Abstract:
The present invention relates to Mycobacterial infections and provides a method of diagnosing infections of  Mycobacterium avium  subsp.  paratuberculosis  (Map), the causative agent of Johne&#39;s disease. In addition, the invention also provides as kits for use in the diagnosis of Map infections and vaccines/immunogenic compositions.

Description:
RELATED APPLICATIONS 
     This application is a 35 U.S.C. §371 national phase entry of PCT Application PCT/GB2009/001364, filed May 29, 2009, and published in English on Dec. 3, 2009, as International Publication No. WO 2009/144478, and which claims the benefit of Great Britain Application No. 0809761.0, filed May 29, 2008, the disclosure of each of which is incorporated herein by reference in its entirety. 
     STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING 
     A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9013-107_ST25.txt, 10,848 bytes in size, generated on Jan. 10, 2013 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures. 
     FIELD OF THE INVENTION 
     The present invention relates generally to Mycobacterial infections and provides a method of diagnosing infections of  Mycobacterium avium  subspecies  paratuberculosis  (Map), the causative agent of Johne&#39;s disease, as well as kits for use in such diagnosis and vaccines. 
     BACKGROUND TO THE INVENTION 
     Johne&#39;s disease, or paratuberculosis, is a fatal chronic granulomatous enteritis of animals caused by Map. The disease is characterised by severe emaciation, loss of body condition and in some species diarrhea. The disease is mainly spread through the ingestion of faeces from an infected animal. Infected animals can also pass on the infection in colostrum or milk and across the placenta to unborn animals. It is generally believed that young animals are more susceptible to infection than adults. Following infection there is a long incubation period of 2 to 4 years during which time the animal may show no signs of clinical disease and may shed Map intermittently. Such animals are often described as “subclinically infected” and act as “carriers” of the disease. The disease is usually introduced to a farm through the purchase of subclinically infected stock. 
     Johne&#39;s disease occurs worldwide and causes considerable economic losses through decreased productivity, increased wastage of adult animals as well as the cost of control, monitoring and diagnosis. There is also some controversy as to whether Map is involved in the development of Crohn&#39;s disease. Contaminated milk would constitute a source of infection and there is a drive towards elimination of Map from the food chain. 
     The diagnosis of Johne&#39;s disease is problematic and there is no single diagnostic test that can detect all stages of the disease. Subclinically infected animals are particularly difficult to diagnose and results of currently available tests may be negative. The most commonly used diagnostic test is the serum Enzyme Linked Immunosorbent Assay (ELISA). This detects circulating antibodies to Map in infected animals. It will detect animals in the later stages of the disease, where clinical symptoms are often present, which are generally those shedding large numbers of Map. It will not, however, detect animals in the early stages of infection, typified as being subclinical where the antibody levels are below the sensitivity threshold of the test. The other most commonly used tests are faecal smears and bacteriological culture. Faecal smears detect the presence of acid-fast bacteria in the faeces and probably only detect a third of the infected animals in a herd/flock. Bacteriological culture is more sensitive and specific but takes 6 weeks or more which is less than desirable. Moreover, low shedders can sometimes be difficult to detect and subclinically infected animals are often missed because they shed intermittently. 
     PCR-based tests are not used routinely as yet but are available for detecting Map in milk, blood or faeces. Also there are particular problems with the application of PCR tests to milk, blood and faecal samples. There is therefore a requirement for a test that can detect early infection and subclinically infected animals. 
     It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages. 
     SUMMARY OF THE INVENTION 
     The present invention is based on the identification of Map antigens which are capable of causing a cell-mediated immune response. 
     In the first aspect there is provided a method of detecting a cell-mediated immune response to one or more purified antigens of Map in an animal, the method comprising the step of: 
     detection of a cell-mediated immune response by measuring the extent of lymphocyte activation and/or proliferation in response to said one or more purified antigens of Map. 
     For example, the cell-mediated immune response may be measured by detecting a level of cytokine such as interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukins (IL-2, IL-6, IL-10, IL-12, IL-17) and granulocyte-macrophage colony-stimulating factor (GM-CSF) using antibodies or indirectly from mRNA by RT-PCR or microarray. Proliferation may be assayed by incorporation of labelled precursors of DNA or protein into the lymphocytes undergoing proliferation. 
     The method may be carried out on a blood sample, or a fraction thereof, isolated from a subject, or may be a skin test, such as a delayed hypersensitivity skin test. 
     Conveniently the blood sample is a whole blood sample obtained from the animal to be tested. Alternatively purified or semi-purified fractions of whole blood, which comprise lymphocytes and monocytes may be employed. Typical amounts of sample required for testing may be 10 μl to 10 ml. Advantageously, a single blood sample would enable testing of an animal&#39;s responsiveness to a wide variety of antigens and therefore diagnosis of other diseases. 
     It will be appreciated that the sample of blood will generally be incubated with said one or more purified Map antigens, for a period of time (e.g. from 1 min to 96 hours but typically from 4 to 72 hours) so as to allow any cell-mediated response (e.g. IFN-γ production), as a result of said one or more purified Map antigens, to develop. IFN-γ is produced from T cells present in the sample of blood which are stimulated to produce IFN-γ by said one or more purified Map antigens. 
     Unlike other similar cell-mediated tests which are known for detecting animals which are infected with, for example,  Mycobacterium bovis  and/or  Mycobacterium tuberculosis  using uncharacterised, what are termed “purified protein derivatives” (PPD) or “protoplasmic antigen” (PPA), the present invention is based upon the use of purified antigens which are expected as being specific. Map-specific antigens are defined here as proteins or fragments thereof that are expressed by Map and elicit an immune response in animals infected by Map which is greater than that elicited by animals infected by other closely related subspecies of  Mycobacterium avium  ( Mycobacterium avium  subspecies  avium  (Maa) and  Mycobacterium avium  subspecies  silvaticum ). In this manner, the present methods may be carried out on animals or herds of animals exposed to other mycobacterial infections including environmental mycobacteria such that a clear distinction can be obtained. Thus, the one or more Map-specific antigens of the present invention will generally be chosen so as to be highly specific for Map and/or to display little or no cross-reactivity with other related species such as other subspecies of  Mycobacterium avium, Mycobacterium bovis  and/or  Mycobacterium tuberculosis.    
     Said one or more purified Map-specific antigens may be identified by way of electrophoretically separating the proteome of a Map isolate and comparing this to a highly related species of  Mycobacterium , such as Maa, in order to identify proteins which are unique to Map and/or differentially expressed. Typically electrophoretic separation may be conducted using a 2-D gel electrophoresis technique well known in the art (see for example Richard Simpson—Proteins and Proteomics: A Laboratory Manual published by Cold Spring Harbor Laboratory Press U.S. 2002) and the proteome of a Map isolate compared to the proteome of the other subspecies. In this manner uniquely and/or differentially expressed proteins can be identified for further study and used as an antigen in accordance with the present invention. 
     In this manner, the present inventors have identified over 30 proteins, which may be of use individually and/or in combination in a method of the present invention. Details of the proteins may be found in attached Table 1. Preferably, the method of the present invention uses two or more Map-specific antigens, such as 3, 4, 5, 6 or more antigens. It is to be understood that a purified antigen may be a whole protein, or immunogenic protein fragment. Such immunogenic protein fragments must be capable of causing a cell-mediated immune response. Once a whole protein has been identified as being capable of eliciting an appropriate cell-mediated immune response, it is straightforward to create protein fragments of said protein and identify whether or not suitable fragments, are also capable of eliciting a cell-mediated immune response. 
     Four particularly preferred Map proteins, of which one or more may be used in the present invention, are identified herein as MAP0268c, MAP1297, MAP1365 and MAP3651c and correspond to proteins having the following NCBI database accession numbers, respectively AAS02585, AAS03614, AAS03682 and AAS06201. The sequences identified in the above accessions are from a Map isolate K-10 (Li et al., 2005) and are shown in  FIGS. 3-6 . It will be understood that minor differences in protein sequence may be identified from one Map isolate to another and as such the proteins identified herein may not be identical in sequence to the corresponding Map K-10 protein identified in the protein database. However, the skilled addressee would be able to easily ascertain if the proteins correspond to one another. In this regard the Map-specific proteins or immunogenic fragments thereof of the present invention will be at least 95%, 98% or 99% identical to a Map K-10 protein or fragment as shown in  FIGS. 3-6 . 
     The present invention extends to a process for the purification of recombinant Map-specific proteins from bacterial or eukaryotic expression systems by the cloning and expression in a host cell such as  Eschericia coli . (see for example Sambrook: Molecular cloning: A laboratory manual (3 rd  Ed) Cold Spring Harbour Laboratory Press. 2001). 
     In a further aspect, the present invention provides use of one or more purified Map-specific proteins or immunogenic fragments thereof in a method of diagnosing Map infection in an animal. 
     Said Map-specific protein or immunogenic fragment thereof is/are preferably one or more of the proteins identified herein, such as those designated MAP0268c, MAP1297, MAP1365 and MAP3651c or immunogenic fragment thereof; or a protein or protein fragment displaying substantial identity with a protein or fragment thereof as shown in  FIGS. 3-6 . 
     There is also provided a kit for carrying out a method of diagnosing a Map infection, the kit comprising one or more purified Map-specific proteins or immunogenic fragments thereof capable of eliciting a cell-mediated immune response. Such a kit may further comprise other reagents for use in the method of diagnosis, such as an anti-IFN-γ antibody. 
     This invention also provides vaccines and immunogenic formulations which may be used to protect animals, particularly cattle, from MAP infection. In one aspect, the present invention provides one or more of the MAP antigens described herein (or fragments, analogues or derivatives thereof) for raising an immune response in an animal. 
     In a yet further aspect, the present invention provides an immunogenic formulation comprising one or more of the MAP antigens described herein (or fragments, analogues or derivatives thereof). 
     In one embodiment, the vaccines and immunogenic formulations described herein comprise combinations of two more of the MAP antigens (or fragments, analogues or derivatives thereof) described herein. 
     One of skill in this field will appreciate that immunogenic formulations of the type suitable for use as a vaccine may be formulated for oral or topical administration. Additionally or alternatively, the immunogenic formulations may be formulated for direct injection, preferably intraperitoneal or intramuscular injection. 
     The present invention also provides a method of vaccinating or immunising an animal against a MAP infection, said method comprising the step of administering one or more of the MAP antigens (or fragments, analogues or derivatives thereof) described herein. 
     In one embodiment, the present invention may extend to vaccines, immunogenic compositions and methods for use in protecting animals against developing diseases caused or contributed to, by Map. Such diseases may include, for example, Johne&#39;s disease. In other embodiments, the vaccines and immunogenic formulations provided by this invention may not protect against Map infection but may reduce the progression of disease, the level of Map colonisation in a host organism and/or the severity of the symptoms of a disease caused or contributed to by Map. 
    
    
     
       DETAILED DESCRIPTION 
       The present invention will now be described by way of example and with reference to the Figures which show: 
         FIG. 1  shows the results of a IFN-γ ELISA showing cell-mediated immune responses to the Map-specific proteins of individual sub-clinical Map infected sheep (P7, P33, W363, W395, PC212, 0269) in comparison to control animals (724A, 329A, 463A, 307A); 
         FIG. 2  shows a plot of the data in  FIG. 1  showing the relative differences between the cell-mediated immune responses to the Map-specific proteins in the infected and control groups with raw means and standard errors; 
         FIG. 3  shows the protein sequence of a Map K-10 isolate protein (AAS02585), corresponding to MAP0268c of the present invention (SEQ ID NO:1); 
         FIG. 4  shows the protein sequence of a Map K-10 isolate protein (AAS03614), corresponding to MAP1297 of the present invention (SEQ ID NO:2); 
         FIG. 5  shows the protein sequence of a Map K-10 isolate protein (AAS03682), corresponding to MAP1365 of the present invention (SEQ ID NO:3); and 
         FIG. 6  shows the protein sequence of a Map K-10 isolate protein (AAS06201), corresponding to MAP3651c of the present invention (SEQ ID NO:4). 
         FIG. 7  shows the enhanced stimulation with cocktails of Map-specific antigens. Blood was stimulated with 113.8 μg/ml of recombinant MAP3651c, 125 μg/ml of 1365 and a mix of 113.8 μg/ml of MAP3651c plus 65 μg/ml of MAP1365. Bovigam IFN-γ ELISA responses to antigens expressed as picogrammes of IFN-γ produced. PC12 and W63 were subclinical ovine paratuberculosis cases resulting from natural Map infection. 
         FIG. 8  shows Bovigam IFN-γ ELISA responses of Map experimentally infected and uninfected calves to antigens (final concentration 4 μg/ml) expressed as optical density (OD). Mean OD calculated for duplicate samples for each group of calves. Note Map-infected calves were inoculated with three different concentrations of Map as given in text. 
         FIG. 9  shows the specificity of the cell-mediated immune response of Map-infected calves compared with Maa-infected calves to recombinant MAP3651c. Blood was stimulated with 7.3 μg/ml of recombinant MAP3651c, 4 μg/ml PPDA and 2 μg/ml PPDJ. Concentrations of the antigens have not been optimised for the diagnostic assay. Bovigam IFN-γ ELISA responses to antigens expressed as optical density (OD). Mean OD calculated for duplicate samples for each group of calves; Maa-infected n=6, Map-infected n=2. 
     
    
    
     EXAMPLES SECTION 
     The methods and Table 1 which follow correspond to information described in a paper submitted by the present inventors to Clinical and Vaccine Immunology (Hughes, V., Bannantine, J. P, Denham, S. et al 2008. Clin. Vaccine Immunol. 15: 1824-1833), the copyright of which is acknowledged.
 
Source and Growth of Microbial Strains
 
     Map strain (JD88/107) was originally isolated from a deer with clinical paratuberculosis. It is mycobactin dependent and IS900 positive. Maa strain (JD88/118) was originally isolated from a deer and is IS901 positive. 
     Routine propagation and maintenance of mycobacteria were carried out as described in Hughes et al. (2007) For propagating mycobacteria for proteome analysis, starter cultures were initiated by inoculating a loopful of mycobacteria (from a 7H11 slope with confluent growth) into a 10 ml volume of Middlebrook 7H9 medium supplemented with 10% (v/v) Middlebrook OADC 0.1% (w/v) Tween-80, 2 μg ml −1  Mycobactin J and 2.5% (v/v) glycerol. The cultures, contained in 50 ml flasks, were stirred continuously using magnetic stirrer bars, on a multiple position low heat transmission magnetic stirrer. After 48 h incubation, 3 ml of the starter culture was transferred to 300 ml of pre-warmed medium in liter flasks. The cultures were incubated at 37° C. with continuous stirring and growth monitored by optical density (OD) at 600 nm as described by Hughes et al. (2007). 
     When cultures were deemed to have reached either exponential or stationary phase and achieved an OD of at least one, they were assessed for contamination by Ziehl Nielsen staining and light microscopy. 
     50 ml aliquots were removed asceptically, cooled on ice and then centrifuged (4000 g, 30 min, 4° C.). The supernatant was decanted and the cell pellet carefully resuspended in 25 ml of ice-cold PBS. The cell suspension was then centrifuged again (4000 g, 30 min, 4° C.), supernatant removed and the pellet stored at −20° C. Cell pellets were stored for no longer than one week before further processing for proteomic analyses. 
     Protein Preparation for 2-D Gel Electrophoresis 
     This was essentially as described previously (Hughes et al 2007). Briefly, cells (0.2-0.3 g wet-weight) were suspended in 2% (w/v) SDS, 0.04 M Tris, 0.06 M DTT. The suspension was layered onto washed 0.1 mm zirconium/silica beads (Biospec Products, Bartlesville, Ok, USA). The mixture was lysed six times using a Fastprep 120 (Qbiogene, Cambridge, UK) at 6.5 beats/sec with 1 min cooling on ice between each round. The resulting suspension was centrifuged (500 g, 30 s, room temperature) to reduce frothing. The supernatant was pipetted into microcentrifuge tubes and heated to 100° C. for 5 min. It was then rapidly cooled and centrifuged (21000 g, 20 min, at room temperature). The protein extract was stored at −70° C. until required. 
     Protein clean-up was performed as described in the protocol accompanying the PlusOne 2-D Clean-Up Kit (Amersham Bioscience, Bucks, UK). 
     The pellet obtained after the clean-up procedure was prepared for isoelectric focussing (IEF) essentially as described by Hughes et al. 2007. Soluble protein extracts were used immediately or stored at −70° C. 
     The concentration of protein extracts was determined using the PlusOne Quant Kit (Amersham Biosciences, Bucks, UK) and was performed essentially as described in the protocol accompanying the product. The results of the assay were routinely confirmed by visual assessment of the protein loaded onto a 1-D SDS polyacrylamide gel. 
     Proteomic Analysis 
     IEF of proteins (150-200 μg for silver stained gels, or 300-500 μg for colloidal coomassie brilliant blue [CBB] stained gels) was performed as described previously (Hughes et al. 2007). Strips were either stored at −70° C. until required or equilibrated prior to electrophoresis in the second dimension. 
     IEF strips were incubated in SDS equilibration buffer (0.05 M Tris, 6 M urea, 0.065 M DTT, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue) for 15 mins with gentle shaking followed by a similar incubation in a fresh aliquot of equilibrium buffer containing iodoacetamide (0.135 M). Polyacrylamide gels (26×20 cm 12.5% (w/v), Amersham Biosciences) were electrophoresed using an Ettan Dalt system (Amersham Biosciences), 1 W per gel, overnight, at 15° C. The power was then increased to 2-4 W per gel until the dye-front was within 1 cm of the bottom of the gel. 
     Gels were stained with either silver using the method described by Morrissey, 1981, with the exception that the glutaraldehyde fix (step 2) was omitted, or colloidal CBB using the method described by Neuhoff et al, 1985. 
     Scanned Gel images were obtained using a Umax PowerLook III Imagescanner coupled with Imagemaster Labscan v 3.01 software (Amersham Bioscience, Bucks, UK). Image analyses and gel comparisons were subsequently performed using Imagemaster Progenesis software (Nonlinear Dynamics. Newcastle-on-Tyne, UK). 
     For each individual spot, the differences in median spot volume between Map and Maa were assessed using a Mann-Whitney test. Because a large number of tests were carried out it was necessary to adjust the P-value from each test to allow for this multiple testing. The False Discovery Rate (FDR) approach of Benjamini and Hochberg, 1995 was used; with an FDR of 5% it would be expected that 5% of the spots identified as differentially expressed would be false positives. 
     Identification of Proteins and Mass Spectroscopy 
     Proteins of interest were excised from gels with either a 15 or 30 mm ‘spot picker’ (The Gel Company, San Francisco, Calif., USA) and cut into small pieces approx 1 to 2 mm in diameter, no excess gel from around the spot was taken. Gel pieces were incubated with 0.1 M ammonium bicarbonate, 50% (v/v) ACN for 15 min at room temperature with at least three changes until the stain had been removed. Finally the gel pieces were dehydrated with ACN 100% (v/v) for 10 min. The ACN was removed and the gel pieces were dried using a speed-vac (Thermo Electron Corporation, MA, USA). The gel pieces were rehydrated in 0.01 M DDT, 0.1 M ammonium bicarbonate and incubated at 56° C. for 1 hr followed by treatment with 0.055 M iodoacetamide, 0.1 M ammonium bicarbonate for 30 mins at room temperature in the dark. Gel pieces were washed with 0.1 M ammonium bicarbonate, ACN 50% (v/v) with at least two changes, dehydrated in ACN 100% (v/v) for 10 mins and then dried on the speed vac for 20 min. Proteolytic digestion was carried out using trypsin solution (10 ng/μl ‘Promega sequencing grade modified’ in 0.025 M ammonium bicarbonate) at 37° C. for at least 16 hr. Protein digest (0.5 μl) was mixed with 0.5 μl of a solution containing 10 mg/ml CHCA, 50% (v/v) ACN, 0.1% (v/v) TFA for analysis by MALDI-TOF on a Voyager-DE Pro mass spectrometer (PerSeptive Biosystems Inc., Framingham, Mass., USA) selecting for a mass range of 600-5000 Da. 
     Silver stained spots were first de-stained using a commercial kit (SilverQuest, Invitrogen, Paisley, UK) prior to the first dehydration step. All subsequent steps were identical to those used for CBB stained spots as described above. 
     Data Explorer was used to create the peak list from the raw data with the smoothing function applied, signal to noise correlation factor was set at 0.7 and the data was baseline corrected with the following parameters: peak width 32, flexibility 0.5 and degree 0.1. The peak height at which centroids were calculated was 50% and peaks were de-isotoped. Resolution for mass spectrometry was greater than 10,000 with a mass accuracy of +/−0.01%. A close-external means of calibrating each spectrum, and no means of exclusion of known contaminant ions (such as keratin) were employed. 
     Bioinformatics 
     The proteome of Map strain K10 (accession numbers NC — 002944, AE016958) contains 4350 predicted open reading frames which were used to compile a protein database and this was queried using Mascot 2.0 (Matrix Science Ltd., London, UK) (Perkins et al., 1999). Searches for trypsin cleavage patterns used a fragment ion mass accuracy of 100 ppm, carbamidomethyl modification was selected and up to one missed cleavage site permitted. 
     Proteins identified using this procedure were characterized using Entrez nr Peptide Sequence data base (National Center For Biotechnology Information [NCBI]) using protein-protein BLAST program. The NCBI Conserved Domain Search service was used to identify domains present in protein query sequences and the Kyoto Encyclopedia of Genes and Genomes was used to identify relevant metabolic pathways. 
     Cloning and Expression of Map-Specific Sequences 
     Maltose binding protein (MBP) fusions of 30 Map predicted coding sequences were produced in  E. coli  by using the pMAL-c2X vector (New England Biolabs, Beverly, Mass., USA). Primers were designed from the reading frame of each coding sequence and contained an XbaI site in the 5′ primer and a HindIII site in the 3′ primer for cloning purposes. Amplifications were performed by using AmpliTaq Gold polymerase (Applied Biosystems, CA, USA) and Map genomic DNA as the template under conditions described previously (Bannantine, et al. 2002), The vector and amplification product were digested with XbaI and HindIII. Ligation of these restricted DNA fragments resulted in an in-frame fusion between the malE gene in the vector and the reading frame of interest. Following ligation, the products were transformed into  E. coli  DH5α and selected on LB agar plates containing 100 μg of ampicillin/ml. (Bannantine et a/l. 2004) 
     Recombinant proteins were prepared essentially as described in the pMAL™ protein fusion and purification system instruction manual (version 5.1, 1/06) with a few minor modifications. 
     Cultures from which recombinant proteins were extracted where grown in medium containing 50 μg/ml Ampicillin. 
     Lysis of  E. coli  was carried out in column buffer containing 2% Tween-80 and protease inhibitor cocktail Set III (Merck cat#539134) using a FastPrep FP120 (speed 5.5 for 20 sec, three times with cooling on ice between each round). 
     MBP fusion protein was prepared from  E. coli  DH5α or TB1 harbouring the parental plasmid pMAL-c2X as described above for Map recombinant proteins and used as a control in all experiments. Purified protein from this control strain consists of an MBP fusion of the LacZ alpha peptide (Bannantine at al. 2004). 
     Preparation and Stimulation of Ovine Peripheral Blood Lymphocytes 20 ml of blood were collected by venipuncture and placed into tubes containing preservative-free heparin. The blood was centrifuged at 638 g (for 15 min at 15° C.). Samples of plasma were removed and stored at −20° C. until analysis by ELISA. Buffy coat was removed and mixed with 10 ml of Hanks/Hep buffer, (Hanks Balanced Salt Solution, w/o Ca 2+  or Mg 2+  with 1% Heparin [Sigma-Aldrich, Poole, England]) and this was then layered over 10 ml of Lymphoprep™ (Axis-Shield, Oslo, Norway) and centrifuged at 1133 g (30 min, 15° C.). The lymphocyte band was aspirated, diluted with 15 ml of Hanks/Hep solution, and centrifuged at 238 g (10 min, 4° C.). The lymphocyte pellet was resuspended in 1 ml lysis buffer (1 part Tris: 9 parts 0.83% Ammonium Chloride, pH 7.2) and incubated on ice for 10 minutes. 9 ml of Hanks/Hep was added and the suspension again centrifuged 238 g (10 min, 4° C.). The cell pellet was resuspended in 1 ml RPMI Complete medium containing RPMI1640 (Gibco, Invitrogen, Paisley, UK), Foetal bovine serum 10% V/V, (Gibco, Invitrogen, Paisley, UK), Glutamine 1 mM (Gibco, Invitrogen, Paisley, UK), 25 mM HEPES (Sigma-Aldrich, Poole, Dorset, UK) 0.08% (V/V) Sodium bicarbonate, 50 μM β-mercaptoethanol (Gibco, Invitrogen, Paisley, UK) and a cocktail of antibiotics (penicillin/streptomycin 100 U/ml (100 μg/ml) gentamycin (100 μg/ml) and the viable cells in this suspension were counted using nigrosin exclusion (20 μl cell suspension mixed with 20 μl 0.1% nigrosin) in a modified Neubauer chamber. The suspension was diluted in RPMI complete to a 2×10 6  cells/ml solution. 50 μl of recombinant protein (110 μg/ml in RPMI complete) and 100 μl Lymphocytes (2×10 X6  in RPMI complete) were added to microtitre wells and incubated in a humidified box at 37° C. in a humidified atmosphere for 96 h. Microtitre plates were centrifuged at 443 g (5 min, 15° C.). 400 ul of supernatant was removed from each well and stored at 4° C. until ELISA analysis.
 
Bovine IFN-γ Assay.
 
     The IFN-γ assay was performed on duplicate supernatants from the lymphocytes as described in the manual from BOVIGAM kit (Prionics A.G., Switzerland). Absorbance readings in ELISA wells were read at 450 nm within 5 min of the reaction being stopped. 
     Stimulation of Whole Blood with Cocktails of Map-Specific Antigens 
     Recombinant antigens were prepared as described previously and diluted in sterile PBS. A cocktail of recombinant Map-specific proteins MAP3651c and MAP1365 was prepared by mixing 113.8 μg/ml of MAP3651c plus 65 μg/ml of MAP1365. The cell-mediated immune responses of sheep were measured using the Bovigam IFN-γ ELISA according to the manufacturer&#39;s instructions with the following modifications. Fifty microliters of antigen preparation in PBS was mixed with 750 microliters of whole blood in a 24 well sterile culture plate. Blood was cultured within one hour of collection. Approximately 400 microliters of supernatant was collected following a 48 hour incubation period. Incubation with the chromogen was carried out for 15 minutes at 37° C. Blood was stimulated with 113.8 μg/ml of recombinant MAP3651c, 125 μg/ml of MAP1365 and the cocktail of MAP3651c and MAP1365.
 
Experimental Infection of Calves
 
Eighteen Holstein male calves were obtained from a herd free from Johne&#39;s disease as determined by ELISA testing and faecal culture. The calves were randomly assigned to three groups of six calves. One group was infected at an average age of 16 weeks (14-17) with an IS901 positive strain of Maa, each calf receiving a single dose of 10 9  organisms. The second group was infected at an average age of 19 weeks (18-21) with Map. Two calves received a single dose of 10 7 , two received 10 8  and two received 10 9  organisms. The third group, the uninfected controls received phosphate buffered saline (PBS). The number of organisms was estimated using the wet weight method and the inocula were given orally as a bacterial suspension in PBS. Blood samples were taken for analyses 54, 57 and 48, 51 weeks post infection for Maa and Map infected calves, respectively.
 
Measurement of Cell-Mediated Responses of Infected Calves
 
The cell-mediated responses of the infected calves were measured using the Bovigam IFN-γ ELISA (Prionics, Schieren, Switzerland) according to the manufacturer&#39;s instructions with the following modifications. The stimulating antigens used were PPDA, PPDJ and recombinant proteins 3651c, 1365, 1297, 0268c at the concentrations given in the figure legends. PPDA was obtained from VLA and dialysed first to remove the phenol. PPDJ was obtained from Douwe Bakker (Central Veterinary Institute of Wageningen University, Lelystad, The Netherlands). Recombinant antigens were prepared as described previously. Twenty five microliters of antigen preparation in PBS was mixed with 200 microliters of whole blood in a 96 well sterile culture plate. Blood was cultured within one hour of collection. Approximately 120 microliters of supernatant was collected following a 24 hour incubation period. For each incubation step in the Bovigam ELISA plates were incubated at 37° C. for one hour. Plates were washed using an automated plate washer six times between steps. Incubation with the chromogen was carried out for ten minutes at 37° C. All antigens were tested in duplicate.
 
     Example 1 
     Proteomic Identification of Map-Specific Proteins 
     Map-specific antigens were identified by comparison of the proteomes of Map and IS901+ Maa, which is the  mycobacterium  most closely related to Map and capable of infecting animals. The rational for this approach was that comparison of the proteomes of the two organisms would identify subspecies-specific proteins, including the products of differential gene regulation that would not be detected by a comparative genomics approach. In order to perform a comparison of IS901 +  Maa and Map, it was necessary first to undertake significant development work to optimise and standardise the experimental and analytical procedures required. The standardized methods are detailed above. Since the proteome changes during the growth cycle, comparisons were made also between the proteomes obtained from the organisms at different stages in the growth cycle (both log and stationary phases were investigated). 
     On comparison of the proteomes, proteins that appeared to be uniquely expressed or upregulated in Map were picked from the gels and subjected to further investigation by MALDI-TOF analyses. It was possible to characterise 32 of these Map proteins and define the corresponding genes following MALDI-TOF and Mascot analyses. The proteomes were compared from different strains of the organisms to ensure that the proteins identified were representative of the subspecies. Comparison with the in vivo proteome of Map confirmed that the proteins were expressed during natural infection of the target species. An example of a Map proteome and a summary of the proteomic data is given in Table 1, which identifies proteins that are specific for Map and potentially suitable antigens for use in the present invention.
 
The genes encoding the Map-specific proteins were amplified by PCR and 30 of the Map-specific proteins were cloned into the pMAL-c2X vector (New England Biolabs Inc.) and expressed as fusion proteins with MBP. Purified recombinant proteins were prepared for use in immunological assays using the procedures described above.
 
     Example 2 
     Recognition of the Map-Specific Proteins by the Cell-Mediated Immune Response of Infected Sheep 
     To determine whether any of the Map-specific proteins would be suitable for use in a cell-mediated immunity assay, it was crucial to determine if they were recognized by the cell-mediated immune response of Map-infected animals. In order to undertake these experiments, it was necessary to identify subclinically infected sheep that were mounting a cell-mediated response to Map. To identify such animals, 35 sheep from a known infected flock were screened using the whole blood Bovigam IFN-γ assay using PPDJ (from Map), PPDA (from Maa) and PPDB (from  Mycobecterium bovis ) as the stimulatory antigens. Concavalin A (ConA) was included as a mitogen control to assess the health status of the cells. Nine animals were identified as having a significant positive response to PPDJ and were selected for further investigation. Control animals were taken from a flock with no history of paratuberculosis and tested in the IFN-γ assay using PPDJ and PPDA for a negative response. Sera from control animals were sent to BioBest Laboratories Ltd. (Pentlands Science Park, Penicuik, Scotland) for testing by ELISA to ensure that the animals did not have circulating antibodies to Map indicative of clinical infection. Faecal smears from the control animals were stained by Ziehl Nielsen staining as a final check to ensure that they were not infected with Map. 
     Blood was taken from the animals and peripheral blood lymphocytes purified following hypotonic lysis and lymphoprep separation (as described above). The IFN-γ assay was performed using 30 recombinant Map-specific proteins, MBP, PPD-J, ConA and medium. OD values were corrected for both medium background and MBP for Map-specific proteins. Taking a cut-off value of a corrected OD &gt;0.1 and a positive result in 2 or more of the subclinically infected sheep, 10 of the 30 recombinant Map-specific proteins appeared to elicit a cell-mediated response. Four of these proteins (MAP3651c, MAP1297, MAP1365 and MAP0268c) were recognised by 50% or more of the animals in the IFN-γ assay and are potential reagents for a cell-mediated immunity test for Map. The experiment was repeated with these four Map-specific proteins using blood from 6 subclinically infected and four control animals. Data from the Bovigam assay carried out in triplicate using these four proteins are given in  FIG. 1  and following statistical analysis in  FIG. 2 . The means of the triplicates were used in subsequent analyses.  FIG. 1  shows the results of the IFN-γ ELISA showing cell-mediated immune responses to the Map-specific proteins of the individual sub-clinical Map infected sheep (P7, P33, W363, W395, PC212, 0269) in comparison to control animals (724A, 329A, 463A, 307A). Box plots were drawn to compare the difference in variability of cell-mediated immune responses to the Map-specific proteins between the groups of infected animals (proteins preceded by I) and the control animals (proteins preceded by C). There was some evidence of a difference in variability between the groups and a clear outlier (observation 20, the value for 0268c for animal W363 in the infected group). The OD value was an order of magnitude higher than the other comparable values and this data point was removed from subsequent analyses. As this was the highest value for an infected animal it would not bias the results in favour of the infected group.  FIG. 2  shows a plot of the data showing the relative differences between the cell-mediated immune responses to the Map-specific proteins in the infected and control groups with raw means and standard errors. Due to variability, the adjusted data (means of triplicates) were analysed using a linear mixed model, with group (control/infected) and protein fitted as categorical fixed effects and animal as a random effect. Ranks of the triplicate means (rather than the raw means) were computed and used in the statistical model. There was evidence of a group×protein interaction (P=0.025) which indicated that there were genuine differences between the control and infected groups for some of the proteins. For all proteins mean levels were higher in the infected group and the differences were markedly different for MAP0268c, MAP1365 and MAP3651c. This supports the raw data plotted in  FIG. 2  and a two sample t-test confirmed MAP0268c and MAP3651c to be significant at the 5% significance level. The P value for MAP1365 is just short of statistical significance (P=0.1)
 
Whilst the present invention is particularly directed to the diagnosis of Map infection in sheep and cattle, the methods of the invention extend to detection of Map infection in any other susceptible animal species including, for example, deer, goats, badger, buffalo, bison, possums, pigs, camels and even man.
 
     Example 3 
     Enhanced Stimulation of Cytokine Production in Ovine Blood with Cocktails of Map-Specific Antigens 
     The sensitivity of immunoassays can often be increased by using a cocktail of antigens. In a small pilot experiment, the cell-mediated immune responses of two sheep subclinically infected with Map were measured in response to single antigen preparations and a cocktail of recombinant Map-specific proteins MAP3651c and MAP1365 as described above. The results are shown in  FIG. 7 . A combination of two Map-specific antigens stimulated an enhanced response in the Bovigam IFN-γ ELISA compared with the single antigen preparations although the magnitude of the response varied between sheep. Different combinations of the Map-specific recombinant antigens will need to be evaluated to determine the optimum mix for diagnostic assays. 
     Example 4 
     Recognition of the Map-Specific Proteins by the Cell-Mediated Response of Experimentally Infected Calves 
     To determine if the Map-specific proteins were recognized by the cell-mediated immune response of another host species a calf model of infection was utilised. Calves were experimentally infected with Map and Maa as described above. Blood was collected, stimulated with preparations of the recombinant antigens and the cell-mediated responses measured using the Bovigam IFN-γ assay as described above. The results showed that the Map infected calves mounted a detectable cell-mediated immune response to all of the antigens compared with the uninfected control calves ( FIG. 8 ).
 
The specificity of the cell-mediated immune response of Map-infected calves compared with Maa-infected calves to recombinant 3651c was evaluated. Blood was analysed from the two calves infected with 10 9  Map and the 6 calves infected with 10 9  Maa 48 and 54 weeks post infection, respectively, using the Bovigam IFN-γ ELISA as described above. Blood was stimulated with recombinant MAP3651c, PPDA and PPDJ. The results are shown in  FIG. 9 . There was a greater cell-mediated response to 3651c in the Map-infected calves than the Maa-infected calves although the magnitude of the response varied between the animals. These results show that MAP3651c can be used to differentiate between Map and Maa-infected animals.
 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                   Mycobacterium avium  subsp.  paratuberculosis  Specific Proteins, 2-D Analysis and Mass Spectrometry Data 
               
             
          
           
               
                   
                   
                   
                 2-D Expression Analysis 
                   
                   
                   
                   
                   
               
             
          
           
               
                   
                   
                 Nom- 
                 Median 
                 Percentage 
                 P Values 
                 Mass Spectrometry Identification 
               
             
          
           
               
                   
                   
                 inal 
                 Spot  
                 Increase in 
                 Mann- 
                 Mascot 
                   
                 Peptide 
                 Masses 
                 Sequence 
               
               
                 Annotation 
                 Protein 
                 Mass 
                 Vol 
                 Expression 
                 Whitney 
                 Score 
                 PI 
                 Count 
                 Searched 
                 Coverage 
               
               
                   
               
             
          
           
               
                 MAP0068 
                 SsB 
                 17.5 
                 0.51 
                 99 
                 0.002 
                 149 
                 5.12 
                 11 
                 28 
                 66 
               
               
                 MAP0139c 
                   
                 23.6 
                 0.05 
                 100 
                 0.002 
                 56 
                 9.95 
                 10 
                 107 
                 54 
               
               
                 MAP0268c 
                   
                 23.8 
                 0.26 
                 100 
                 0.002 
                 106 
                 4.96 
                 7 
                 16 
                 39 
               
               
                 MAP0334 
                   
                 34.5 
                 0.09 
                 100 
                 0.002 
                 147 
                 5.34 
                 10 
                 17 
                 33 
               
               
                 MAP0494* 
                   
                 38.5 
                 0.36 
                 89 
                 0.002 
                 180 
                 5.85 
                 11 
                 16 
                 47 
               
               
                 MAP1012c 
                   
                 37.5 
                 0.07 
                 100 
                 0.003 
                 118 
                 4.6 
                 13 
                 57 
                 48 
               
               
                 MAP1160c 
                   
                 29.4 
                 0.38 
                 58 
                 NS 
                 84 
                 5.44 
                 7 
                 27 
                 33 
               
               
                 MAP1293 
                 HisD 
                 49.4 
                 0.04 
                 89 
                 0.002 
                 94 
                 4.92 
                 12 
                 33 
                 27 
               
               
                 MAP1297 
                 HisA 
                 25.4 
                 0.09 
                 100 
                 0.002 
                 106 
                 4.73 
                 8 
                 29 
                 58 
               
               
                 MAP1365 
                 ArgF 
                 33.6 
                 0.04 
                 100 
                 0.002 
                 71 
                 4.9 
                 6 
                 26 
                 30 
               
               
                 MAP1564c 
                   
                 23.1 
                 0.12 
                 100 
                 NS 
                 110 
                 5.66 
                 8 
                 23 
                 43 
               
               
                 MAP1754c 
                 Usp 
                 30.8 
                 0.03 
                 100 
                 0.002 
                 131 
                 5.72 
                 10 
                 24 
                 38 
               
               
                 MAP2541c 
                 MDH 
                 34.5 
                 0.09 
                 100 
                 0.002 
                 208 
                 4.87 
                 13 
                 18 
                 44 
               
               
                 MAP2685 
                   
                 21.3 
                 0.20 
                 39 
                 NS 
                 130 
                 4.84 
                 7 
                 11 
                 57 
               
               
                 MAP2872c 
                 FabG5_2 
                 26.7 
                 0.37 
                 75 
                 NS 
                 86 
                 5.65 
                 10 
                 71 
                 40 
               
               
                 MAP2878c 
                 DapB 
                 25.6 
                 0.38 
                 58 
                 NS 
                 89 
                 5.52 
                 7 
                 27 
                 39 
               
               
                 MAP3175c 
                 PrfB 
                 41.5 
                 0.07 
                 100 
                 0.003 
                 68 
                 4.73 
                 8 
                 57 
                 28 
               
               
                 MAP3205 
                 NuOE 
                 26.9 
                 0.11 
                 39 
                 NS 
                 66 
                 4.66 
                 6 
                 31 
                 31 
               
               
                 MAP3385 
                   
                 32.3 
                 0.11 
                 39 
                 NS 
                 57 
                 4.62 
                 5 
                 18 
                 24 
               
               
                 MAP3457 
                 MetC 
                 47.6 
                 0.07 
                 68 
                 NS 
                 153 
                 5.25 
                 12 
                 28 
                 46 
               
               
                 MAP3491 
                   
                 28.2 
                 0.05 
                 100 
                 NS 
                 124 
                 5.45 
                 8 
                 17 
                 49 
               
               
                 MAP3540c 
                   
                 25.1 
                 0.03 
                 100 
                 0.002 
                 124 
                 5.18 
                 9 
                 23 
                 38 
               
               
                 MAP3567 
                   
                 30.1 
                 0.40 
                 88 
                 0.002 
                 217 
                 5.7 
                 14 
                 33 
                 66 
               
               
                 MAP3627 
                   
                 23.1 
                 0.18 
                 100 
                 0.002 
                 92 
                 5.4 
                 7 
                 22 
                 34 
               
               
                 MAP3651c 
                 FadE3_2 
                 43.9 
                 0.47 
                 93 
                 0.002 
                 273 
                 6.15 
                 19 
                 29 
                 49 
               
               
                 MAP3692c 
                 FabG4 
                 47.1 
                 0.14 
                 23 
                 NS 
                 102 
                 5.79 
                 8 
                 19 
                 30 
               
               
                 MAP3693* 
                 FadA2 
                 46.7 
                 0.11 
                 73 
                 NS 
                 165 
                 6.21 
                 14 
                 31 
                 42 
               
               
                 MAP3841 
                 GrpE 
                 23.7 
                 0.23 
                 71 
                 NS 
                 151 
                 4.58 
                 11 
                 24 
                 42 
               
               
                 MAP3857 
                 UmpA 
                 18.7 
                 0.06 
                 100 
                 0.002 
                 103 
                 6.06 
                 7 
                 19 
                 49 
               
               
                 MAP3932c 
                 MoA3 
                 41.4 
                 0.04 
                 89 
                 0.002 
                 56 
                 5.01 
                 6 
                 28 
                 24 
               
               
                 MAP4147 
                   
                 42.2 
                 0.07 
                 100 
                 0.003 
                 125 
                 4.73 
                 15 
                 57 
                 49 
               
               
                 MAP 4233 
                 Rpo 
                   
                 0.07 
                 100 
                 0.003 
                 125 
                 4.73 
                 15 
                 57 
                 49 
               
               
                   
               
               
                 *Clones containing the sequence of these proteins were unobtainable after three attempts to amplify or clone the sequence. Median Spot vol is that of the  M. avium  subsp.  paratuberculosis  population. Percentage increase in expression is the difference of the spot volume medians expressed as a proportion of the median spot volume recorded for  M. avium  subsp.  paratuberculosis   
               
             
          
         
       
     
     REFERENCES 
     
         
         Bannantine, J. P., E. Baechler, Q. Zhang, L. Li, and V. Kapur. 2002. Genome scale comparison of  Mycobacterium avium  subsp.  paratuberculosis  with  Mycobacterium avium  subsp.  avium  reveals potential diagnostic sequences. J. Clin. Microbiol. 40: 1303-1310. 
         Bannantine J. P., J. K. Hansen, M. L. Paustian, A. Amonsin, L-L. Li, J. R. Stabel, and V. Kapur. 2004. Expression and Immunogenicity of Proteins Encoded by Sequences Specific to  Mycobacterium avium  subsp.  paratuberculosis . J. Olin, Microbiol. 42: 106-114. 
         Benjamini, Y. and Y. Hochberg. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc., Ser. B. 57: 289-300. 
         Hughes, V., J. P. Bannantine, S. Denham, S. Smith, A. Garcia-Sanchez, J. Sales, M. Paustian, K. McLean, K. Stevenson. 2008. Proteome-determined  Mycobacterium avium  paratuberculosis-specific proteins: Immunogenicity in ovine paratuberculosis. Clin. Vaccine Immunol. 15: 1824-1833. 
         Hughes, V., S. Smith, A. Garcia-Sanchez, Sales, J., and K. Stevenson. 2007. Proteomic comparison of  Mycobacterium avium  subspecies  paratuberculosis  grown in vitro and isolated from clinical cases of ovine paratuberculosis. Microbiol. 153: 196-205. 
         Li, L., J. P. Bannantine, Q. Zhang, A. Amonsin, B. J., May, D. Alt, N. Banerji, S. Kanjilal and V. Kapur. 2005. The complete genome sequence of  M. avium  subsp.  paratuberculosis . Proc. Natl. Acad. Sci. USA. 102 12344-12349. 
         Morrissey, J. H. 1981. Silver stain for proteins in polyacrylamide gels-a modified procedure with enhanced uniform sensitivity. Annal. Biochem. 117: 307-310. 
         Neuhoff, V., R. Stamm, and H. Eibl. 1985. Clear background and highly sensitive protein staining with coomassie blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6:427-448. 
         Perkins, D. N., D. J. Pappin, D. M. Creasy, and J. S. Cottrell. 1999. Probability-based protein identification by searching sequence data bases using mass spectrometry data. Electrophoresis. 20: 3551-3567.