solanacearum;
Deinococcus radiodurans
Mycobacterium leprae; Mycobacterium tuberculosis;
Leishmania major
Ralstonia solanacearum; Leishmania major; Pseudomonas
aeruginosa; Deinococcus radiodurans; Magnetospirillum
magnetotacticum; Streptomyces coelicolor; Oryza sativa
Homo sapiens; Mycobacterium tuberculosis; Leishmania
major; Mesorhizobium loti; Oryza sativa; Deinococcus
radiodurans; Chlamydomonas reinhardtii
Mycobacterium avium subsp. avium
Escherichia coli
Bifidobacterium longum; Rattus norvegicus
Rhodococcus sp.; Mesorhizobium loti
Mycobacterium tuberculosis; Novosphingobium
aromaticivorans; Caulobacter crescentus; Streptomyces
coelicolor
Mycobacterium tuberculosis; Streptomyces coelicolor;
Azotobacter vinelandii; Ralstonia solanacearum; Oryza
sativa; Homo sapiens; Chloroflexus aurantiacus;
Ralstonia metallidurans; Burkholderia fungorum;
Desulfitobacterium hafniense; Zea mays; Mus musculus
Mycobacterium tuberculosis; Mycobacterium leprae;
Giardia microti; Xanthomonas axonopodis; Paracoccus
denitrificans; Streptomyces coelicolor; Caulobacter
crescentus; Giardia lamblia; Magnetospirillum
magnetotacticum; Pseudomonas syringae; Giardia
intestinalis
Mycobacterium avium subsp. avium
With respect to the organisms identified in Table 1, some of them represent multiple species, subspecies, or strains. To test whether or not particular reagents distinguish between M. paratuberculosis and such species, subspecies, or strains, it may be desirable to test a representative number of species, subspecies, or strains, respectively. In cases where the genetic variation is minimal within the species, subspecies, or strains, it may not be necessary to test more than one or two species, subspecies, or strains, respectively. In other cases, multiple species, subspecies, or strains may need to be tested, although initial testing can focus on the most genetically distant species, subspecies, or strains, respectively.
In another aspect, the invention provides for vectors comprising a nucleic acid of the invention. Host cells comprising such a vector are further provided by the invention.
In yet another aspect, the invention provides for isolated polypeptides encoded by the nucleic acids of the invention. For example, the nucleic acid molecule having the sequence of SEQ ID NO:1 can encode a polypeptide having an amino acid sequence of SEQ ID NO:24; the nucleic acid molecule having the sequence of SEQ ID NO:2 can encode a polypeptide having an amino acid sequence of SEQ ID NO:25; the nucleic acid molecule having the sequence of SEQ ID NO:3 can encode a polypeptide having an amino acid sequence of SEQ ID NO:26; the nucleic acid molecule having the sequence of SEQ ID NO:4 can encode a polypeptide having an amino acid sequence of SEQ ID NO:27; the nucleic acid molecule having the sequence of SEQ ID NO:5 can encode a polypeptide having an amino acid sequence of SEQ ID NO:28; the nucleic acid molecule having the sequence of SEQ ID NO:6 can encode a polypeptide having an amino acid sequence of SEQ ID NO:29; the nucleic acid molecule having the sequence of SEQ ID NO:7 can encode a polypeptide having an amino acid sequence of SEQ ID NO:30; the nucleic acid molecule having the sequence of SEQ ID NO:8 can encode a polypeptide having an amino acid sequence of SEQ ID NO:31; the nucleic acid molecule having the sequence of SEQ ID NO:9 can encode a polypeptide having an amino acid sequence of SEQ ID NO:32; the nucleic acid molecule having the sequence of SEQ ID NO:10 can encode a polypeptide having an amino acid sequence of SEQ ID NO:33; the nucleic acid molecule having the sequence of SEQ ID NO:11 can encode a polypeptide having an amino acid sequence of SEQ ID NO:34; the nucleic acid molecule having the sequence of SEQ ID NO:12 can encode a polypeptide having an amino acid sequence of SEQ ID NO:35; the nucleic acid molecule having the sequence of SEQ ID NO:13 can encode a polypeptide having an amino acid sequence of SEQ ID NO:36; the nucleic acid molecule having the sequence of SEQ ID NO:14 can encode a polypeptide having an amino acid sequence of SEQ ID NO:37; the nucleic acid molecule having the sequence of SEQ ID NO:15 can encode a polypeptide having an amino acid sequence of SEQ ID NO:38; the nucleic acid molecule having the sequence of SEQ ID NO:16 can encode a polypeptide having an amino acid sequence of SEQ ID NO:39; the nucleic acid molecule having the sequence of SEQ ID NO:17 can encode a polypeptide having an amino acid sequence of SEQ ID NO:40; the nucleic acid molecule having the sequence of SEQ ID NO:18 can encode a polypeptide having an amino acid sequence of SEQ ID NO:41; the nucleic acid molecule having the sequence of SEQ ID NO:19 can encode a polypeptide having an amino acid sequence of SEQ ID NO:42; the nucleic acid molecule having the sequence of SEQ ID NO:20 can encode a polypeptide having an amino acid sequence of SEQ ID NO:43; the nucleic acid molecule having the sequence of SEQ ID NO:21 can encode a polypeptide having an amino acid sequence of SEQ ID NO:44; and the nucleic acid molecule having the sequence of SEQ ID NO:22 can encode a polypeptide having an amino acid sequence of SEQ ID NO:45.
In another aspect, the invention provides articles of manufacture that include one or more polypeptides of the invention. In still another aspect of the invention, there are provided antibodies that have specific binding affinity for a polypeptide of the invention.
In another aspect, the invention provides for methods for detecting the presence or absence of M. paratuberculosis in a biological sample. Such methods include contacting the biological sample with one or more of the nucleic acids of the invention (e.g., SEQ ID NOs:1-23 and 110-1342) under standard amplification conditions, wherein an amplification product is produced if M. paratuberculosis nucleic acid is present in the biological sample; and detecting the presence or absence of the amplification product. Generally, the presence of the amplification product indicates the presence of M. paratuberculosis in the biological sample, and the absence of the amplification product indicates the absence of M. paratuberculosis in the biological sample. Representative animals from which the biological sample can be derived include a cow, a sheep, a goat, a rabbit, a deer, an antelope, a bison, or a human. Representative biological samples include a fecal sample and a milk sample. Further, representative nucleic acids that can be used in the above-described methods include those having the sequence of SEQ ID NO:46-101 and 1343-1354.
In another aspect, the invention provides methods for detecting the presence or absence of M. paratuberculosis in a biological sample. Such methods include contacting the biological sample with one or more of the nucleic acids of the invention (e.g., SEQ ID NOs:1-23 and 110-1342) under hybridization conditions, wherein a hybridization complex is produced if M. paratuberculosis nucleic acid molecules are present in the biological sample; and detecting the presence or absence of the hybridization complex. Generally, the presence of the hybridization complex indicates the presence of M. paratuberculosis in the biological sample, and the absence of the hybridization complex indicates the absence of M. paratuberculosis in the biological sample. Typically, nucleic acids present in the biological sample are electrophoretically separated. Such electrophoretically separated nucleic acids can be attached to a solid support. Representative solid supports include nylon membranes and nitrocellulose membranes. Further, one or more nucleic acids can be labeled. Representative biological samples include a fecal sample, a milk sample, and a blood sample.
In another aspect, the invention provides methods for detecting the presence or absence of M. paratuberculosis in a biological sample. Such methods include contacting the biological sample with a polypeptide of the invention (e.g., SEQ ID NOs:24-45), wherein a polypeptide-antibody complex is produced if an antibody having specific binding affinity for the polypeptide is present in the sample; and detecting the presence or absence of the polypeptide-antibody complex. Typically, the presence of the polypeptide-antibody complex indicates the presence of M. paratuberculosis in the biological sample, and the absence of the polypeptide-antibody complex indicates the absence of M. paratuberculosis in the biological sample. Polypeptides used in the above-described method can be attached to a solid support. Further, representative biological samples include a blood sample and a milk sample.
In yet another aspect, the invention provides for methods for detecting the presence or absence of M. paratuberculosis in a biological sample. Such methods include contacting the biological sample with an antibody of the invention (e.g., an antibody having specific binding affinity for a polypeptide having an amino acid sequence of SEQ ID NOs:24-45), wherein an antibody-polypeptide complex is produced if a polypeptide is present in the biological sample for which the antibody has specific binding affinity, and detecting the presence or absence of the antibody-polypeptide complex. Generally, the presence of the antibody-polypeptide complex indicates the presence of M. paratuberculosis in the biological sample, and the absence of the antibody-polypeptide complex indicates the absence of M. paratuberculosis in the biological sample. Antibodies used in the above-described methods can be bound to a solid support. Representative biological samples that can be used in the above-described methods include a blood sample and a milk sample.
In still another aspect of the invention, there are provided methods of preventing infection by M. paratuberculosis in an animal. Such methods include administering a compound to the animal, wherein the compound comprises a polypeptide of the invention (e.g., SEQ ID NOs:24-45). Alternatively, such methods include administering a compound to the animal, wherein the compound comprises a nucleic acid of the invention (e.g., a nucleic acid comprising a nucleic acid molecule having at least 75% sequence identity to SEQ ID NOs:1-23, 110-1341, or 1342). Typically, the compound immunizes the animal against M. paratuberculosis.
In another aspect of the invention, there is provided a composition comprising a first oligonucleotide primer and a second oligonucleotide primer, wherein the first oligonucleotide primer and the second oligonucleotide primer are each 10 to 50 nucleotides in length, and wherein the first and second oligonucleotide primers, in the presence of M. paratuberculosis nucleic acid, generate an amplification product under standard amplification conditions, but do not generate an amplification product in the presence of M. avium subsp. avium nucleic acid. The invention further provides for an article of manufacture comprising such a composition.
In yet another aspect of the invention, there is provided an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid molecule greater than 10 nucleotides in length. Such a molecule has at least 75% sequence identity to SEQ ID NO:1355 or to the complement of SEQ ID NO:1355, and hybridizes under stringent conditions with M. paratuberculosis nucleic acid but does not hybridize with M. avium subsp. avium nucleic acid under the same hybridization conditions. Any of the sequences disclosed herein (e.g., SEQ ID NOs:1-23, 110-1341, or 1342) can be used to design one or more oligonucleotide primers.
In still another aspect of the invention, there is provided articles of manufacture. Articles of manufacture of the invention can include one or more isolated nucleic acids of the invention.
In yet another aspect of the invention, the reagents and methods disclosed herein are used by a diagnostic service provider (e.g., a diagnostic testing laboratory) to obtain and report test results. For example, a provider could send out, or otherwise provide, a collection vial or other container to a customer (recipient) desiring the provider's diagnostic testing services. The container can be part of a collection kit including sterile plastic ware components, for example. The collection kit further can include instructions for collecting a biological sample (e.g., feces, or milk), and for returning the container, with the sample, to the provider. Upon return to the provider, the provider can perform a diagnostic assay on the sample using the reagents and methods disclosed herein. Following completion of the assay, the provider can send or otherwise communicate results of the assay to the recipient.
Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.
The close genetic relationship between M. paratuberculosis and M. avium has made difficult the identification of nucleic acids and polypeptides specific to M. paratuberculosis that can be used with high sensitivity and specificity to detect M. paratuberculosis. The present invention provides nucleic acid molecules that are unique to M. paratuberculosis. The invention also provides the M. paratuberculosis-specific polypeptides encoded by the nucleic acid molecules of the invention, and antibodies having specific binding affinity for the M. paratuberculosis-specific polypeptides. The nucleic acid molecules, polypeptides, and antibodies of the invention can be used in methods of the invention to detect M. paratuberculosis in a sample. The invention additionally provides methods of preventing a M. paratuberculosis infection in an animal.
Isolated M. paratuberculosis-Specific Nucleic Acid Molecules
The present invention is based, in part, on the identification of nucleic acid molecules that are unique to M. paratuberculosis. These nucleic acid molecules are herein referred to as “M. paratuberculosis-specific” nucleic acid molecules. Particular nucleic acid molecules of the invention include the sequences shown in SEQ ID NOs:1-23 and 110-1342. As used herein, the term “nucleic acid molecule” can include DNA molecules and RNA molecules and analogs of the DNA or RNA molecule generated using nucleotide analogs. A nucleic acid molecule of the invention can be single-stranded or double-stranded, and the strandedness will depend upon its intended use.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NOs:1-23 and 110-1342. Nucleic acid molecules of the invention include molecules that are at least 10 nucleotides in length and that have at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 95%, or 99% sequence identity) to any of SEQ ID NOs:1-23 and 110-1342. The full-length sizes of each of the novel M. paratuberculosis-specific nucleic acid molecules having the sequences shown in SEQ ID NOs:1-23 are indicated in Table 2. Nucleic acid molecules that differ in sequence from the nucleic acid sequences shown in SEQ ID NOs:1-23 and 110-1342 can be generated by standard techniques, such as site-directed mutagenesis or PCR-mediated mutagenesis. In addition, nucleotide changes can be introduced randomly along all or part of the M. paratuberculosis-specific nucleic acid molecule, such as by saturation mutagenesis. Alternatively, nucleotide changes can be introduced into a sequence by chemically synthesizing a nucleic acid molecule having such changes.
In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It will be appreciated that a single sequence can align differently with other sequences and hence, can have different percent sequence identity values over each aligned region. It is noted that the percent identity value is usually rounded to the nearest integer. For example, 78.1%, 78.2%, 78.3%, and 78.4% are rounded down to 78%, while 78.5%, 78.6%, 78.7%, 78.8%, and 78.9% are rounded up to 79%. It is also noted that the length of the aligned region is always an integer.
The alignment of two or more sequences to determine percent sequence identity is performed using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389-3402) as incorporated into BLAST (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performed to determine percent sequence identity between a M. paratuberculosis-specific nucleic acid molecule of the invention and any other sequence or portion thereof aligned using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When utilizing BLAST programs to calculate the percent identity between a sequence of the invention and another sequence, the default parameters of the respective programs are used. Sequence analysis of the M. paratuberculosis-specific nucleic acid sequences as performed herein used BLAST version 2.2.2 (updated on Dec. 14, 2001).
The sequences of representative nucleic acids of the invention having 75%, 80%, 85%, 90%, 95%, and 99% sequence identity to SEQ ID NO:1 are shown in
Nucleic acid molecules of the invention between about 10 and about 30 nucleotides in length will, under standard amplification conditions, generate an amplification product in the presence of M. paratuberculosis nucleic acid using an appropriate second nucleic acid molecule (e.g., an oligonucleotide primer that specifically anneals to M. paratuberculosis nucleic acid) but will not generate an amplification product in the presence of nucleic acid from an organism other than M. paratuberculosis using an appropriate third nucleic acid molecule (e.g., an oligonucleotide primer that specifically anneals to nucleic acid from the other organism). As used herein, “standard amplification conditions” refer to the basic components of an amplification reaction mix, and cycling conditions that include multiple cycles of denaturing the template nucleic acid, annealing the oligonucleotide primers to the template nucleic acid, and extension of the primers by the polymerase to produce an amplification product (see, for example, U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188). The basic components of an amplification reaction mix generally include, for example, about 10-25 nmole of each of the four deoxynucleoside triphosphates, (e.g., dATP, dCTP, dTTP, and dGTP, or analogs thereof), 10-100 pmol of primers, template nucleic acid, and a polymerase enzyme. The reaction components are generally suspended in a buffered aqueous solution having a pH of between about 7 and about 9. The aqueous buffer can further include one or more co-factors (e.g., Mg2+, K+) required by the polymerase. Additional components such as DMSO are optional. Template nucleic acid is typically denatured at a temperature of at least about 90° C., and extension from primers is typically performed at a temperature of at least about 72° C.
The annealing temperature can be used to control the specificity of amplification. The temperature at which primers anneal to template nucleic acid must be below the Tm of each of the primers, but high enough to avoid non-specific annealing of primers to the template nucleic acid. The Tm is the temperature at which half of the DNA duplexes have separated into single strands, and can be predicted for an oligonucleotide primer using the formula provided in section 11.46 of Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Non-specific amplification products are detected as bands on a gel that are not the size expected for the correct amplification product. The annealing temperature used in amplification reactions described herein to demonstrate that the claimed nucleic acid molecules are M. paratuberculosis-specific was 55° C. and 60° C. for nucleic acids isolated from bacteria or from a biological sample, respectively. It can be appreciated by those of skill in the art that appropriate positive and negative controls should be performed with every set of amplification reactions to avoid uncertainties related to contamination and/or non-specific annealing of oligonucleotide primers and extension therefrom.
An appropriate second nucleic acid molecule is generally an oligonucleotide primer that specifically anneals to M. paratuberculosis nucleic acid, and that can act in combination with a nucleic acid molecule of the invention, specifically for example a 10- to 30-, or 40-, or 50-nucleotide-long nucleic acid molecule of the invention, under appropriate amplification conditions to generate an amplification product in the presence of M. paratuberculosis nucleic acid. In order for a second nucleic acid molecule to act in combination with a nucleic acid molecule of the invention to generate an amplification product, the two molecules must anneal to opposite strands of the template nucleic acid, and should be an appropriate distance from one another such that the polymerase can effectively polymerize across the region and such that the amplification product can be readily detected using, for example, electrophoresis. Oligonucleotide primers can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) to assist in designing primers that have similar melting temperatures. Typically, oligonucleotide primers are 10 to 30 or 40 or 50 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length), but can be longer or shorter if appropriate amplification conditions are used.
Representative pairs of oligonucleotide primers that were used to amplify each of the M. paratuberculosis-specific nucleic acid molecules of the invention are shown in Tables 3, 6, and 12 (SEQ ID NOs:46-101 and 1343-1354). Alternatively, the nucleic acid molecules having the sequences shown in SEQ ID NOs:1-23 and 110-1342 can be used to design a pair of oligonucleotide primers. Oligonucleotides of the invention can be obtained by restriction enzyme digestion of M. paratuberculosis-specific nucleic acid molecules or can be prepared by standard chemical synthesis and other known techniques.
As used herein, an organism other than M. paratuberculosis refers to any organism that is not M. paratuberculosis. Generally, only relevant organisms are used in amplification reactions to examine the specificity of a 10 or more nucleotide-long nucleic acid molecule of the invention. Particularly relevant organisms include, without limitation, Ralstonia solanacearum, Sinorhizobium meliloti, Homo sapiens, Mesorhizobium loti, Oryza sativa, Drosophila melanogaster, Rhizobium leguminosarum, Xylella fastidiosa, Deinococcus radiodurans, Achromobacter cycloclastes, Candida cylindracea, Streptomyces lavendulae, Streptococcus pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Sus scrofa, Mycobacterium leprae, Streptomyces coelicolor, Pseudomonas sp. (e.g., strain CA-10), Thauera aromatica, Brucella melitensis, Streptomyces noursei, Rhizobium meliloti, Synechococcus elongates, Rhodobacter capsulatus, Agrobacterium tumefaciens, Mycobacterium smegmatis, Drosophila virilis, Mus musculus, Leishmania major, Botrytis cinerea, Caulobacter crescentus, Rhodobacter sphaeroides, Spermatozopsis similes, Giardia intestinalis, Triticum aestivum, Bovine herpesvirus, Streptomyces sp. (e.g., strain MA-6548), Streptomyces peucetius, Rhizobium sp. (e.g., strain NGR-234), Haloferax volcanii, Streptomyces viridochromogenes, Felis catus, Xanthomonas campestris, Thermotoga maritime, Thermotoga neapolitana, Frankia alni, Halobacterium NRC-1 (ATCC Accession No. 700922), Glycine max, Leishmania tarentolae, Neisseria meningitides, Escherichia coli, Caenorhabditis elegans, Leishmania mexicana, Zea mays, Ictalurid herpesvirus, Rattus norvegicus, Arabidopsis thaliana, Pseudomonas fluorescens, Pantoea agglomerans, Erwinia uredovora, Pantoea ananatis, Streptomyces hygroscopicus, Rickettsia typhi, Pseudomonas cruciviae, Xanthomonas albilineans, Halobacterium salinarium, Micromonospora griseorubida, Pseudomonas paucimobilis, Streptomyces lividans, Pyrobaculum aerophilum, Sinorhizobium meliloti, Mesorhizobium loti, Bacillus halodurans, Trypanosoma rangeli, Trypanosoma minasense, Trypanosoma leeuwenhoeki, and Brassica napus. A 10 or more nucleotide-long nucleic acid molecule of the invention in combination with an appropriate third nucleic acid molecule (e.g., a third oligonucleotide primer) will not generate an amplification product from nucleic acid of one or more of these other organisms.
As used herein, an “isolated” nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are usually associated with the isolated nucleic acid molecule. Thus, an “isolated” nucleic acid molecule includes, without limitation, a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such an isolated nucleic acid molecule is generally introduced into a vector (e.g., a cloning vector, or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule. In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule. A nucleic acid molecule existing among hundreds to millions of other nucleic acid molecules within, for example, a nucleic acid library (e.g., a cDNA, or genomic library) or a portion of a gel (e.g., agarose, or polyacrylamine) containing restriction-digested genomic DNA is not to be considered an isolated nucleic acid.
Isolated nucleic acid molecules of the invention can be obtained using techniques routine in the art. For example, isolated nucleic acids within the scope of the invention can be obtained using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid molecule of the invention. Isolated nucleic acids of the invention also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. In addition, isolated nucleic acid molecules of the invention also can be obtained by mutagenesis. For example, and isolated nucleic acid that shares identity with an art known M. paratuberculosis-specific nucleic acid sequence can be mutated using common molecular cloning techniques (e.g., site-directed mutagenesis). Possible mutations include, without limitation, deletions, insertions, substitutions, and combinations thereof.
Vectors containing M. paratuberculosis-specific nucleic acid molecules also are provided by the invention. Vectors, including expression vectors, suitable for use in the present invention are commercially available and/or produced by recombinant DNA technology methods routine in the art. A vector containing a M. paratuberculosis-specific nucleic acid molecule can have elements necessary for expression operably linked to such a M. paratuberculosis-specific nucleic acid, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene), and/or those that can be used in purification of a M. paratuberculosis-specific polypeptide (e.g., 6×His tag).
Elements necessary for expression include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. One example of an element necessary for expression is a promoter sequence, for example, a M. paratuberculosis-specific promoter (e.g., from the same coding sequence being expressed or from a different coding sequence) or a non-M. paratuberculosis-specific promoter. Elements necessary for expression also can include introns, enhancer sequences, response elements, or inducible elements that modulate expression of a M. paratuberculosis-specific nucleic acid. Elements necessary for expression can be of bacterial, yeast, insect, mammalian, or viral origin and vectors can contain a combination of elements from different origins. Elements necessary for expression are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, Calif. As used herein, operably linked means that a promoter and/or other regulatory element(s) are positioned in a vector relative to a M. paratuberculosis-specific nucleic acid in such a way as to direct or regulate expression of the M. paratuberculosis-specific nucleic acid. Many methods for introducing nucleic acids into cells, both in vivo and in vitro, are well known to those skilled in the art and include, without limitation, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer.
Another aspect of the invention pertains to host cells into which a vector of the invention, e.g., an expression vector, or an isolated nucleic acid molecule of the invention has been introduced. The term “host cell” refers not only to the particular cell but also to the progeny or potential progeny of such a cell. A host cell can be any prokaryotic or eukaryotic cell. For example, M. paratuberculosis-specific nucleic acids can be expressed in bacterial cells such as E. coli, or in insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vectors containing representative nucleic acid molecules unique to M. paratuberculosis were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard Manassas, Va. 20110, on Apr. 3, 2002, and assigned Accession Numbers PTA-4199, and PTA-4200. Each deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Purified M. paratuberculosis Polypeptides
One aspect of the invention pertains to purified M. paratuberculosis-specific polypeptides, as well as polypeptide fragments. A “M. paratuberculosis-specific polypeptide” refers to a polypeptide encoded by a nucleic acid molecule that is unique to M. paratuberculosis (e.g., M. paratuberculosis-specific nucleic acid molecules, for example, those having the sequence shown in SEQ ID NOs:1-23 and 110-1342). Predicted amino acid sequences encoded by novel M. paratuberculosis-specific nucleic acids of the invention are shown in SEQ ID NOs:24-45.
The term “purified” polypeptide as used herein refers to a polypeptide that has been separated or purified from cellular components that naturally accompany it. Typically, the polypeptide is considered “purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the proteins and naturally occurring molecules with which it is naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separated from the components that naturally accompany it, a synthetic polypeptide is “purified.”
M. paratuberculosis-specific polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A purified M. paratuberculosis-specific polypeptide also can be obtained by expressing a M. paratuberculosis-specific nucleic acid in an expression vector, for example. In addition, a purified M. paratuberculosis-specific polypeptide can be obtained by chemical synthesis. The extent of purity of a M. paratuberculosis-specific polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
In addition to naturally-occurring M. paratuberculosis-specific polypeptides, the skilled artisan will further appreciate that changes can be introduced into a nucleic acid molecule (e.g., those having the sequence shown in SEQ ID NOs:1-23, 110-1341, or 1342) as discussed herein, thereby leading to changes in the amino acid sequence of the encoded polypeptide. For example, changes can be introduced into M. paratuberculosis-specific nucleic acid coding sequences leading to conservative and/or non-conservative amino acid substitutions at one or more amino acid residues. A “conservative amino acid substitution” is one in which one amino acid residue is replaced with a different amino acid residue having a similar side chain. Similarity between amino acid residues has been assessed in the art. For example, Dayhoff et al. (1978, in Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, pp 345-352) provides frequency tables for amino acid substitutions that can be employed as a measure of amino acid similarity. A non-conservative substitution is one in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain.
The invention also provides for chimeric or fusion polypeptides. As used herein, a “chimeric” or “fusion” polypeptide includes a M. paratuberculosis-specific polypeptide operatively linked to a heterologous polypeptide. A heterologous polypeptide can be at either the N-terminus or C-terminus of the M. paratuberculosis-specific polypeptide. Within a chimeric or fusion polypeptide, the term “operatively linked” is intended to indicate that the two polypeptides are encoded in-frame relative to one another. In a fusion polypeptide, the heterologous polypeptide generally has a desired property such as the ability to purify the fusion polypeptide (e.g., by affinity purification). A chimeric or fusion polypeptide of the invention can be produced by standard recombinant DNA techniques, and can use commercially available vectors.
A polypeptide commonly used in a fusion polypeptide for purification is glutathione S-transferase (GST), although numerous other polypeptides are available and can be used. In addition, a proteolytic cleavage site can be introduced at the junction between a M. paratuberculosis-specific polypeptide and a non-M. paratuberculosis-specific polypeptide to enable separation of the two polypeptides subsequent to purification of the fusion polypeptide. Enzymes that cleave such proteolytic sites include Factor Xa, thrombin, or enterokinase. Representative expression vectors encoding a heterologous polypeptide that can be used in affinity purification of a M. paratuberculosis polypeptide include pGEX (Pharmacia Biotech Inc; Smith & Johnson, 1988, Gene, 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.).
Anti-M. paratuberculosis-Specific Antibodies
Another aspect of the invention relates to anti-M. paratuberculosis-specific antibodies. The term “anti-M. paratuberculosis-specific antibodies” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules that have specific binding affinity for a M. paratuberculosis-specific polypeptide. The invention provides polyclonal and monoclonal antibodies that have specific binding affinity for M. paratuberculosis-specific polypeptides. The sequences of numerous M. paratuberculosis-specific polypeptides that can be used to generate anti-M. paratuberculosis-specific antibodies are disclosed herein (e.g., SEQ ID NOs:24-45). Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments, which can be generated by treating an immunoglobulin molecule with an enzyme such as pepsin. As used herein, an antibody that has “specific binding affinity” for a M. paratuberculosis-specific polypeptide is an antibody that binds a M. paratuberculosis-specific polypeptide but does not bind a non-M. paratuberculosis-specific polypeptides. A non-M. paratuberculosis-specific polypeptide as used herein refers to a polypeptide that may or may not be found in M. paratuberculosis, but is found in at least one other organism besides M. paratuberculosis.
A purified M. paratuberculosis-specific polypeptide or a fragment thereof can be used as an immunogen to generate polyclonal or monoclonal antibodies that have specific binding affinity for M. paratuberculosis-specific polypeptides. Such antibodies can be generated using standard techniques as described herein. Full-length M. paratuberculosis-specific polypeptides (see Table 2) or, alternatively, antigenic fragments of M. paratuberculosis-specific polypeptides can be used as immunogens. An antigenic fragment of a M. paratuberculosis-specific polypeptide usually includes at least 8 (e.g., 10, 15, 20, or 30) amino acid residues of a M. paratuberculosis-specific polypeptide (e.g., having the sequence shown in SEQ ID NOs:23-45), and encompasses an epitope of a M. paratuberculosis-specific polypeptide such that an antibody (e.g., polyclonal or monoclonal) raised against the antigenic fragment has specific binding affinity for a M. paratuberculosis-specific polypeptide.
Antibodies are typically prepared by first immunizing a suitable animal (e.g., a rabbit, a goat, a mouse or another mammal) with an immunogenic preparation. An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized M. paratuberculosis-specific polypeptide, of a fragment thereof. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable animal with an immunogenic M. paratuberculosis-specific polypeptide preparation induces a polyclonal anti-M. paratuberculosis-specific antibody response.
The titer of the anti-M. paratuberculosis-specific antibody in the immunized animal can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using immobilized M. paratuberculosis-specific polypeptides. If desired, the antibody molecules directed against M. paratuberculosis-specific polypeptides can be isolated from the animal (e.g., from the blood) and further purified by well-known techniques such as protein A chromatography to obtain the IgG fraction.
At an appropriate time after immunization, e.g., when the anti-M. paratuberculosis-specific antibody titers are highest, antibody-producing cells can be obtained from the animal and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler & Milstein (1975, Nature, 256:495-497), the human B cell hybridoma technique (Kozbor et al., 1983, Immunol. Today, 4:72), or the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). The technology for producing various monoclonal antibody hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.). Briefly, an immortal cell line (e.g., a myeloma cell line) is fused to lymphocytes (e.g., splenocytes) from an animal immunized with an immunogenic M. paratuberculosis-specific polypeptide as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that has specific binding affinity for the M. paratuberculosis-specific polypeptide.
Any of the well-known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-M. paratuberculosis-specific monoclonal antibody (see, e.g., Current Protocols in Immunology, supra; Galfre et al., 1977, Nature, 266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner, 1981, Yale J. Biol. Med., 54:387-402). Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods that also would be useful. Typically, the immortal cell line is derived from the same species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of ATCC-available myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (PEG). Hybridoma cells resulting from the fusion are then selected using HAT medium. Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind M. paratuberculosis-specific polypeptides, e.g., using a standard ELISA assay.
As an alternative to preparing monoclonal antibody-secreting hybridomas, an anti-M. paratuberculosis-specific monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with M. paratuberculosis-specific polypeptides. Immunoglobulin library members that have specific binding affinity for M. paratuberculosis-specific polypeptides can be isolated from such libraries. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/20791; PCT Publication No. WO 93/01288; Hay et al., 1992, Hum. Antibod. Hybridomas, 3:81-85; Griffiths et al., 1993, EMBO J., 12:725-734; and references therein.
Additionally, recombinant anti-M. paratuberculosis-specific antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent (EP) Application 184,187; U.S. Pat. No. 4,816,567; Better et al., 1988, Science, 240:1041-1043; Shaw et al., 1988, J. Natl. Cancer Inst., 80:1553-1559); U.S. Pat. No. 5,225,539; Verhoeyan et al., 1988, Science, 239:1534; Beidler et al., 1988, J. Immunol., 141:4053-4060; and references therein.
An anti-M. paratuberculosis-specific antibody (e.g., a monoclonal antibody) can be used to isolate M. paratuberculosis-specific polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-M. paratuberculosis-specific antibody can facilitate the purification of natural M. paratuberculosis-specific polypeptides from cells and of recombinantly-produced M. paratuberculosis-specific polypeptides expressed in host cells. Moreover, an anti-M. paratuberculosis-specific antibody can be used to detect M. paratuberculosis-specific polypeptides (e.g., in a cellular lysate or cell supernatant) in order to evaluate the presence or absence of the M. paratuberculosis-specific polypeptides. Anti-M. paratuberculosis-specific antibodies can be used diagnostically to detect M. paratuberculosis-specific polypeptides, and hence, M. paratuberculosis, in a biological sample, e.g., to determine the infection status of an animal, or to determine the efficacy of a given treatment regimen.
Methods of Detecting M. paratuberculosis
The M. paratuberculosis-specific nucleic acid molecules and polypeptides, and the anti-M. paratuberculosis-specific antibodies described herein can be used in diagnostic assays for the detection of M. paratuberculosis. Diagnostic assays for determining the presence or absence of M. paratuberculosis are performed using a biological sample (e.g., fecal, blood, milk) to determine whether an animal has been exposed to or is infected with M. paratuberculosis. An exemplary method for detecting the presence or absence of M. paratuberculosis in a biological sample involves obtaining a biological sample from an animal and contacting the biological sample with an appropriate agent capable of detecting M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies.
The term “biological sample” is intended to include cells and biological fluids obtained from an animal. In one embodiment, a biological sample contains polypeptides from the animal. Alternatively, the biological sample can contain nucleic acid molecules from the animal, or the biological sample can contain antibodies from the animal. It should be understood that any biological sample in which M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies may be present can be utilized in the methods described herein.
In one embodiment, an agent for detecting the presence or absence of M. paratuberculosis in a biological sample is an isolated M. paratuberculosis-specific nucleic acid molecule of the invention. The presence of M. paratuberculosis-specific nucleic acids in a sample indicates the presence of M. paratuberculosis in the sample. Methods for detecting nucleic acids include, for example, PCR and nucleic acid hybridizations (e.g., Southern blot, Northern blot, or in situ hybridizations). Specifically, an agent can be one or more oligonucleotides (e.g., oligonucleotide primers) capable of amplifying M. paratuberculosis-specific nucleic acids using PCR. PCR methods generally include the steps of collecting a biological sample from an animal, isolating nucleic acid (e.g., DNA, RNA, or both) from the sample, and contacting the nucleic acid with one or more oligonucleotide primers that hybridize(s) with specificity to M. paratuberculosis-specific nucleic acid under conditions such that amplification of the M. paratuberculosis-specific nucleic acid occurs if M. paratuberculosis is present. In the presence of M. paratuberculosis, an amplification product corresponding to the M. paratuberculosis-specific nucleic acid is produced. Conditions for amplification of a nucleic acid and detection of an amplification product are known to those of skill in the art (see, e.g., PCR Primer: A Laboratory Manual, 1995, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188). Modifications to the original PCR also have been developed. For example, anchor PCR, RACE PCR, or ligation chain reaction (LCR) are additional PCR methods known in the art (see, e.g., Landegran et al., 1988, Science, 241:1077-1080; and Nakazawa et al., 1994, Proc. Natl. Acad. Sci. USA, 91:360-364).
Alternatively, an agent for detecting M. paratuberculosis-specific nucleic acids can be a labeled oligonucleotide probe capable of hybridizing to M. paratuberculosis-specific nucleic acids on a Southern blot. An oligonucleotide probe can be, for example, a M. paratuberculosis-specific nucleic acid molecule such as a nucleic acid molecule having the sequence shown in SEQ ID NO:1-22, 110-1341, or 1342, or a fragment thereof. In the presence of M. paratuberculosis, a hybridization complex is produced between M. paratuberculosis nucleic acid and the oligonucleotide probe. Hybridization between nucleic acid molecules is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57).
For oligonucleotide probes less than about 100 nucleotides, Sambrook et al. discloses suitable Southern blot conditions in Sections 11.45-11.46. The Tm between a sequence that is less than 100 nucleotides in length and a second sequence can be calculated using the formula provided in Section 11.46. Sambrook et al. additionally discloses prehybridization and hybridization conditions for a Southern blot that uses oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.52). Hybridizations with an oligonucleotide greater than 100 nucleotides generally are performed 15-25° C. below the Tm. The Tm between a sequence greater than 100 nucleotides in length and a second sequence can be calculated using the formula provided in Sections 9.50-9.51 of Sambrook et al. Additionally, Sambrook et al. recommends the conditions indicated in Section 9.54 for washing a Southern blot that has been probed with an oligonucleotide greater than about 100 nucleotides.
The conditions under which membranes containing nucleic acids are prehybridized and hybridized, as well as the conditions under which membranes containing nucleic acids are washed to remove excess and non-specifically bound probe can play a significant role in the stringency of the hybridization. Such hybridizations can be performed, where appropriate, under moderate or high stringency conditions. Such conditions are described, for example, in Sambrook et al. section 11.45-11.46. For example, washing conditions can be made more stringent by decreasing the salt concentration in the wash solutions and/or by increasing the temperature at which the washes are performed. In addition, interpreting the amount of hybridization can be affected, for example, by the specific activity of the labeled oligonucleotide probe, by the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and by the amount of exposure of an autoradiograph or other detection medium.
It will be readily appreciated by those of ordinary skill in the art that although any number of hybridization and washing conditions can be used to examine hybridization of a probe nucleic acid molecule to immobilized target nucleic acids, it is more important to examine hybridization of a probe to target nucleic acids, for example, from M. paratuberculosis and at least one organism other than M. paratuberculosis, under identical hybridization, washing, and exposure conditions. Preferably, the target nucleic acids (e.g., nucleic acids from M. paratuberculosis and at least one organism other than M. paratuberculosis) are on the same membrane. Representative Southern blot conditions are described in Example 3.
A nucleic acid molecule is deemed to hybridize to M. paratuberculosis nucleic acids but not to nucleic acids from an organism other than M. paratuberculosis if hybridization to nucleic acid from M. paratuberculosis is at least 5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater than hybridization to nucleic acid from an organism other than M. paratuberculosis. The amount of hybridization can be quantitated directly on a membrane or from an autoradiograph using, for example, a PhosphorImager or a Densitometer (Molecular Dynamics, Sunnyvale, Calif.). It can be appreciated that useful primers and probes of the invention include primers and probes that anneal and hybridize, respectively, to nucleic acids of organisms other than M. paratuberculosis provided that such nucleic acids are not typically present in the relevant test animals. For example, the fact that a particular primer or probe anneals or hybridizes, respectively, to human nucleic acid does not diminish the value of that primer or probe for detecting the presence or absence of M. paratuberculosis in ruminants, since ruminants typically are not contaminated with human nucleic acid.
In addition, anti-M. paratuberculosis-specific antibodies provided by the invention can be used as agents to detect the presence or absence of M. paratuberculosis-specific polypeptides in a biological sample. The presence of M. paratuberculosis-specific polypeptides is an indication of the presence of M. paratuberculosis in the sample. Techniques for detecting M. paratuberculosis-specific polypeptides include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody of the invention can be polyclonal or monoclonal, and usually is detectably labeled. An antibody having specific binding affinity for a M. paratuberculosis-specific polypeptide can be generated using methods described herein. The antibody can be attached to a solid support such as a microtiter plate using methods known in the art (see, for example, Leahy et al., 1992, BioTechniques, 13:738-743). In the presence of M. paratuberculosis, an antibody-polypeptide complex is formed.
In addition, M. paratuberculosis-specific polypeptides of the invention can be used as an agent to detect the presence or absence of anti-M. paratuberculosis-specific antibodies in a biological sample. The presence of anti-M. paratuberculosis-specific antibodies in a sample indicates that the animal from which the sample was obtained mounted an immune response toward M. paratuberculosis. Given the etiology of M. paratuberculosis in its host animals, an animal that has detectable levels of anti-M. paratuberculosis-specific antibodies is likely infected with M. paratuberculosis. Alternatively, an animal that is positive for anti-M. paratuberculosis-specific antibodies may have resisted infection following a previous exposure to M. paratuberculosis, or may possess maternally-transmitted anti-M. paratuberculosis-specific antibodies. Techniques for detecting anti-M. paratuberculosis-specific antibodies in a biological sample include ELISAs, Western blots, immunoprecipitations, and immunofluorescence. A M. paratuberculosis-specific polypeptide can be attached to a solid support such as a microtiter plate by known methods (Leahy et al., supra). In the presence of M. paratuberculosis, a polypeptide-antibody complex is formed.
Detection of an amplification product, a hybridization complex, an antibody-polypeptide complex, or a polypeptide-antibody complex is usually accomplished by detectably labeling the respective agent. The term “labeled” with regard to an agent (e.g., an oligonucleotide, a polypeptide, or an antibody) is intended to encompass direct labeling of the agent by coupling (i.e., physically linking) a detectable substance to the agent, as well as indirect labeling of the agent by reactivity with another reagent that is directly labeled with a detectable substance. Detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H. Examples of indirect labeling include using a fluorescently labeled secondary antibody to detect an appropriate agent (e.g., a primary antibody), or end-labeling an agent with biotin such that it can be detected with fluorescently labeled streptavidin.
In another embodiment, the methods further involve obtaining a biological sample from an animal known to be infected with M. paratuberculosis (positive control) and a non-infected (negative control) animal, contacting the control samples with an agent capable of detecting M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies, such that the presence or absence of M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies in the samples is determined. The presence or absence of M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies in the control samples should correlate with the presence and absence of M. paratuberculosis in the positive and negative control samples, respectively.
Methods of Preventing a M. paratuberculosis Infection
In one aspect, the invention provides methods for preventing a disease or condition associated with infection by M. paratuberculosis (e.g., Johne's disease) in an animal by administering a compound to the animal that immunizes the animal against M. paratuberculosis infection. Animals at risk for M. paratuberculosis infection can be administered the compound prior to the manifestation of symptoms that are characteristic of a M. paratuberculosis infection, such that a M. paratuberculosis infection is prevented or delayed in its progression.
In one embodiment, a compound that immunizes an animal can be a M. paratuberculosis-specific polypeptide. The sequences of representative M. paratuberculosis-specific polypeptides are disclosed herein (e.g., SEQ ID NOs:24-45) and can be produced using methods described herein. An M. paratuberculosis-specific polypeptide can be a fusion polypeptide, for example a M. paratuberculosis-specific polypeptide-immunoglobulin fusion polypeptide in which all or part of a M. paratuberculosis-specific polypeptide is fused to sequences derived from a member of the immunoglobulin family. An M. paratuberculosis-specific polypeptide or fusion polypeptide of the invention can be used as an immunogen to elicit anti-M. paratuberculosis-specific antibodies in an animal, thereby immunizing the animal.
In another embodiment, a compound that immunizes an animal can be a M. paratuberculosis-specific nucleic acid molecule. A M. paratuberculosis-specific nucleic acid molecule used to immunize an animal can include one of the M. paratuberculosis-specific nucleic acid molecules having the sequence shown in SEQ ID NOs:1-23, 110-1341, or 1342. M. paratuberculosis-specific nucleic acid coding sequences (e.g., full-length or otherwise) can be introduced into an appropriate expression vector such that a M. paratuberculosis-specific polypeptide or fusion polypeptide is produced in the animal upon appropriate expression of the expression vector. Expression of the M. paratuberculosis-specific nucleic acid molecule and production of a M. paratuberculosis-specific polypeptide in an animal thereby elicits an immune response in the animal and thereby immunizes the animal.
Compounds that can be used in immunogenic compositions of the invention (e.g., M. paratuberculosis-specific nucleic acid molecules or M. paratuberculosis-specific polypeptides) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule or polypeptide, and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion or inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)), fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), glycerine, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged administration of the injectable compositions can be brought about by including an agent that delays absorption. Such agents include, for example, aluminum monostearate and gelatin. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be liquid, or can be enclosed in gelatin capsules or compressed into tablets. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of an oral composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for an animal to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms of the invention are dependent upon the amount of a compound necessary to immunize the animal. The amount of a compound necessary to immunize an animal can be formulated in a single dose, or can be formulated in multiple dosage units. Immunization of an animal may require a one-time dose, or may require repeated doses.
For polypeptide vaccines, the dose typically is from about 0.1 mg/kg to about 100 mg/kg of body weight (generally, about 0.5 mg/kg to about 5 mg/kg). Modifications such as lipidation (Cruikshank et al., 1997, J. Acquired Immune Deficiency Syndromes and Human Retrovirology, 14:193) can be used to stabilize polypeptides and to enhance uptake and tissue penetration. For nucleic acid vaccines, the dose administered will depend on the level of expression of the expression vector. Preferably, the amount of vector that produces an amount of a M. paratuberculosis-specific polypeptide from about 0.1 mg/kg to about 100 mg/kg of body weight is administered to an animal.
Articles of Manufacture
The invention encompasses articles of manufacture (e.g., kits) for detecting the presence of M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies in a biological sample (a test sample). Such kits can be used to determine if an animal has been exposed to, or is infected with, M. paratuberculosis. For example, a kit of the invention can include an agent capable of detecting M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies in a biological sample (e.g., a M. paratuberculosis-specific oligonucleotide, an anti-M. paratuberculosis-specific antibody, or a M. paratuberculosis-specific polypeptide, respectively).
For antibody-based kits to detect M. paratuberculosis-specific polypeptides, the kit can include, for example, a first antibody (e.g., attached to a solid support) that has specific binding affinity for a M. paratuberculosis-specific polypeptide and, optionally, a second antibody which binds to M. paratuberculosis-specific polypeptides or to the first antibody and is detectably labeled. For oligonucleotide-based kits to detect M. paratuberculosis-specific nucleic acids, the kit may comprise, for example, one or more oligonucleotides. For example, a kit of the invention can include a detectably labeled oligonucleotide probe that hybridizes to a M. paratuberculosis-specific nucleic acid molecule or a pair of oligonucleotide primers for amplifying a M. paratuberculosis-specific nucleic acid molecule. Such oligonucleotides provided in a kit of the invention can be detectably labeled or, alternatively, the components necessary for detectably labeling an oligonucleotide can be provided in the kit. Polypeptide-based kits for detecting anti-M. paratuberculosis-specific antibodies in a biological sample can contain a M. paratuberculosis-specific polypeptide as disclosed herein (e.g., attached to a solid support) and, optionally, an antibody which binds to M. paratuberculosis-specific polypeptides or to an anti-M. paratuberculosis-specific antibody and is detectably labeled.
Kits can include additional reagents (e.g., buffers, co-factors, or enzymes) as well as reagents for detecting the agent (e.g., labels or other detection molecules), as well as instructions for using such agents and reagents to detect the presence or absence of M. paratuberculosis-specific nucleic acids or polypeptides, or anti-M. paratuberculosis-specific antibodies. The kit can also contain a control sample or a series of control samples that can be assayed and compared to the biological sample. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package.
The invention also encompasses articles of manufacture (e.g., vaccines) for preventing M. paratuberculosis infection in an animal. Articles of manufacture of the invention can include pharmaceutical compositions containing either a M. paratuberculosis-specific nucleic acid molecule or a M. paratuberculosis-specific polypeptide. Such nucleic acid molecules or polypeptides are formulated for administration as described herein, and are packaged appropriately for the intended route of administration. Pharmaceutical compositions of the invention further can include instructions for administration.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Mycobacteria used in this study are listed in Table 3. All mycobacteria were cultured in Middlebrook 7H9 media with 0.05% Tween 80 and oleic acid albumin dextrose complex (Becton Dickinson Microbiology, Sparks, Md.). Cultures containing M. paratuberculosis isolates were supplemented with 2 mg/L ferric mycobactin J (Allied Monitor Inc., Fayette, Mo.). All growth flasks were incubated at 37° C. without shaking.
M. avium subsp.
paratuberculosis
M. avium subsp. avium
M. phlei
M. smegmatis
M. intracellulare
M. scrofulaceum complex 6
M. fortuitum
M. bovis
M. tuberculosis TB 14323
aDates of isolation (month/day/year) are in parentheses;
bATCC, American Type Culture Collection; NADC, National Animal Disease Center: UMN, University of Minnesota
The sequencing and assembly strategies used herein for M. paratuberculosis were as described for Pasteurella multocida (see May et al., 2001, Proc. Natl. Acad. Sci. USA, 98:3460-5). For these studies, assembled M. paratuberculosis contig fragments greater than 10 kb were chosen. Predicted coding sequences were identified using ARTEMIS software and TB-parse, a program used to identify coding sequences in the M. tuberculosis genome (Cole et al., 1998, Nature, 393:537-44). The results were compared and verified manually in ARTEMIS. A putative ribosome-binding site (RBS) was also evaluated for each coding sequence. The presence of an AG-rich sequence approximately 30-bp upstream of the start codon was scored as a putative RBS sequence. Similarities were identified with BLASTP analysis by using GenBank and a local database constructed by the Computational Biology Center at the University of Minnesota (cbc.umn.edu on the World Wide Web).
ARTEMIS and ACT are funded by the Wellcome Trust's Beowulf Genomics initiative and are available free on the internet at sanger.ac.uk/Software/. Sequence alignments between M. paratuberculosis and M. avium were compared and visualized with ACT software. M. avium is being sequenced by The Institute for Genomic Research (TIGR; at tigr.org/cgi-bin/BlastSearch/blast.cgi?organism=m_avium on the World Wide Web). Sequence alignments to produce figures or schematic illustrations were performed with AssemblyLIGN™ software (Accelrys, Princeton, N.J.).
The nucleotide sequence of each M. paratuberculosis gene described in this study was deposited individually in the GenBank/EMBL Nucleotide Sequence Data Library and were assigned GenBank Accession Nos. AF445420 through AF445446.
Genomic DNA was extracted from several species of mycobacteria using a modified method from that described by Whipple et al. (Whipple et al., 1987, J. Clin. Microbial., 25:1511-15). Briefly, one liter of Middlebrook 7H9 cultured mycobacteria was incubated at 37° C. until an OD540 of between 0.50 and 0.56 was reached. D-Cycloserine was added to the media at a final concentration of 0.5 mg/ml and incubated an additional 24 h. Mycobacteria were harvested by centrifugation at 8,000 rpm for 15 min and the pellet was resuspended in 11 ml of Qiagen buffer B1 containing 1 mg/ml Qiagen RNase A. Lipase was added (450,000 Units, Sigma Catalog No L4384) to digest mycobacterial cell wall lipids. Following incubation for 2 h at 37° C., 20 mg of lysozyme was added and incubation proceeded for an additional 3 h at 37° C. 500 μl of Qiagen proteinase K (20 mg/ml) was added and incubated for 1.5 h at 37° C. Qiagen buffer B2 (4 ml) was added and the slurry mixed and incubated 16 h at 50° C. The remaining cellular debris was removed by centrifugation at 10,000 rpm for 20 min. The supernatant was poured over a pre-equilibrated Qiagen 500/G genomic tip. The loaded column was washed and processed according to the instructions of the manufacturer. PstI restricted DNA fragments were separated on a 1% agarose gel. DNA-containing gels were depurinated, denatured, and neutralized as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). DNA was transferred by capillary action to BrightStar-Plus membranes (Ambion, Austin, Tex.) and probes were labeled using [α-32P]dCTP (ICN, Cost Mesa, Calif.) by random priming. Hybridization was performed in a AUTOBLOT hybridization oven (Bellco Biotechnology, Vineland, N.J.) at 45° C. for 16 h in ExpressHyb hybridization solution (Clontech, Palo Alto, Calif.). Probed blots were washed sequentially with solutions increasing in stringency as follows: 2 washes at room temp in 2×SSC, 0.1% SDS; 2 washes at room temp in 0.2×SSC, 0.1% SDS; and 2 washes at room temp in 0.16×SSC, 0.1% SDS. Detection was by autoradiography at room temp using BioMax MR film (Kodak, Rochester, N.Y.) with a Kodak intensifying screen for less than 16 hours.
Oligonucleotide primers listed in Table 4 were designed using the M. paratuberculosis-specific nucleic acid sequences identified herein. PCR amplification of M. paratuberculosis-specific nucleic acid molecules was performed as follows. A PCR master mix was generated that contained (each in final concentration) 1× AmpliTaq Gold buffer (Perkin-Elmer), 5% dimethylsufoxide (DMSO, Sigma), 20 mM each nucleotide (Roche Biochemicals), 10 ng genomic DNA, and 1 Unit of AmpliTaq Gold DNA polymerase (Perkin-Elmer). Primers were added to individual PCR tubes containing 25 μl of master mix. The PCR reaction conditions were as follows: a 5 min denaturing step at 94° C., followed by 35 cycles of: 94° C. for 45 sec, 55° C. for 1 min, and 72° C. for 2 min. At the end of 35 cycles, there was a 7 min incubation at 72° C. and a hold at 4° C. High fidelity Pwo polymerase (Boehringer Ingelheim Pharmaceutical Inc., Ridgefield, Conn.) was used in amplifications to generate probes used in Southern hybridization experiments. All other amplifications used Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, Ind.).
Primers used to amplify the #7 sequence for a probe in Southern hybridizations were
The complete genome of M. paratuberculosis K-10, a field isolate recovered from a cow with clinical Johne's disease, is currently being sequenced (cbc.umn.edu/ResearchProjects/AGAC/Mptb/Mptbhome.html on the World Wide Web). The genome size is estimated to be >5 Mb based on assembled sequence data, and by July, 2001, 2.65 Mb were contained in contig fragments greater than 10 kb. Those Contigs above 10 kb were annotated using ARTEMIS and represent 48% of the total genome. The average size of the annotated contigs is 25 kb with one contig over 70 kb. Each gene within the annotated contig set was also checked manually and confirmed by TB-parse. These contigs were aligned with M. avium sequence data generated at TIGR. TIGR has 612 contigs that total 5,867,714 bp in the Jul. 8, 2001 data set.
M. avium and M. paratuberculosis display a high degree of similarity at the nucleotide level as well as local gene order conservation. An analysis of an 11-kb region surrounding the origin of replication for each of these genomes shows 98% sequence identity at the nucleotide level. The sequence similarity between orthologs in M. paratuberculosis and M. avium was greater than between M. paratuberculosis and other mycobacterial species. A more global comparison shows that these strong nucleotide identities are present throughout both genomes. Despite this strong genetic similarity, a total of 27 genes from the annotated M. paratuberculosis contigs were identified that did not align with the unfinished M. avium genome by computerized alignments. Of these, three contained weak similarity to proteins in other mycobacterial species or proteins in GenBank. This left 24 genes that have no significant similarity to any known proteins. Since only about half of the M. paratuberculosis genome was used in these analyses, a complete genome analysis may reveal an estimated 50 unique M. paratuberculosis genes.
Some M. paratuberculosis sequences that did not align with M. avium sequences, either in silico or experimentally, contain similarity to other mycobacterial species. One such sequence, designated #7, was tested by PCR and Southern hybridization with two M. avium isolates and two M. paratuberculosis strains. An amplified PCR fragment was produced only with M. paratuberculosis genomic DNA as template. Likewise, DNA hybridization on Southern blots detected only M. paratuberculosis sequences, not M. avium. However, BLASTP analysis of the #7 sequence revealed strong similarity to hypothetical proteins in the M. tuberculosis genome.
PCR amplification was performed on several mycobacterial species, strains and isolates to experimentally determine the specificity for 26 of the 27 sequences (Table 5). Gene 128 was not included in these analyses because it had the lowest expect value (highest similarity to a sequence in GenBank) of the 27 sequences by BLASTP analysis. These data show that primers designed from all 26 M. paratuberculosis-specific genes from isolate K-10 could produce an amplified product in all 10 M. paratuberculosis strains or isolates tested. In addition, despite an absence of any homologous sequences in public databases, PCR products of the correct size were obtained for five genes using template from other mycobacterial species. Following this analysis a core group of 21 genes remained that are present only in M. paratuberculosis (Table 5).
Eight genes were present on contig fragment 1614. These eight genes are arranged in tandem, span a total of 4.4 kb at the end of the 1614 contig (
M. paratuberculosis
M. avium
M. phlei
M. smegmatis
M. intracellulare
M. fortuitum
M. bovis BCG
M. bovis 95-1398
M. tuberculosis
M. paratuberculosis
M. avium
M. phlei
M. smegmatis
M. intracellulare
M. fortuitum
M. bovis BCG
M. bovis 95-1398
M. tuberculosis
A TBLASTX analysis was performed on the 9.4-kb sequence (designated contig 1398-trimmed in
To confirm experimentally that #481 is present only in M. paratuberculosis, three arbitrarily chosen genes of the #481 sequence (251, 253, and 255) were radiolabeled and used as probes in DNA hybridization with several mycobacterial species including M. fortuitum, M. bovis, M. intracellulare, M. avium, and M. paratuberculosis. Following Southern blotting, only a M. paratuberculosis fragment greater than 9.5 kb was detected by each of the three probes.
The characteristics of M. paratuberculosis-specific polypeptides shown in Table 6 were obtained using MacVector sequence analysis software (Oxford Molecular).
To confirm coding predictions of novel M. paratuberculosis genes and assess their immunogenicity, coding sequences were amplified from the genome by PCR and cloned into the pMAL-c2 E. coli expression plasmid. These proteins were expressed as a fusion with E. coli maltose binding protein (MBP) to enable affinity purification on an amylase resin column. An immunoblot was probed with a monoclonal antibody that binds MBP, which identified each fusion protein. A duplicate immunoblot was probed with polyclonal sera from a rabbit immunized with heat-killed preparation of M. paratuberculosis. Only the fusion protein containing the M. paratuberculosis specific polypeptide produced from gene 253 was detected by the rabbit sera, indicating that the polypeptide encoded by gene 253 was produced by M. paratuberculosis. The MBP protein was not detected by the polyclonal sera.
The polypeptide produced from gene 253 was termed psp-1 (paratuberculosis-specific protein). To determine if psp-1 is recognized during infection of cattle, the purified MBP/psp-1 fusion was evaluated further by immunoblot with sera from cattle with overt signs of Johne's disease. Sera from all three Johne's cows examined recognized the MBP/psp-1 fusion protein but did not recognize MBP alone. Another M. paratuberculosis-MBP fusion protein using gene 251 was also evaluated in this experiment, but the fusion protein produced therefrom was only weakly detected.
Immunoblot analysis of psp-1 was further expanded to include additional sera from Johne's cattles as well as control cattle housed at NADC and a local Iowa diary herd. The polypeptide designated psp-1 was not detected by sera from 7 control cows, but was detected by 14 of 16 Johne's cows tested.
Coding sequences within M. paratuberculosis-specific DNA fragments are cloned into E. coli expression vectors (e.g., containing a sequence encoding a 6×His tag). Heterologously expressed mycobacterial proteins are affinity purified from E. coli lysates by a polyhistidine tag. These purified proteins are then evaluated serologically with a panel of sera from infected and control cows to determine if the protein is recognized by sera from infected animals.
Specifically, each open reading frame identified as unique to M. paratuberculosis is amplified from genomic DNA, cloned into the pCRT7 expression vector (Invitrogen), and transformed into E. coli DH5-α. Each of the constructs are verified by DNA sequence analysis. The level of expression of the gene of interest is evaluated by loading the recombinant E. coli lysates onto SDS-PAGE gels and staining them in Coomassie blue. Expressed proteins are purified from E. coli lysates using the vector-encoded polyhistidine tag that has affinity for metal ions. Column purification using TALON metal resin (Clontech) is used. The fusion alone is used as a negative control. Comparisons of the reactivity of a collection of cattle antisera with the fusion proteins are conducted using a slot-blotting device (BioRad). Lysates of recombinant E. coli are loaded onto preparative 12% (w/v) polyacrylamide gels and transferred to nitrocellulose. After blocking, these filters are placed into the slot-blot device. Individual cattle antisera, each diluted 1:200, is added to independent slots. The rest of the procedure is carried out using standard immunoblot protocols. Protein G-peroxidase diluted 1:25,000 or anti-bovine IgG-peroxidase diluted 1:20,000 are used for detection of bound antibody.
All expressed and purified M. paratuberculosis-specific polypeptides are used to immunize both BALB/c mice and New Zealand white rabbits. Standard immunization regimens are used in each instance. TiterMax or Freund's incomplete serve as the adjuvant. Splenic lymphocytes from the immunized mice are hybridized with myeloma cells for the production of monoclonal antibodies. ELISA is the method used to assay secreting hybridomas for reactivity to purified antigens. Hybridomas in positive wells are cloned and expanded using standard methods. Rabbit antisera is collected following boost injections of isolated polypeptide until a sufficient titer is obtained.
Improvement in the specificity of the ELISA test for detection of animals with Johne's disease has always been a major goal. The only test commercially available in the US is a direct test that uses a protoplasmic antigen preparation (Dubash et al., 1995, J. Vet. Diag. Invest., 7:347-51; Collins & Sockett, 1993, J. Am. Vet. Med. Assoc., 203:1456-63). Efforts to amplify antigen/antibody reactions focus on the use of an indirect biotin/avidin system. The purified M. paratuberculosis-specific polypeptide to be evaluated is diluted in PBS and added to 96-well microtiter plates. Plates with bound polypeptide are blocked in PBS containing 1% gelatin and then washed three times with PBS containing 0.05% Tween. Test cattle sera is diluted 1:400 in PBS, added to individual wells, and processed as a standard ELISA. Mouse anti-bovine IgM or mouse anti-bovine IgG is the second antibody in these assays. Results show that the use of a biotinylated second antibody followed by streptavidin/alkaline phosphatase and enzyme detection enhances test sensitivity 8 to 16-fold (based on antibody titers) as compared to the standard direct ELISA.
The method described herein using a M. paratuberculosis-specific polypeptide is compared to the commercially available direct ELISA by determining antibody titers of sera from clinically affected animals. Sera selected for these evaluations will include samples from both clinical and subclinical animals at NADC and from a nearby diary herd (State Center, Iowa) shown to have Johne's disease. For all evaluations, it is necessary to include samples from known negative animals to assess specificity. In addition, because of potential cross-reactivity that may be encountered with other bacteria, especially other mycobacteria, sera from animals known to be naturally or experimentally infected with other mycobacterial, particularly M. avium, are included. These controls determine whether the ELISA test detects only M. paratuberculosis-infected cattle.
Histopathologic analysis of tissues from infected animals is a rapid method of detecting M. paratuberculosis. Biopsy tissue or tissue samples taken at necropsy are stained for acid-fastness to determine the presence of M. paratuberculosis. However, this method is non-specific and does not distinguish among mycobacterial species. Therefore, bovine tissues from M. paratuberculosis-, M. bovis-, M. avium-infected and uninfected animals are tested by histopathologic analysis using high-titer antibodies directed at M. paratuberculosis-specific polypeptides. Briefly, samples from the ileum and mesenteric lymph node of cows are fixed in buffered formalin, processed routinely, and embedded in paraffin wax. 6 μm cut sections are stained with hematoxylin and eosin or Ziehl-Neelsen by conventional methods. Replicate unstained sections will be prepared for immunohistochemistry. Sections that are immunostained are deparaffinized, rehydrated and blocked using routine methods (Stabel et al., 1996, J. Vet. Diagn. Invest., 8:469-73). Blocked sections are incubated with M. paratuberculosis-specific antibodies developed in the above-described studies. Depending on the nature of the primary antibody, either goat anti-rabbit biotinylated antibody or goat anti-mouse biotinylated antibody is added followed by washing instreptavidin-alkaline phosphatase solution. The tissue is stained with chromogen, and Histomark Red. Results are visualized under a bright-field microscope. Staining intensities are quantitatively compared among the different infected and uninfected tissues.
Detection of M. paratuberculosis using oligonucleotide primers complementary to M. paratuberculosis-specific genes 93, 135, 218, 228, 240, and 251 or oligonucleotide primers complementary to IS900 nucleic acid sequences was examined by PCR. IS900 primer sequences were as follows: 5′-AAT CAA CTC CAG CAG CGC GGC CTC G-3′ (SEQ ID NO:108) and 5′-CCG CTA ATT GAG AGA TGC GAT TGG-3′ (SEQ ID NO:109). Fourteen fecal samples were processed from cattle in various stages of shedding. The bacterial load being shed by each animal was determined by culture on 7H10 slants.
To detect M. paratuberculosis by amplification of nucleic acids from a biological sample, a PCR master mix was generated similar to that described in Example 4 with the addition to the master mix of 10 mM MgCl. The PCR reaction conditions for amplification of nucleic acids from a biological sample were as follows: a 10 min denaturing step at 94° C., followed by 50 cycles of: 94° C. for 59 sec, 60° C. for 30 sec, and 72° C. for 1 min. At the end of 50 cycles, there was a 10 min incubation at 72° C. followed by a hold at 4° C.
Results of the PCR assays are as follows. Seven cattle identified as shedding heavily were all positive for M. paratuberculosis nucleic acid using either IS900 or MP228 primers. Out of 5 cattle identified as medium shedders, primers directed toward IS900 detected M. paratuberculosis nucleic acid in 1 animal, while primers directed toward MP228 detected M. paratuberculosis-specific nucleic acid in 2 animals. Out of 2 cattle identified as low shedders, primers directed toward IS900 detected M. paratuberculosis nucleic acid in 1 animal, while MP228 primers didn't detect M. paratuberculosis-specific nucleic acid in any animal. In titrations of M. paratuberculosis genomic DNA (isolate K-10), IS900 nucleic acids were detectable in 1 fg of nucleic acid, while each of the M. paratuberculosis-specific nucleic acids were detectable in 10 fg of nucleic acid.
A shotgun strategy was adopted to sequence the genome of M. paratuberculosis strain K-10. To create a small (1.5- to 3.0-kb) insert library, genomic DNA was isolated using a chloroform/cetyltrimethylammonium bromide-based method and DNA was sheared by nebulization and cloned into a pUC18 plasmid vector for shotgun sequence analyses essentially as described (May et al., 2001, Proc. Natl. Acad. Sci., USA, 98:3460-5). Approximately 24,000 clones were sequenced from both ends using Dye-terminator chemistry on ABI 3700 and 3100 (Applied Biosystems) sequencing machines and a total of 45,653 sequences (representing ˜7.8-fold coverage of the genome) were generated in this manner for inclusion in the final sequence assembly. Sequence assembly and verification were accomplished by using the phredPhrap and Consed suite of software (genome.washington.edu on the internet). In order to close the final ˜400 gaps at the end of the shotgun phase, several methods were used, including primer walking and random PCR. The final sequence showed that the M. paratuberculosis genome was a single circular chromosome of 4,830,869 bp and an average GC content of 69.3%.
The sequence of the entire M. paratuberculosis genome (SEQ ID NO:1355) is shown in Table 8 (contained on the appended compact disc, which has been incorporated by reference herein).
The resulting approximately 24,000 nucleic acid segments were analyzed as follows. Each of the 24,155 segments was compared to the M. avium genome using BLASTN (released May 14, 2002). 23,056 segments had homology to M. avium sequences, while 1,099 segments had no homology with sequences in the M. avium database. Of the 23,056 segments having homology to M. avium sequences, 22,558 segments had >50% sequence identity to M. avium sequences, while 498 segments possessed <50% sequence identity to M. avium sequences. The 498 segments having <50% sequence identity to M. avium sequences were then compared to sequences in the GenBank database (having a release date of Dec. 28, 2002) using BLASTN. Of the 498 segments used in the BLASTN comparison, 130 segments were identified as having <50% sequence identity with sequences in the GenBank database, while 277 segments had no sequence identity with sequences in the GenBank database. Those 407 segments (277 segments+130 segments) were considered to be M. paratuberculosis-specific nucleic acids. The 1,099 segments that had no homology to M. avium sequences were then compared to the GenBank database using the BLASTN. 702 segments had no homology with sequences in GenBank, while 397 segments possessed homology with sequences in the GenBank database. Of those 397 segments, 29 segments exhibited <50% sequence identity with sequences in the GenBank database, while 95 segments exhibited sequence identity with M. paratuberculosis sequences that were previously submitted to the GenBank database. Those 825 segments (702 segments+29 segments+95 segments) were also considered to be M. paratuberculosis-specific nucleic acids.
The positions of these M. paratuberculosis-specific nucleic acids are schematically shown in
Potential coding sequences (CDSs) in the genome were predicted by using GLIMMER, and ARTEMIS, and the results were compared and verified manually in ARTEMIS. Homology studies using the predicted polypeptide sequence were completed with BLASTP analysis by using customized sequence databases constructed by the Computational Biology Center at the University of Minnesota (cbc.umn.edu on the World Wide Web). Table 9 (contained on the appended compact disc, which has been incorporated by reference herein) describes the annotation of the M. paratuberculosis genomic sequences, and Table 10 (contained on the appended compact disc, which has been incorporated by reference herein) describes the predicted amino acid sequences encoded by each identified coding sequence.
A total of 161 bacterial isolates were used in these studies, including M. paratuberculosis (n=118), M. avium (n=21), and other mycobacterial and non-mycobacterial species (n=22). See Table 11. M. paratuberculosis strain K-10 was used as the standard strain. M. paratuberculosis isolates were grown on Middlebrook 7H9 broth or 7H11 agar (Difco Laboratories, Detroit, Mich.) with OADC supplement (Becton Dickinson, Sparks, Md.) and mycobactin J (2 mg/100 ml), and cultures were incubated at 37° C. for 4-6 months until colonies were observed. M. avium isolates were grown on Middlebrook 7H9 broth without mycobactin J and cultures were incubated at 37° C. for 2 weeks. Other mycobacterial and non-mycobacterial species were grown on LB medium and incubated overnight at 37° C.
DNA was isolated from bacterial cultures using the QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, Calif.). Briefly, bacteria were pelleted and resuspended in 180 μl Buffer ALT. Proteinase K (20 μl) was added, and the samples were vortexed and incubated at 56° C. for 10 minutes. After addition of 200 μl Buffer AL, the samples were incubated at 70° C. for 10 minutes. Ethanol (200 μl) was added and the samples were vortexed and loaded onto spin columns, which were subjected to centrifugation at 8000 rpm for 1 minute. The columns were washed with 500 μl Buffer AW and the DNA was eluted in 50 μl distilled water.
Mycobacterium isolates tested
M. paratuberculosis
M. avium
M. intracellulare
M. scrofulaceum
M. phlei
M. smegmatis
M. sylvaticum
M. fortuitum
Salmonella spp.
S. aureus
The QIAamp DNA Stool Mini Kit (QIAGEN Inc.) was used to isolate DNA from stool samples. Briefly, 1.4 ml Buffer ASL was added to 200 mg of sample (1 gm of sample can be used with 10× Buffer ASL). The mixtures were vortexed, heated at 95° C. for 5 minutes, and pelleted to remove stool particles, and 1.2 ml of each resulting supernatant was transferred to a new 2 ml tube. An InhibitEX tablet was added to each sample, and the tubes were vortexed and then incubated for 1 minute at 25° C. The mixtures were pelleted and 15 μl of Proteinase K was added to 200 μl of each supernatant. Buffer AL (200 μl) was added and the samples were incubated at 70° C. for 10 minutes. After addition of 200 μl ethanol, samples were vortexed and loaded onto spin columns. The columns were subjected to centrifugation at 8000 rpm for 1 minute and then washed two times with 500 μl Buffer AW1 and Buffer AW2. DNA was eluted in 50 μl distilled water.
A PCR master mix was prepared containing the following: 1× TaqMan Buffer A (Perkin Elmer), 5.0 mM MgCl2, 1.25 units per reaction Amplitaq Gold, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 400 μM dUTP, 5% DMSO, 0.01 units per reaction UNG, 100 μM of each primer, and 150 μM of each probe. Five μl of template DNA was placed in each PCR reaction tube, and 45 μl of Master mix was added. PCR samples were subject to initial denaturation at 50° C. for 10 minutes and then at 95° C. for 10 minutes; 40 amplification cycles of 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute; a final extension at 72° C. for 7 minutes; and a soak at 25° C. Specific PCR products were detected using the ABI Prism 7700 or 7900HT Sequence Detection System (Applied Biosystems, Inc.). Results were recorded as Delta-RQ, which is the difference in the Rn values from the samples and the no-template control. The Rn values are the ratio of reporter emission to quencher emission. Agarose gel electrophoresis with ethidium bromide staining was performed to verify the results of the TaqMan assay. All assays were performed in duplicate.
To evaluate the sensitivity of the assay, ten-fold dilutions of M. paratuberculosis strain K-10 cells were spiked into a negative fecal sample collected from a known M. paratuberculosis-free dairy farm. M. paratuberculosis DNA amounts ranged from 100 ng to 1 fg, equivalent to 112900 to 0.0011 molecules/reaction. DNA was extracted from the spiked samples using a QIAamp DNA Stool Mini Kit, the sensitivity of the assay for detecting M. paratuberculosis in fecal samples was assessed by PCR as described above.
The specificity of the assay was evaluated using template DNA from other mycobacteria (n=48), and non-mycobacterial spp. (n=3). In addition, the TaqMan assay was compared to conventional PCR, which was performed using primer sequences complimentary to SR134 (see Table 12).
A real-time PCR assay was developed for detection and quantitation of M. paratuberculosis. Primers and probes were designed based on a novel unique sequence, SR134 (Table 11). SR134 is a sequence unique to M. paratuberculosis and is present in 6 copies in the genome. To increase sensitivity, two sets of primer-probe combinations were tested and used in the TaqMan assay as a multiplex strategy to amplify 211 bp and 215 bp fragments of the M. paratuberculosis SRI 34 sequence. Assay conditions were optimized for MgCl2, primer, and probe concentrations in the reaction mix; optimal concentrations were found to be 5.0 mM MgCl2, 100 μM each primer, and 150 nM each probe.
To quantitate standard M. paratuberculosis, curves resulting from amplification of SRI 34 from known amounts of M. paratuberculosis DNA (100 ng to 1 fg) were generated. A regression line was generated from the data points, and the correlation coefficient (R2) value was determined to be 0.99. The ability to employ the TaqMan approach for quantitation of M. paratuberculosis also was determined. For example, a sample containing a “blinded” number of M. paratuberculosis cells had a Ct value of 24.16, which was equivalent to 0.082 ng DNA or 96 cell equivalents, and closely approximates the 100 cell equivalents that were spiked into the sample.
Known amounts of M. paratuberculosis K-10 genomic DNA were used to test the sensitivity of the assay. DNA concentrations ranging from 100 ng to 1 fg resulted in Ct values of 15.18 to 39.09. The cut-off point for accurate detection of M. paratuberculosis K-10 DNA was approximately 100 fg of DNA (35.04 Ct), which represents 0.11 cell equivalents of M. paratuberculosis. Ten-fold dilutions of M. paratuberculosis K-10 cells spiked in feces also were used to determine the sensitivity of the assay. The assay was reliably able to detect 1 cell of M. paratuberculosis per PCR reaction.
The specificity of the TaqMan assay was tested using 118 M. paratuberculosis isolates from different animal species including bovine, ovine, murine, and humans, isolates representing 7 other mycobacterial species (n=27) including the closely related Mycobacterium avium, atypical mycobacteria (n=4), and uncharacterized non-MAP (n=9) (Table 11). The SRI 34 TaqMan assay was able to detect all but two M. paratuberculosis isolates, whereas no amplification was observed with any of the other mycobacterial and non-mycobacterial species. Thus, this assay was 100% specific for amplification of M. paratuberculosis DNA.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation and claims benefit of priority to International Application No. PCT/IB2003/006509, having an International Filing Date of Mar. 6, 2003, which claims benefit of priority to U.S. application Ser. No. 10/137,113, filed Apr. 30, 2002, now U.S. Pat. No. 7,074,559 issued Jul. 11, 2006, which claims benefit under 35 U.S.C. §119(e) of U.S. application Ser. No. 60/362,396, filed Mar. 6, 2002.
The U.S. Government may have certain rights in this invention pursuant to Grant Nos. 00-35201-9200, 58-3625-0-137, and 00-02215 awarded by the U.S. Department of Agriculture.
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| Number | Date | Country | |
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| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2003/006509 | Mar 2003 | US |
| Child | 10934893 | US |
