Abstract:
An  M. paratuberculosis  gene referred to as hspX provides a useful target region for the construction of suitable probes and primers that are species-specific for distinguishing  M. paratuberculosis  from related mycobacteria in a test sample.  M. paratuberculosis  is the causative agent of Johne&#39;s disease and has been isolated from human patients with Crohn&#39;s disease.

Description:
This application is a division of application Ser. No. 09/108,051, filed Jun. 30, 1998, now U.S. Pat. No. 5,985,576. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
       Paratuberculosis  poses a significant economic and health problem worldwide, especially in the cattle industry. 1,2    Mycobacterium avium  subspecies  paratuberculosis   3  ( M. paratuberculosis  ) is the etiologic agent of  paratuberculosis  (Johne&#39;s disease), a chronic granulomatous enteritis of both domestic and wild ruminants. This organism is an intracellular pathogen that replicates within macrophage of both the gastrointestinal tract and associated lymphatic tissues. 4  The disease can be transmitted in utero, to nursing calves, or via infected fecal contamination of food. Diagnosis of subclinical  paratuberculosis  is problematic because infection progresses slowly and infected animals often do not show signs of the disease for years. Once the disease is established in a herd, there is no cure. Annual economic losses to the dairy industry are in the billions of dollars worldwide, primarily as a result of reduced milk production, decreased reproductive efficiency, and death. 
     In humans,  M. paratuberculosis  has been isolated from patients with Crohn&#39;s disease, a chronic enteritis with clinical symptoms similar to animals with  paratuberculosis.   5,6    M. paratuberculosis  has been implicated as a possible cause of Crohn&#39;s disease, however, the etiology of this disease remains unknown. 
     This invention relates to a species-specific genetic target element useful for identifying  M. paratuberculosis  and for distinguishing this organism from related bacteria by various diagnostic techniques. Probes and primer sets are disclosed for detecting target sequence in laboratory and clinical samples containing  M. paratuberculosis.    
     2. Description of the Prior Art 
     Cattle shed  M. paratuberculosis  in their feces during the subclinical and clinical stages of infection. Currently, the most sensitive test available for subclinical  paratuberculosis  requires a prolonged 8-12 week fecal culturing of the organism. Existing immunological diagnostic tests are rapid but have disadvantages resulting from poor specificity of the antigens used in the assays. 
     Nucleic acid diagnostic methodology is used as a rapid and sensitive way to identify specific species of mycobacteria. 7,8,9  Some mycobacterial species are genetically very closely related to  M. paratuberculosis  according to DNA-DNA hybridization analysis. 10  Genome homology ranging from 50% to nearly 100% has been reported between the ATCC 19698 reference strain of  M. paratuberculosis  and species of the  Mycobacterium avium  complex (MAC) which includes the incompletely separated  Mycobacterium avium  (subspecies avium [ M. avium ] and subspecies silvaticum [ M. silvaticum ] and  Mycobacterium intracellulare  as well as other strains not assigned to either species. 11,12,13    M. paratuberculosis  DNA is also related to DNA of other mycobacteria, such as  Mycobacterium bovis, Mycobacterium leprae,  and  M. tuberculosis.   14,15  The high percentage of genetic relatedness of  M. paratuberculosis  with other mycobacterial species requires the cloning, sequencing, and characterization of unique genetic markers (genetic elements or genes) to differentiate these closely related species. Species-specific genetic markers are useful tools for the development of new molecular diagnostic tests. 
     Only two species-specific genetic elements have been identified in the  M. paratuberculosis  genome. 16,17  DNA probes derived from these genetic elements have been used to detect  M. paratuberculosis  infection. One genetic element, IS900, is a 1.45 kbp insertion element found at approximately 20 copies per chromosome. 16  Similar insertion elements, which have sequences related to IS900, have been identified in closely related mycobacteria, such as  M. avium  (IS901) 18,19  and  M. silvaticum  (IS902). 20  The now commercially available IS900 DNA diagnostic kit (IDEXX Corp.), which was used in studies conducted in a  M. paratuberculosis  control program, yields an 89% specificity and a 13% sensitivity. 21    
     The other  M. paratuberculosis  species-specific genetic element that has been identified, F57, is a 620 bp DNA fragment not related to any known sequence, including the IS900 insertion element. 17  Southern hybridization analysis using the F57 fragment suggests that this genetic element is single-copy in the  M. paratuberculosis  genome. The F57 genetic element is currently being used as a diagnostic tool to identify  M. paratuberculosis  infection in both cattle and humans (patients with Crohn&#39;s disease). 10,17  However, the cloning and sequencing of additional  M. paratuberculosis -specific genetic elements or genes is needed to improve or develop new rapid and sensitive nucleic acid diagnostic tests for the differentiation of  paratuberculosis  infection. 
     Currently, there is a need for an accurate, rapid and reliable detection of  M. paratuberculosis  infection. 
     SUMMARY OF THE INVENTION 
     We have now discovered a  M. paratuberculosis  gene, hereafter referred to as hspX, that is present as a single-copy gene in the  M. paratuberculosis  genome. This gene provides a useful target region for the construction of suitable probes and primers that are species-specific for distinguishing  M. paratuberculosis  from related mycobacteria in a test sample. Diagnostic assays for  M. paratuberculosis  could also be based on expressed protein products of the hspX gene, such as in an ELISA assay. 
     In accordance with this discovery, it is an object of the invention to provide a sensitive, specific, and rapid diagnostic tool for positively identifying  M. paratuberculosis  in a clinical or laboratory sample. 
     It is also an object of the invention to provide a target region for constructing probes and primer sets tailored to the desired specificity for detecting  M. paratuberculosis  infections. 
     Another object of the invention is to provide an improved method for diagnosing Johne&#39;s disease in ruminant animals. 
     Other objects and advantages of the invention will become readily apparent from the ensuing description. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic diagram of BamHI restriction fragments containing the hspX and recA open reading frames in  Mycobacterium avium  subspecies genomes. The positions of a  M. paratuberculosis  species-specific DNA probe (MP probe) in the hspX gene of the invention and of the generic mycobacterial DNA probe (M probe) in the recA gene are shown. 
     FIG. 2 is the hspX open reading frame target sequence of the invention. Bold italics letters depict the MP probe, the ATG start codon is underlined, the TGA stop codon is double underlined, and the deduced amino acid sequence is indicated (amino acid shown below the first nucleotide of each codon). 
    
    
     DEFINITIONS 
     As used herein, the expression “test sample” is intended to mean any clinical, laboratory, environmental or other collected sample of material that is suspected of containing the intended target nucleic acid which is to be detected. Exemplary test samples include swabs, scrapings or collections of food, bacteriologic cultures, body fluids, tissues or other sources of mycobacterial infection or contamination. 
     The expression “target nucleic acid” or “target sequence” is intended to include the sequence within the hspX gene (FIG. 2, SEQ ID NO:1) given below, DNA or RNA sequences complementary to SEQ ID NO:1, and any portion of the aforementioned DNA or RNA sequences that is of sufficient size to permit the desired level of identification. 
     The term “probe” is used herein in the broadest sense to refer to either a labeled or an unlabeled, single-stranded nucleic acid that will hybridize under predetermined conditions of stringency to the target nucleic acid. Such probes may be DNA or RNA and will typically be at least about 15 bases in length, and preferably about 20-100 bases in length. When used in a hybridization assay, hybrids formed from the probes and the target sequence are usually detected by means of a detectable label affixed directly to the probe. Alternatively, probes can be used as helper probes to facilitate binding of a separate labeled probe to the target nucleotide. It is understood that for hybridization to occur, the probe may or may not be exactly complementary to the-target sequence, provided that the hybridization conditions are appropriately selected to permit hybridization even when there are a limited number of mismatches between the respective sequences. 
     The term “primer” is used herein in its usual sense to be descriptive of an oligonucleotide (DNA or RNA), usually about 15-30 nucleotides in length, and preferably about 17-26 bases in length, that will participate in a primer extension reaction when catalyzed by a polymerase. These reactions are more commonly referred to as “polymerase chain reactions” (“PCR”). Contemplated herein as primers are only those nucleotides that are properly oriented so as to amplify a region within the target sequence. 
     The expression “substantial equivalent thereof” in reference to any target sequence or to the sequence of a probe or primer is intended to mean that minor additions, deletions, or mismatches can be present in the sequence to the extent that such variations do not prevent the hybridization or annealing of the nucleic acids essential to the assay. 
     “Stringency” refers to the conditions under which hybridization takes place. At high stringency, only exact matches of DNA and/or RNA will hybridize stably. Under low stringency, 80-90% homologous sequences may still hybridize. 
     Unless otherwise indicated, the term “species-specific” is used herein to indicate specificity for the subspecies  M. paratuberculosis.  The expression “sequence-specific oligonucleotide” is used herein to refer to probes or primers having a hybridizing region that is exactly complementary to a segment of the target region. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The target sequence for use in the invention is the  M. paratuberculosis  hspX gene, the complement of said gene, or an RNA transcript of this gene. These target sequences lend themselves to the development of species-specific DNA- or RNA-based diagnostic assays for  M. paratuberculosis  because they are unique to this subspecies. 
     To initially identify the target sequence,  M. paratuberculosis  genomic DNA was digested with PstI, size fractionated by agarose gel electrophoesis, and screened using an “in-gel” hybridization method with eight 15 bp DNA probes (SEQ ID NOS:5-12) that contained in one open reading frame (ORF) the nucleotide sequence encoding the arginine-glycine-aspartic acid (RGD) peptide adhesion motif. Purified PstI fragments of size ˜3.2 kbp and ˜3.6 kbp were cloned into the phagemid pBluescript II SK+multiple cloning site and the recombinant plasmids were used to transform  E. coli  ElectroMAX DH10B™ cells by electroporation using standard protocols.  25  Plasmids from about 50 random transformants were screened by DNA hybridization using the degenerate RGD probes to yield two positive recombinant plasmid DNAs designated pBpst101 and pBpst102. These clones were subjected to nucleotide sequence analysis for the purpose of identifying all potential open reading frames. 
     The pBpst101 clone contains part of an open reading frame (ORF) of 507 nucleotides coding for 169 amino acids (aa) residues from the putative  M. paratuberculosis  recA gene. Comparison of nucleotide and deduced amino acid sequences with known sequences using the BLASTN and BLASTX search algorithms revealed that this partial ORF has significant homology to the conserved 5′ terminus of the known ORFs of the recA genes cloned from  M. tuberculosis  and  Mycobacterium leprae.  The closest similarities were found to be the nucleotides of the  M. tuberculosis  and  M. leprae,  89% and 85% similar, respectively (data not shown). Nucleotide sequence analysis also revealed that the recA portion of this pBpst101 clone was 80% similar to the recA homologue cloned from  Streptomyces venezuelae  ISP5230. From the recA portion of the pBpst101 clone, a 33 bp mycobacterial DNA probe (SEQ ID NO:3), designated M (FIG.  1 ), was synthesized and used as an internal control probe for detection of mycobacterial genomic DNA. 
     Clone pBpst102 contains an ORF of 432 nucleotides encoding for 144 amino acid residues (SEQ ID NO:2) for a putative heat-shock-like protein, designated hspX, which is unique to  M. paratuberculosis.  Residues 60-64, arginyl-aspartic acid-glycyl-aspartic acid-aspartic acid (RDGDD) form a conserved peptide motif found in four cloned dnaJ genes including the  Bacillus subtilis  and  M. tuberculosis  dnaJ genes. The BLASTX analysis revealed that the sequences flanking the region encoding the RDGDD motif were unique to  M. paratuberculosis.  From these flanking regions a 30 bp  M. paratuberculosis  species-specific DNA probe (SEQ ID NO:4), designated MP (FIGS.  1  and  2 ), was selected and synthesized. 
     When the M probe derived from the recA is tested against genomic DNA extracted from various mycobacteria such as shown in Table I using Southern hybridization analysis, it is able to differentiate mycobacterial species from other pathogenic bacterial species as discussed in further detail in Example 2. The MP probe of the invention, derived from the hspX genomic sequence, is able to differentiate  M. paratuberculosis  from other species (and subspecies) belonging to the  M. avium  complex as also discussed in further detail in Example 2. The observation that the MP sequence is only found in  M. paratuberculosis  species suggests that the open reading frame containing this sequence encodes a protein that is specific to  M. paratuberculosis.    
     The strategy for identifying other useful probes or PCR primer sets based on the hspX target region sequence would be in accord with standard guidelines as well-known in the art. Of course, particularly for a short oligomer, the primary consideration would be sequence distinctness within the region being assayed. Other considerations would include the length and the melt temperature (T m ) of the selected target region. The methods for construction and use of probes and primers are well-established in the art. 
     A strategy for constructing an oligonucleotide useful as a probe or primer is initiated by predetermining the length of the oligonucleotide. As previously indicated, probes will typically be at least about 15 bases, and preferably about 20-100 bases, in length. Primers are more typically about 15-30 bases, and preferably about 17-26 bases, in length. The nucleotide sequence complementary to the target DNA or RNA transcript is determined, and the oligodeoxyribo-nucleotide or oligoribonucleotide is synthesized as the inverse of the complementary sequence. In this way, the probe or primer is in the correct orientation for binding to native nucleic acid in the target sample. Exemplary oligonucleotides useful for purposes of the invention include the 30 bp MP probe described below in Example 2 and the  M. avium  subspecies  paratuberculosis  primers described in Example 3. 
     In practice of the invention, various assays for  M. paratuberculosis  could be performed. For example, a clinical sample obtained from the test subject would first be cultured under suitable conditions to expand the  M. paratuberculosis  organism, and then a test sample of the resultant culture would be subjected to polymerase chain reaction (PCR). PCR using the hspX primers could also be performed directly on nucleic acids eluted from frozen biopsy tissue sections and/or formalin-fixed paraffin-embedded tissue sections. Amplified nucleic acid fragments can then be detected, for example, by Southern hybridization. Alternatively, clinical samples would be used in a hybridization assay with a labeled probe to indicate the presence of the hspX gene or its RNA transcript. 
     In an alternative embodiment, the protein expressed by the hspX gene may be used as an immunodiagnostic reagent for binding and detecting antibodies in the serum of an animal. Detection of antibodies against the hspX protein or fragments thereof in the sera of animals may be used for monitoring and detecting animals which are carriers of  M. paratuberculosis  but which do not show outward signs of infection, as well as identifying animals previously exposed or infected with the mycobacterium. A variety of conventional immunoassays are suitable for use herein, although ELISA is preferred. For example, in such an ELISA test, the purified hspX protein may be used as an antigen bound to the wells of a microtiter plate. Following contact of the test animal sera with the adsorbed antigen, bound anti-hspX antibodies may then be detected. 
     The hspX protein may also be covalently bonded to a non-related fusion protein as described in greater detail hereinbelow. The invention also encompasses substantial equivalents of this protein which retain the ability to elicit antibody production in an animal against  M. paratuberculosis.  The practitioner of ordinary skill in the art will recognize that slight deviations of the amino acid sequences may be made without affecting the immunogenicity of the protein. Substantial equivalents of the above protein include conservative substitutions of amino acids with other amino acids, including either naturally occurring or non-conventional amino acids, which maintain substantially the same charge and hydrophobicity as the original amino acid. Conservative substitutions include for example, replacement of glycine for alanine, valine for isoleucine, leucine for isoleucine, aspartic acid for glutamic acid, lysine for arginine, asparagine for glutamine, phenylalanine for tryptophan, and tryptophan for tyrosine. Examples of conservative substitutions with non-conventional amino acids are described in Rosenberg et al. (U.S. Pat. No. 5,679,782) the contents of which are incorporated by reference herein. 
     In use, it is envisioned that the isolated protein will typically be formulated in conjunction with a suitable inert carrier or vehicle as known in the art. The skilled practitioner will recognize that such carriers should of course be compatible with the protein. The concentration and amount of the protein in the final composition may vary depending upon the desired use and type of response needed, and the host animal. In any event, the protein should be employed in an amount effective to induce the preferred response as determined by routine testing. 
     When the protein is used to elicit antibody production against  M. paratuberculosis,  the proteins may be formulated with a physiologically acceptable diluent or carrier such as phosphate buffered saline. The proteins may be administered to a target animal by any convenient route, including intramuscularly, intraperitonealy or preferably subcutaneously, in a single dose or in a plurality of doses. The protein may also be administered in combination with optional stabilizers and immunopotentiating agents or adjuvants. Typical stabilizers include, for example, sucrose, an alkali metal hydrogen phosphate salt, glutamate, serum albumin, gelatin, or casein. A variety of adjuvants are suitable for use herein, although a mixture of alhydrogel and amphigen is preferred. Other conventional adjuvants which may be suitable for use herein include those described by Davis et al. (ed.) (Microbiology, second edition, Harper &amp; Row, Hagerstown, Md., 1973, pp. 480-482), the contents of which are incorporated by reference herein. The proteins may be stored under refrigeration or in frozen or lyophilized form. 
     In a preferred embodiment, the objective of antibody production is the protection of cattle against  M. paratuberculosis  by eliciting antibody production and/or an immediate-type hypersensitivity in the animal. Generally, the proteins are administered to the target animal in an amount effective to elicit either or both of these responses in a subject animal as compared to an untreated control. The effective amount will vary with the particular target animal, its age and size, and may be readily determined by the practitioner skilled in the art. Without being limited thereto, typical doses for treatment of cattle may be greater than 5 μg/animal/dose, preferably between 5 to 25 μg/animal/dose administered by subcutaneous or intramuscular injection. 
     The antigenic proteins of the invention are produced by growing host cells transformed by the expression vectors described above under conditions whereby the antigen is produced. The antigens are then isolated from the host cells. Depending on the host cell used, transformation is done using standard techniques. For example, the calcium treatment employing calcium chloride, as described by Cohen (1972, Proc Natl Acad Sci USA, 69:2110), or the RbC1 method described in Maniatis et al. (ibid, p. 254) may be used for procaryotes or other cells which contain substantial cell wall barriers. Infection with  Agrobacterium tumefaciens  such as described by Shaw (1983, Gene, 23:315) may be used for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology, 52:546) may be used. Transformations into yeast may be conducted, for example, according to the method of Van Solingen, et al., (1977, J. Bacter., 130:946), and Hsiao et al. (1979, Proc Natl Acad Sci USA, 76:3829). 
     In general, after construction of a suitable expression system, the system is transfected into the appropriate host and successful transformants may be selected by markers contained on the expression vectors. Successfully transformed colonies are then cultured in order to produce the protein. Optionally, a promoter which can be controlled by regulating conditions in the environment may be used such that the cells can be grown under conditions where the gene encoding the desired protein of the invention is not expressed, but production of the protein may be induced by appropriate manipulation of conditions, as described in U.S. Pat. No. 5,670,339. This protocol may be used to prevent premature accumulation of the protein which may be harmful to the growth of the cell. 
     The protein may be produced intracellularly, or in secreted form by construction of vectors wherein the peptide is preceded by a signal peptide workable in the appropriate host. The recombinant protein may then be recovered from the medium or from the cells using suitable techniques generally known in the art, and purified by, for example, ion exchange chromatography, ammonium sulfate precipitation, or gel permeation chromatography. 
     In a variation of the above embodiment, the antibodies so-produced in the host animal or monoclonal antibodies raised to the hspX protein may be recovered for use in a diagnostic assay for the identification of  M. paratuberculosis.  A variety of conventional immunoassay techniques are suitable for use herein, including RIA, or ELISA, or double antibody sandwich immunoassays. 
     The following examples are intended to further illustrate the invention. 
     EXAMPLE 1 
     Bacterial Strains, Growth Conditions, and Mycobacterial Identification. 
     The origin and source of the 41 mycobacterial strains, including 28 mycobacterial strains, used in this study are listed in Table I, below. 
     Primary  Mycobacterium avium  complex isolates were obtained by conventional bacteriological culture and were identified by growth characteristics and mycobactin dependence (with all  M. paratuberculosis  isolates being mycobactin J-dependent). The cultures were passed and grown at 37° C. to late exponential phase (A 540 =0.2) in 150-cm 2  tissue culture flasks containing 75 ml of Middlebrook 7H9 liquid medium (pH 5.9) supplemented with Dubos oleic albumin complex enrichment, 0.05% Tween 80, and ferric mycobactin J. Subsequently, D-cycloserine (1 mg/ml final concentration) was added to each flask, mixed thoroughly, and cultures were incubated for an additional 24 h. The cell cultures were then harvested by centrifugation (11,000×g for 30 min at 10° C.). 
       Escherichia coli  ElectroMAX DH10B™ cells (Gibco BRL: Life Technologies, Inc.) were used as the host strain for recombinant plasmids. Working cultures of  E. coli  were grown in Luria-Bertani broth (LB) and maintained on LB agar plates. 22  Stock cultures were stored in LB supplemented with 20% glycerol at −80° C. 
     DNA Isolation, Plasmids, and Cloning Procedures. 
     Genomic DNA was extracted from mycobacteria ( M. avium, M. intracellulare, M. paratuberculosis  and  M. silvaticum ) by the method described by Whipple et al. 23 , as modified by Bauerfeind et al. 24  Mycobacterial cells were harvested (˜100 mg, wet weight), washed in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), and incubated in TE buffer containing 16,000 U/ml (final concentration) of lipase for 2 h at 37° C. Lysozyme (5 mg/ml) was then added to the solution and incubation was continued for an additional 2 h at 37° C. The samples were then treated with proteinase K (2 mg/ml) and sodium dodecyl sulfate (10 mg/ml) and incubated for an additional 15 h at 50° C. Following the incubation, ½ volume of 7.5 M potassium acetate was gently mixed into each sample, the samples were placed on ice for 10 min, and centrifuged for 10 min at 4° C. Subsequently, DNA was purified from the supernatant by repeated phenol-chloroform-isoamyl alcohol (25:24:1; vol/vol/vol) extraction and was precipitated by adding 2 volumes of 95% ethanol. Precipitated DNA was washed with 70% ethanol, dried, and resuspended in sterile ultrapure water. 
       M. paratuberculosis  genomic DNA was digested with PstI, size fractionated by agarose gel electrophoresis, and screened using an “in-gel” hybridization method with eight 15 bp DNA probes (5′-GCACGGGGCGACGTC-3′, 5′-GCACGGGGGGACGTC-3′, 5′-GCACGCGGCGACGTC-3′, 5′-GCACGAGGCGACGTC-3′, 5′-GCACGCGGGGACGTC-3′, 5′-GCACGGGGGGACGTC-3′, 5′-GCAAGAGGGGACGTC-3′, 5′-GCAAGGGGGGACGTC-3′; SEQ ID NOS:5-12, respectively) that contained in one open reading frame the nucleotide sequence encoding the RGD peptide adhesion motif. 25  PstI restriction fragments of size ˜3.2 kb and ˜3.6 kb were isolated and purified by the 1% agarose “gel trough” method. 19  Purified PstI fragments were cloned into the phagemid pBluescript II SK+multiple cloning site and the recombinant plasmids were used to transform  E. coli  ElectroMAX DH10B™ cells by electroporation using standard protocols. 25  Plasmids from ˜50 random transformants were screened by DNA hybridization using the degenerate RGD probes. The positive recombinant plasmid DNAs were isolated and purified by using alkaline-lysis/polyethylene glycol (PEG) precipitation 22  and were designated pBpst101 and pBpst102, respectively. 
     DNA Sequence Analysis and Identification of Mycobacterial Genes. 
     Nucleotide sequences were determined by dye chain termination reactions on Applied Biosystems instrument and sequence scan software at the Iowa State University Nucleic Acid Sequencing Facility (Ames, Iowa). Sequencher™ (Gene Codes, Ann Arbor, Mich.) software was used to align all sequences into a contiguous DNA fragment and to determine all potential open reading frames. Sequence data analysis was done by screening National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md., databases using BLASTN and BLASTX algorithms 26  via the BLAST Network Service. 
     EXAMPLE 2 
     Detection of the Genus Mycobacterium and the Species  M. paratuberculosis  by Dioligonucleotide Hybridization analysis. 
     Purified bacterial genomic DNA from the bacterial strains in Table I was digested with BamHI, BglII, PstI, PvuII, XbaI, or XhoI at 37° C. for 3 h. DNA fragments were separated by electrophoresis-through (11×14 cm or 20×25 cm) 0.8% agarose gels in Tris-borate-EDTA buffer (pH 8.3) at 25 V·18 h or 45 V·20 h, respectively. Hybridization was carried out at 65° C. in 5×SSC containing 0.1% (w/v) N-lauroylsarcosine and 0.02% (w/v) sodium laurylsulfate (1×SSC is 0.15 M NaCl and 0.015 M sodium citrate); the membrane was washed twice in 0.5×SSC−0.1% SDS at 25° C. for 15 min followed by two washes in 0.1×SSC−0.1% SDS at 65° C. for 15 min. 
     The M Probe 
     The M probe (33 bp, 5′-GACACCGATTCGCTGCTGGTCAGCCAG CCGGAC-3′ (SEQ ID NO:3), mycobacterial recA probe) end-labeled with digoxigenin detectable by chemiluminescence was used in Southern hybridization analysis to identify sequences present in various mycobacterial species. In the initial experiments, the M probe was tested against genomic DNA extracted from various mycobacteria (Table 1). The DNA was digested with BamHI or PvuII (positive controls, only  M. bovis  and  M. tuberculosis ) and hybridized in Southern blots with the M probe. When genomic DNA isolated from  M. bovis  and  M. tuberculosis  was digested with PvuII and hybridized with the M probe only the 2.7 kbp restriction fragment carrying the entire recA gene was present. However, when genomic DNA isolated from  M. bovis  and  M. tuberculosis  was digested with BamHI and hybridized with the M probe only the expected 1.4 kbp restriction-fragment was identified. Only the 1.4 kbp restriction fragment was present because the BamHI restriction sites are internal to the recA ORF and the M probe was designed to be positioned between BamHI restriction sites of the cloned  M. tuberculosis  recA gene. 27  When genomic DNA isolated from  M. avium, M. intercellulare, M. paratuberculosis,  and  M. silvaticum  was digested with BamHI and hybridized with the M probe, restriction fragments of sizes 3.6 kbp, 4.0 kbp, 3.6 kbp, and 3.6 kbp, respectively, were present. 
     The MP Probe 
     The MP probe (30 bp, 5′-CCGTCGTGGTATCTGAATCTGCAAGCC AAT-3′ (SEQ ID NO:4),  M. paratuberculosis  species-specific probe) end-labeled with digoxigenin detectable by chemiluminescence was used in Southern hybridization analysis to identify sequences present only in the  M. paratuberculosis  genome. In this experiment the MP probe was tested against genomic DNA extracted from various mycobacteria (Table 1). The DNA was digested with BamHI or PvuII and hybridized in Southern blots with the MP probe. When genomic DNA isolated from  M. avium, M. bovis, M. intercellulare, M. silvaticum,  and  M. tuberculosis  digested with BamHI or PvuII was hybridized with the MP probe no restriction fragments carrying this sequence was present. However, when genomic DNA isolated from  M. paratuberculosis  was digested with BamHI and hybridized with the MP probe, a 1.8 kbp restriction fragment was present. 
     Dioligonucleotide hybridization (M probe and MP probe in the same hybridization solution) was used in Southern analysis to identify sequences present in mycobacteria, specifically sequences in the  M. paratuberculosis  genome. In the initial experiments the dioligonucleotide hybridization (dOH) solution was tested against genomic DNA extracted from various mycobacteria (Table 1). When genomic DNA isolated from  M. avium, M. bovis, M. intercellulare,  and  M. tuberculosis  was hybridized with the dOH solution only the expected restriction fragments carrying the M probe sequence were present. However, when genomic DNA isolated from  M. paratuberculosis  was digested with BamHI and hybridized with the dOH solution, restriction fragments of sizes 3.6 kbp (M probe) and 1.8 kbp (MP probe) were present. 
     EXAMPLE 3 
     Detection of the Species  Mycobacterium avium  and the Subspecies  Paratuberculosis  by PCR Analysis 
     Purified mycobacterial genomic DNA listed in Table 2, below, was amplified by PCR using oligonucleotide primers (SEQ ID NO:13 and SEQ ID NO:14) derived from the 16S rRNA ( M. avium  species) sequence and to the unique  M. avium  subspecies  paratuberculosis  hspX gene sequence (SEQ ID NO:15 and SEQ ID NO:16). Amplified DNA fragments were separated by electrophoresis through 20×25 cm 1.5% agarose gels in Tris-borate-EDTA buffer (pH 8.3) at 115 V for 2 h and the DNA product was detected by staining with ethidium bromide. Briefly, amplification products were analyzed by electrophoresing a 12.5 μl —sample in 1.5% agarose gel and staining the DNA with ethidium bromide, the amplified product size was compared with positive control DNA; and if the product was the same molecular weight the amplified DNA product was considered positive. DNA samples that had bands at any other molecular weight position or that had no bands were considered negative. Amplification reactions were performed using a hot start method with a standard 50 μl buffer (pH 8.0) containing the following: 1.5 mM or 2.5 mM Mg2+, 20 pM of each primer, 1.25 U DNA polymerase, and 0.2 mM nucleotides. The oligonucleotide primers used to identify species  M. avium  and the subspecies  paratuberculosis  were derived from 16S rRNA ( M. avium ) sequence and the hspX gene ( paratuberculosis ) sequence, respectively. Primers used to identify the species  M. avium  produced a 180-bp fragment from 16S rRNA sequence and the primers used to identify the subspecies  paratuberculosis  produced a 271-bp fragment from the hspX sequence. Amplification conditions for the 16S rRNA primers included an initial denaturation at 94° C. for 10 min, 50 cycles of 60 sec at 94° C., 15 sec at 65° C. and 60 sec at 72° C., and a final 10-min extension at 72° C. Amplification conditions for the hspX primers included an initial denaturation at 94° C. for 10 min, 50 cycles of 60 sec at 94° C., 60 sec at 60° C. and 60 sec at 72° C., and a final 10-min extension at 72° C. 
     PCR analysis using the primers derived from the 16S rRNA sequence identified 20/20 (100%)  M. avium  and the primers derived from the hspX gene sequence specifically identified 14/14 (100%) reference (ATCC 19698), bovine, and human isolates of the subspecies  paratuberculosis  (Table 2). The  M. paratuberculosis -specific primers distinguished  M. paratuberculosis  isolates from related mycobacteria, including all closely related members of the  Mycobacterium avium  subspecies tested in this study. The experiments indicate that the PCR analysis is a useful diagnostic tool to detect mycobacterial infection, specifically  M. paratuberculosis.    
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Bacterial strains used to determine the specificity of the M probe 
               
               
                 and the MP probe 
               
             
          
           
               
                   
                   
                   
                 M 
                 MP 
               
               
                 Bacteriai strain (Serotype) 
                 Source a   
                 Origin b   
                 Probe 
                 Probe 
               
               
                   
               
               
                   M. paratuberculosis  19698 c   
                 ATCC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  BEN 43544 
                 ATCC 
                 Human 
                 + 
                 + 
               
               
                   M. paratuberculosis  LINDA 43015 
                 ATCC 
                 Human 
                 + 
                 + 
               
               
                   M. paratuberculosis  1003 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1004 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1010 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1018 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1026 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1036 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1113 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1425 
                 NADC 
                 Ovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1434 
                 NADC 
                 Ovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  4090 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  KAY 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. avium  18 (Ser. 2) d   
                 NADC 
                 Bovine 
                 + 
                 − 
               
               
                   M. avium  25291 (Ser. 2) c   
                 ATCC 
                 Chicken 
                 + 
                 − 
               
               
                   M. avium  2264 (Ser. 8) 
                 NVSL 
                 Bovine 
                 + 
                 − 
               
               
                   M. avium  subsp.  silvaticum  M21 
                 NADC 
                 Wood 
                 + 
                 − 
               
               
                   
                   
                 Pigeon 
               
               
                   M. avium  23667-2625 
                 NVSL 
                 Bovine 
                 + 
                 − 
               
               
                   M. avium  27183-3013 
                 NVSL 
                 Swine 
                 + 
                 − 
               
               
                   M. bovis  1145 
                 NVSL 
                 Bovine 
                 + 
                 − 
               
               
                   M. bovis  2045 
                 NVSL 
                 Bovine 
                 + 
                 − 
               
               
                   M. bovis  TMC401 35720 
                 ATCC 
                 Bovine 
                 + 
                 − 
               
               
                   M. bovis  BCG Pasteur 35734 
                 ATCC 
                 Bovine 
                 + 
                 − 
               
               
                   M. intracellulare  35772 (Ser. 19) c   
                 ATCC 
                 Human 
                 + 
                 − 
               
               
                   M. intracellulare  35764 (Ser. 20) 
                 ATCC 
                 Human 
                 + 
                 − 
               
               
                   M. tuberculosis  H37Rv 27294  
                 ATCC 
                 Human 
                 + 
                 − 
               
               
                   M. tuberculosis  H37Ra 25177 
                 ATCC 
                 Human 
                 + 
                 − 
               
               
                   B. bronchiseptica  Human 1 
                 NADC 
                 Human 
                 − 
                 − 
               
               
                   B. bronchiseptica  Dog 1 
                 NADC 
                 Canine 
                 − 
                 − 
               
               
                   B. bronchiseptica  Rat 1 
                 NADC 
                 Rat 
                 − 
                 − 
               
               
                 
                   B. pertussis 
                 
                 NADC 
                 Human 
                 − 
                 − 
               
               
                   B. abortus  2308 
                 NADC 
                 Bovine 
                 − 
                 − 
               
               
                   E. coli  O157:H7 3081 
                 NADC 
                 Bovine 
                 − 
                 − 
               
               
                   E. coli  O157:H7 3100 
                 NADC 
                 Bovine 
                 − 
                 − 
               
               
                   E. coli  O157:H7 43888 
                 ATCC 
                 Human 
                 − 
                 − 
               
               
                 
                   L. interrogans 
                 
                 NADC 
                 Bovine 
                 − 
                 − 
               
               
                 
                   S. aureus 
                 
                 NADC 
                 NA 
                 − 
                 − 
               
               
                   P. haemolytica  D153 
                 NADC 
                 Ovine 
                 − 
                 − 
               
               
                 
                   P. multocida 
                 
                 NADC 
                 NA 
                 − 
                 − 
               
               
                 
                   Y. enterocolitica 
                 
                 NADC 
                 NA 
                 − 
                 − 
               
               
                   
               
               
                   a Source of bacterial strains were as follows: ATCC, American Type Culture Collection (Rockville, MD); NADC, National Animal Disease Center (Ames, IA); NVSL, National Veterinary Services Laboratory (Ames, IA).  
               
               
                   b Origin: NA, Not Available.  
               
               
                   c ATCC bacterial type strain.  
               
               
                   d Formerly  M. paratuberculosis  18.  
               
               
                 M., Mycobacterium; B., Bordetella;  B. abortus, Brucella abortus;  E., Escherichta; L., Leptospira; S., Staphylococcus; P., Pasteurella; Y., Yersina.  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Bacterial strains used to determine the specificity of  M. avium   
               
               
                 subspecies [16S rRNA] primers and the  M. avium  subspecies-specific 
               
               
                   paratuberculosis  [hspX] primers. 
               
             
          
           
               
                   
                   
                   
                 16S 
                   
               
               
                   
                   
                   
                 rRNA 
                 hspX 
               
               
                 Bacterial Strain (Serotype) 
                 Source a   
                 Origin 
                 Primers 
                 Primers 
               
               
                   
               
               
                   M. paratuberculosis  19698 b   
                 ATCC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  BEN 43544 
                 ATCC 
                 Human 
                 + 
                 + 
               
               
                   M. paratuberculosis  LINDA 43015 
                 ATCC 
                 Human 
                 + 
                 + 
               
               
                   M. paratuberculosis  1003 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1004 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1010 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1018 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1026 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1036 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1113 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1425 
                 NADC 
                 Ovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  1434 
                 NADC 
                 Ovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  4090 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. paratuberculosis  KAY 
                 NADC 
                 Bovine 
                 + 
                 + 
               
               
                   M. avium  18 (Ser. 2) c   
                 NADC 
                 Bovine 
                 + 
                 − 
               
               
                   M. avium  25291 (Ser. 2) b   
                 ATCC 
                 Chicken 
                 + 
                 − 
               
               
                   M. avium  2264 (Ser. 8) 
                 NVSL 
                 Bovine 
                 + 
                 − 
               
               
                   M. avium  subsp.  silvaticum  M21 
                 NADC 
                 Wood 
                 + 
                 − 
               
               
                   
                   
                 Pigeon 
               
               
                   M. avium  23667-2625 
                 NVSL 
                 Bovine 
                 + 
                 − 
               
               
                   M. avium  27183-3013 
                 NVSL 
                 Swine 
                 + 
                 − 
               
               
                   M. bovis  1145 
                 NVSL 
                 Bovine 
                 − 
                 − 
               
               
                   M. bovis  2045 
                 NVSL 
                 Bovine 
                 − 
                 − 
               
               
                   M. bovis  TMC401 35720 
                 ATCC 
                 Bovine 
                 − 
                 − 
               
               
                   M. bovis  BCG Pasteur 35734 
                 ATCC 
                 Bovine 
                 − 
                 − 
               
               
                   M. intracellulare  35772 (Ser. 19) b   
                 ATCC 
                 Human 
                 − 
                 − 
               
               
                   M. intracellulare  35764 (Ser. 20) 
                 ATCC 
                 Human 
                 − 
                 − 
               
               
                   M. tuberculosis  H37Rv 27294 b   
                 ATCC 
                 Human 
                 − 
                 − 
               
               
                   M. tuberculosis  H37Ra 25177 
                 ATCC 
                 Human 
                 − 
                 − 
               
               
                   
               
               
                   a Source of bacterial strains were as follows: ATCC, American Type Culture Collection (Rockville, MD); NADC, National Animal Disease Center (Ames, IA); NVSL, National Veterinary Services Laboratory (Ames, IA).  
               
               
                   b ATCC bacterial type strain.  
               
               
                   c Formerly  M. paratuberculosis  18.  
               
             
          
         
       
     
     REFERENCES 
     1. McNab, W. B, Meek, A. H., Duncan, J. R., Brooks, B. W., Sugden, E. A. (1991a). An evaluation of selected screening tests for bovine paratuberculosis.  Canadian Journal of Veterinary Research  55, 252-259. 
     2. McNab, W. B, Meek, A. H., Martin, S. W., &amp; Duncan, J. R. (1991b). Associations between dairy production indices and lipoarabinomannan enzyme-immunoassay results for paratuberculosis.  Canadian Journal of Veterinary Research  55, 356-361. 
     3. Thorel, M., Krichevsky, M. &amp; Vincent Levy-Frebault V. (1990). Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of  Mycobacterium avium  subsp.  avium  subsp. nov.,  Mycobacterium avium  subsp.  paratuberculosis  subsp. nov., and  Mycobacterium avium  subsp.  silvaticum  subsp. nov. Int.  J. Syst. Bacteriology  40, 254-260. 
     4. Chiodini, R. J., Kruiningen, H. J., &amp; Merkal, R. S. (1984). Ruminant  paratuberculosis  (Johne&#39;s disease): the current status and future prospects.  Cornell Vet.  74, 218-262. 
     5. Chiodini, R. J., Kruiningen, H. J., Thayer, W. R., &amp; Coutu, J. A. (1986). The spheroplastic phase of mycobacteria isolated from patients with Crohn&#39;s disease.  J. Clin. Microbiol.  24, 357-363. 
     6. McFadden, J. J., Butcher, P. D., Chiodini, R., &amp; Hermon-Taylor, J. (1987). Crohn&#39;s disease-isolated mycobacteria are identical to  Mycobacterium paratuberculosis,  as determined by DNA probes that distinguish between mycobacterium species.  J. Clin. Microbiol.  25, 796-801. 
     7. Vary, C. P. H., Anderson, P. R., Green, E., Hermon-Taylor, J., &amp; McFadden, J. J. (1990). Use of highly specific DNA probes and the polymerase chain reaction to detect  Mycobacterium paratuberculosis  in Johne&#39;s disease.  J. Clin. Microbiol.  28, 268-275. 
     8. Mazurek, G. H., Hartman, S., Shang, Y., Brown, B. A., Hector, J. S. R., Murphy, D., &amp; Wallace Jr., R. H. (1993). Large DNA restriction fragment polymorphism in the  Mycobacterium avium -M. intracellulare complex: a potential epidemiological tool.  J. Clin. Microbiol.  31, 390-394. 
     9. Collins, D. M., Erasmuson, S. K., Stephens, D. M., Yates, G. F., &amp; De Lisle, G. W. (1993). DNA fingerprinting of  Mycobacterium bovis  strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110.  J. Clin. Microbiol.  31, 1143-1147. 
     10. Cocito, C., Gilot, P., Coene, M., De Kessel, M., Poupart, P., &amp; Vannuffel, P. (1994).  Paratuberculosis. Clin. Microbiol. Rev.  7, 328-345. 
     11. Inderlied, C. B., Kemper, C. A., &amp; Bermudez, L. E. M. (1993). The  Mycobacterium avium  complex.  Clin. Microbiol. Rev.  6, 266-310. 
     12. McFadden, J. J., Butcher, P. D., Chiodini R., &amp; Hermon-Taylor, J. (1987). Crohn&#39;s disease-isolated mycobacteria are identical to  Mycobacterium paratuberculosis,  as determined by DNA probes that distinguish between mycobacterial species.  J. Clin. Microbiol.  25, 796-801. 
     13. Yoshimura, H. H., &amp; Graham, D. Y. (1988). Nucleic acid hybridization studies of mycobactin-dependent mycobacteria.  J. Clin. Microbiol.  26, 1309-1312. 
     14. Hurley, S. S., Splitter, G. A., &amp; Welch, R. A. (1988). Deoxyribonucleic acid relatedness of  Mycobacterium paratuberculosis  to other members of the family Mycobacteriaceae.  Int. J. Syst. Bacteriol.  38, 143-146. 
     15. Imaeda, T., Browslawski, G., &amp; Imaeda, S. (1988). Genomic relatedness among mycobacterial species by nonisotopic blot hybridization.  Int. J. Syst. Bacteriol.  38, 151-156. 
     16. Green, E. P., Tizard, M. L., Moss, M. T., Thompson, J., Winterbourne, D. J., McFadden, J. J., &amp; Herman-Taylor, J. (1989). Sequence and characteristics of IS900, an insertion element identified in a human Crohn&#39;s disease isolate of  Mycobacterium paratuberculosis. Nucleic Acids Res.  17, 9063-9073. 
     17. Poupart, P., Coene, M., Van Hueverswyn, H. &amp; Cocito, C. (1993). Preparation of a specific RNA probe for detection of  Mycobacterium paratuberculosis  and diagnosis of Johne&#39;s disease.  J. Clin. Microbiol.  31, 1601-1605. 
     18. Kunze, Z. M., Portaels, F., &amp; McFadden, J. J. (1992). Biologically distinct subtypes of  Mycobacterium avium  differ in possession of insertion sequence IS901.  J. Clin. Microbiol.  30, 2366-2372. 
     19. Kunze, M. F., Wall, S., Appelberg, R., Silva, M. T., Portaels, F., &amp; McFadden, J. J. (1991). IS901, a new member of a wide-spread class of atypical insertion sequence, is associated with pathogenesis in  Mycobacterium avium. Mol. Microbiol.  5, 2265-2272. 
     20. Moss, M. T., Malik, Z. P., Tizard, M. L. V., Green, E. P., Sanderson, J. D., &amp; Hermon-Taylor, J. (1992). IS902, an insertion element of the chronic-enteritis-causing  Mycobacterium avium  subsp.  silvaticum. J. Gen. Microbiol.  138, 139-145. 
     21. Van der Griessen, J. W. B., Haring, R. M., Vauclare, E., Eger, A., Haagsma, J., &amp; van der Zeijst, B. A. M. (1992). Evaluation of the abilities of three diagnostic tests based on the polymerase chain reaction to detect  Mycobacterium paratuberculosis  in cattle: application to control program.  J. Clin. Microbiol.  30, 1216-1219. 
     22. Sambrook, J., Fritsch, E. F., &amp; Maniatis, T. (1989). Molecular Cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. 
     23. Whipple, D. L., Le Febvre, R. B., Andrews, R. E., &amp; Thiermann, A. B. (1987). Isolation and analysis of restriction endonuclease digestive patterns of chromosomal DNA from  Mycobacterium paratuberculosis  and other Mycobacterium species.  J. Clin. Microbiol.  25, 1511-1515. 
     24. Bauerfeind, R., Benazzi, S., Weiss, R., Schliesser, T., Willems, H., &amp; Galjer, G. (1996). Molecular characterization of  Mycobacterium paratuberculosis  isolates from sheep, goats, and cattle by hybridization with a DNA probe to insertion element IS900.  J. Clin. Microbial.  34, 1617-1621. 
     25. Wells, J. M., Ellingson, J. L. E., Catt, D. M., Berger, P. J., &amp; Karrer, K. M. (1994). A small family of elements with long repeats is located near sites of developmentally regulated DNA rearrangement in  Tetrahymena thermophila. Mol. Cell. Biol.  14, 5939-5949. 
     26. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., &amp; Lipman, D. J. (1990). Basic local alignment search tool.  J. Mol. Biol.  215, 403-410. 
     27. Davis, E. O., Sedgwick, S. G. &amp; Colston, M. J. (1991). Novel structure of the recA locus of  Mycobacterium tuberculosis  implies processing of the gene product.  J. Bacteriol.  173, 5653-5662. 
     
       
         
           
             16 
           
           
             
               435 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             
               CDS 
                1..432 
             
             1
ATG TCT GAA CCC GGC TAC ACA CCG CCC GAC CTG ATG CTG GTC GGC GAC       48
Met Ser Glu Pro Gly Tyr Thr Pro Pro Asp Leu Met Leu Val Gly Asp
  1               5                  10                  15
GAC CAC GTG CGC GCA TAC CGC GAA ACC GGC GGC GAG ACC GGC TAT CTG       96
Asp His Val Arg Ala Tyr Arg Glu Thr Gly Gly Glu Thr Gly Tyr Leu
             20                  25                  30
TGG AAC GGC GTT CCG ATC TTG CTG CTC ACG GTG ACC GGG CGT CGC ACC      144
Trp Asn Gly Val Pro Ile Leu Leu Leu Thr Val Thr Gly Arg Arg Thr
         35                  40                  45
GGC CGC GCA CTC ACG TCG GCG CTG ATC TTC GGC CGC GAC GGC GAC GAC      192
Gly Arg Ala Leu Thr Ser Ala Leu Ile Phe Gly Arg Asp Gly Asp Asp
     50                  55                  60
TAT CTG GTG GTG GCC TCC ATG GGC GGC GCG CCG CGG CAC CCG TCG TGG      240
Tyr Leu Val Val Ala Ser Met Gly Gly Ala Pro Arg His Pro Ser Trp
 65                  70                  75                  80
TAT CTG AAT CTG CAA GCC AAT CCG GCG GCC GGA ATT CAG GTG CAA GCC      288
Tyr Leu Asn Leu Gln Ala Asn Pro Ala Ala Gly Ile Gln Val Gln Ala
                 85                  90                  95
GAC GAG TTG GCG GTC GTG GCG CGC ACC GCG TCG GCC GCC GAG AAG CCG      336
Asp Glu Leu Ala Val Val Ala Arg Thr Ala Ser Ala Ala Glu Lys Pro
            100                 105                 110
CGG TTT TGG AAG ATC ATG ACT GAC GTG TGG CCC AAC TAC GAC GTC TAC      384
Arg Phe Trp Lys Ile Met Thr Asp Val Trp Pro Asn Tyr Asp Val Tyr
        115                 120                 125
CAG TCA CGA ACC GAC CGC GAC ATT CCC GTC GTT GTA CTC ACA CCG GCA      432
Gln Ser Arg Thr Asp Arg Asp Ile Pro Val Val Val Leu Thr Pro Ala
    130                 135                 140
TGA                                                                  435 
           
           
             
               144 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               not provided 
             
             2
Met Ser Glu Pro Gly Tyr Thr Pro Pro Asp Leu Met Leu Val Gly Asp
  1               5                  10                  15
Asp His Val Arg Ala Tyr Arg Glu Thr Gly Gly Glu Thr Gly Tyr Leu
             20                  25                  30
Trp Asn Gly Val Pro Ile Leu Leu Leu Thr Val Thr Gly Arg Arg Thr
         35                  40                  45
Gly Arg Ala Leu Thr Ser Ala Leu Ile Phe Gly Arg Asp Gly Asp Asp
     50                  55                  60
Tyr Leu Val Val Ala Ser Met Gly Gly Ala Pro Arg His Pro Ser Trp
 65                  70                  75                  80
Tyr Leu Asn Leu Gln Ala Asn Pro Ala Ala Gly Ile Gln Val Gln Ala
                 85                  90                  95
Asp Glu Leu Ala Val Val Ala Arg Thr Ala Ser Ala Ala Glu Lys Pro
            100                 105                 110
Arg Phe Trp Lys Ile Met Thr Asp Val Trp Pro Asn Tyr Asp Val Tyr
        115                 120                 125
Gln Ser Arg Thr Asp Arg Asp Ile Pro Val Val Val Leu Thr Pro Ala
    130                 135                 140 
           
           
             
               33 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             3
GACACCGATT CGCTGCTGGT CAGCCAGCCG GAC                                  33 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             4
CCGTCGTGGT ATCTGAATCT GCAAGCCAAT                                      30 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             5
GCACGGGGCG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             6
GCACGGGGGG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             7
GCACGCGGCG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             8
GCACGAGGCG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             9
GCACGCGGGG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             10
GCACGGGGGG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             11
GCAAGAGGGG ACGTC                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             12
GCAAGGGGGG ACGTC                                                      15 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium 
             
             13
AGAGTTTGAT CCTGGCTCAG                                                 20 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium 
             
             14
ACCAGAAGAC ATGCGTCTTG                                                 20 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             15
GACCGGCTAT CTGTGGAAC                                                  19 
           
           
             
               18 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Mycobacterium avium subspecies
               paratuberculosis 
             
             16
CTCGTCGGCT TGCACCTG                                                   18