crude extract ( ce ): the map type strain atcc19698 was grown as a surface pellicle at 39 ° c . in mycobactin j - supplemented synthetic sauton medium to stationary phase as described previously ( 1 ). cells were harvested by centrifugation were washed three times . an equivalent volume of 106 μm glass beads was then added and the sample homogenized for 2 minutes in a mini - beadbeater ( biospec product , bartlesville , usa ). after one freeze / thaw cycle and a 2 minute sonication , centrifugation was performed to recover the supernatant . culture filtrates ( cf ): map strain atcc19698 was grown as a surface pellicle on mycobactin j - supplemented sauton medium for 4 weeks at 39 ° c . culture filtrates were separated from bacteria by filtration and proteins were recovered by ammonium sulphate precipitation . protein concentration in culture filtrates and extracts was determined using the bio - rad protein assay kit ( bio - rad laboratories , usa ). the map secretome from cf was analysed by sds - page and by two - dimensional gel electrophoresis ( 2 - de ), followed by the systematic identification of all coomassie blue - stained protein bands / spots by mass spectrometry . for sds - page , cf samples were diluted in laemmli sample buffer and 50 μg of proteins were electrophoretically separated on a 12 % vertical acrylamide gel ( hoefer , amersham bioscience ) at 250 v , 40 ma . for 2 - de , map cf proteins were precipitated a second time using tca , and pellets were solubilized in a minimal volume of sample buffer ( 7 m urea , 2 m thiourea , 4 % ( w / v ) chaps and 50 mm dtt ), and cleared by centrifugating at 18000 × g . for the first dimension , 500 μg of proteins were subjected to isoelectric focusing on immobilized ph gradient ( ipg ) strips ( ph 3 - 10 ; nl ; 11 cm ; amersham pharmacia biotech , sweden ). the first - dimensional isoelectric focusing was carried out as previously described ( 2 ). the second dimension vertical slab sds - page was run about 4 hours at 30 ma / gel using the criterion apparatus ( bio - rad laboratories , usa ) and pre - cast gradient gels ( 10 - 20 %). the sds - page and 2 - de gels were stained with coomassie brilliant blue r - 250 ( amresco , solon , ohio , usa ). protein bands / spots were excised and submitted to trypsinolysis as described previously ( 2 ). for maldi - tof analysis , 1 μl of each sample was mixed with 1 μl of matrix ( 5 mg / ml α - cyano - 4 - hydroxycinnamic acid and 0 . 5 μmol / μl rennin as internal standard , in 25 % ( v / v ) ethanol , 25 % ( v / v ) acetonitrile , 0 . 05 % ( v / v ) tfa ), then spotted onto a maldi sample plate and allowed to air dry . maldi - tof was performed using a maldi ™ mass spectrometer ( micromass , manchester , uk ) equipped with a 337 nm nitrogen laser . the resulting peptide masses were automatically searched for in a local copy of the swiss - prot , trembl databases using the proteinlynx global server and the protein probe ( micromass ltd ., manchester , uk ) and / or mascot ( http & lt ;:& gt ;// www & lt ;.& gt ; matrixscience & lt ;.& gt ; com ) search engines . for electro - spray ionization mass spectrometry ( esi - ms ) and collision - induced dissociation mass spectrometry ( ms / ms ) analysis , peptides were extracted from gel pieces in 25 mm nh 3 hco 3 , 50 % ( v / v ) ch 3 cn , 5 % ( v / v ) formic acid , and dried in a speed vacuum . after reconstitution in 5 % ( v / v ) formic acid , esi - ms and ms / ms were performed with a q - tof 2 mass spectrometer ( micromass , manchester , uk ) equipped with a z - spray nanoflow electrospray ion ( nanoesi ) source and a high - pressure collision cell . amino acid sequences were manually deduced with the assistance of micromass &# 39 ; peptide sequencing program pepseq ( biolynx , micromass ltd ., manchester , uk ). searches for protein identity from sequence data were performed using the blastp algorithm using the swissprot or trembl databases . the search was carried out in all species . positive reference sera used in the elisa and immunoproteomics tests were from 21 naturally infected cows shedding map at the time of sampling , as shown by faecal culture ( see table 7 ). immunoproteomic analysis was performed with two of the 21 positive sera and one 11 - month - old calf infected intravenously with 10 8 cfu of map atcc 19698 as described previously ( 1 ). all three animals tested at post - mortem , were positive in bacterial culture and presented strong seroconversions in m . phlei - adsorbed lam - based ( 9 ), and commercial crude cell extract based , elisas ( pourquier , france ). three sera , originating from animals experimentally infected by m . bovis , were used for the specificity selection . these sera originated from cattle infected intra - tracheally with 106 cfu of a low passage field strain of m . bovis , sampled and confirmed infected at week 13 post - infection by post - mortem and bacterial culture , and presenting at that time strong serological responses in the lam elisa , as described previously . 100 μg of map cf or ce were separated by 2 - de as described above and blotted on nitrocellulose membrane ( hybond ecl ; ap biotech ) after wash in pbs and saturation using bsa , membrane was incubated overnight with primary antiserum preabsorbed on map lipidoarabinomannan . membrane was rinsed before incubation with the secondary antibody ( rabbit anti - bovine , hrp - conjugated ; pierce ). immunoreactive proteins were detected by a chemiluminescence detection kit ( lumi - light western blotting substrate , roche ) following manufacturer &# 39 ; s instructions and identified by mass spectrometry . in this context , three positive sera were used : two came from naturally and one from experimentally map infected animals tested positive in faecal culture test and presenting a high respond in a commercial elisa test ( pourquier , france ). negative sera used for specificity selection came from three animals experimentally infected intratracheally with 10 6 cfu of a low passage field strain of m . bovis ( 3 ). candidate antigens were selected based on two criteria : prospective specificity in blast searches , and antigenicity in immunoproteomic analysis . specific proteins selected in the cf database were blasted against the complete m . bovis and the unfinished m . avium subsp . avium genomes ( tigr server ). antigenic proteins were selected by immunoproteomic approach in map cf and ce . only proteins recognized by at least two of the three map - positive sera and by none of the three sera of m . bovis infected animals , were selected . genes encoding candidate antigenic proteins were amplified by pcr from map genomic dna using primers derived from the map genomic sequence . amplified fragments were purified by agarose gel separation followed by purification using a qiakit pcr kit ( qiagen ). purified amplicons were ligated into a pqe - 80l ( qiagen ) expression vector . culture of e . coli transformants containing the selected construct was grown to an optical density of 0 . 6 . recombinant protein expression was then induced by adding 1 mm iptg in overnight culture at 37 ° c . cells were harvested by centrifuging 15 minutes at 5000 g at 4 ° c . harvested cells were lysed in lysis buffer ( 50 mm tris - hcl , ph 7 . 5 , 500 mm nacl , 8 m urea , 10 mm imidazole ), and lysozyme ( novagen ) was added . samples were sonicated and subsequently incubated for 15 minutes . nucleic acids were digested by addition of 5 μl benzonase ( 25 . 0 u / μl , novagen ). supernatants were finally clarified by ultracentrifugation at 110000 × g at 4 ° c . for 40 min . a his - select column ( 6 . 4 ml , sigma ) was used with a constant flow rate of 3 ml / min and a sample collector ( fc250 , gilson ) programmed to collect 3 - ml fractions . non - binding proteins were removed by washing with 45 ml of lysis buffer . a 10 to 300 mm linear gradient of imidazole in a total volume of 100 ml was used for protein elution . contents of collected fractions were analyzed by sds - page followed by coomassie brilliant blue staining . fractions containing the recombinant protein were pooled and extensively dialysed against 10 mm pbs , ph7 . 2 , containing 0 . 1 m urea . after concentration by ultrafiltration ( ultracel 5 kda , amicon , millipore , usa ) and protein quantification in an assay ( bio - rad protein assay kit , bio - rad laboratories , usa ), samples were stored at − 80 ° c . until use . flat - bottom 96 - well plates ( maxisorp , nunc ) were coated overnight with 50 μl of each recombinant protein diluted to 5 μg / ml in 37 % formaldehyde . after pbst wash ( 100 mm pbs , ph 7 . 2 , 0 . 05 % ( v / v ) tween 20 ) and uncoated sites blocking with 5 % ( w / v ) casein hydrolysate in pbst , 50 μl of primary antibodies ( bovine sera diluted 250 × in pbst , 1 % ( w / v ) casein ) were then added to the plate and incubated for 1 hour at 37 ° c . after wash with pbst , 50 μl of secondary antibody ( hrp - conjugated goat anti - bovine immunoglobulin , sigma , diluted 25000 × in pbst , 1 % ( w / v ) casein ), were added to each well for 1 hour at 37 ° c . after wash with pbst , peroxidase activity was detected by adding 75 μl of tmb ( 3 , 3 ′, 5 , 5 ′- tetramethylbenzidine , liquid substrate system , sigma ). after 10 minutes , reaction was stopped by addition of 35 μl 1n h 2 so 4 , and od was read on a bio - tek plate reader at 450 nm . m . avium subsp . paratuberculosis ( map ) atcc 19698 was purchased from the american tissue culture collection and was grown at 39 ° c . for 4 weeks as a surface pellicle on synthetic sauton medium supplemented with mycobactin j ( 2 μg / ml ) ( synbiotics europe ) as described previously ( 4 ). culture filtrate ( cf - p ) was separated from the bacteria ; cf proteins were precipitated by ammonium sulphate and extensively dialyzed against phosphate - buffered saline ( pbs ). cf - b of m . bovis ( an5 ) was obtained from m . bovis cultures grown as surface pellicle for 2 weeks at 37 ° on synthetic sauton medium . ppd - b from m . bovis vallée was kindly given to us by the late dr . j . nyabenda ( wiv - pasteur institute brussels ). ppd - p was prepared from 6 - 8 week old cultures of m . avium subsp paratuberculosis strain atcc 19698 as described before ( 8 ). preparation of genomic dna from m . avium subsp . paratuberculosis atcc 19698 . genomic dna of m . avium subsp . paratuberculosis atcc 19698 was prepared as described previously by tanghe et al . for m . ulcerans ( 5 ). a six histidine tag was inserted in 3 ′ position of the bglii restriction site of the v1j . ns - tpa vector ( merck research laboratories , pa ., usa ). after this modification , genes encoding 5 map proteins were amplified by pcr and cloned in directional sense bglii 5 ′ and ecori 3 ′. primers used for pcr are disclosed in table 4 . genes were amplified from m . avium subsp . paratuberculosis atcc19698 genomic dna using primers ( proligo ) derived from sequences of map k - 10 . pcr fragments were purified on column (“ qiaquick pcr purification ”, qiagen ), and ligated into a v1 . jns - tpa - his vector predigested with ecori / bglii and dephosphorylated with the shrimp phosphatase ( roche ). after ligation ( t4 dna ligase , fermentas ) and transformation into dh5 - α chemically competent e . coli cells ( invitrogen ), positive clones were selected on lb - kanamycin medium ( 50 μg / ml ) and confirmed by restriction enzyme digestion . v1j . ns - tpa - his vectors encoding these proteins were purified with purelink ™ hipure plasmid dna gigaprep kit ” ( invitrogen ). recombinant map proteins were expressed and purified using pqe - 80l vector . recombinant proteins were expressed as his - tagged proteins in top - 10f ′ e . coli after iptg induction and purified by affinity chromatography on immobilized nickel - chelate ( ni - nta ) column as described before ( 4 ). coding sequences were subcloned by pcr amplification ( expand high fidelity pcr system , roche ) from v1 . jns - tpa - his - map0586c , v1 . jns - tpa - his - map4308c , v1 . jns - tpa - his - map2677c , v1 . jns - tpa - his - map3199 and v1 . jns - tpa - his - map1693c vector using primers derived from sequences of map k - 10 . genes encoding for map0907 and map1168c were isolated by pcr from map genomic dna or by pcr on colony for amplification of map3547c gene as described before ( 6 ). primers sequences are given in table 5 . pcr fragments were purified on agarose gel ( qtakit pcr kit , qiagen ), and ligated into a pqe - 80l ( qiagen ) expression vector predigested with bamhi / hindiii . after ligation ( t4 dna ligase , fermentas ) and transformation into top - 10f ′ chemically competent e . coli cells ( invitrogen ) for expression , positive clones were screened on lb - ampicillin medium ( 100 μg / ml ) and confirmed by restriction enzyme digestion . female balb / c and c57bl / 6 ( b6 ) mice were bred in the animal facilities of the wiv - pasteur institute in brussels , belgium , from breeding couples originally obtained from bantin & amp ; kingman ( united kingdom ). all animals were 6 to 8 weeks old at the start of the experiments . mice were anesthetized with ketamine / xylazine and injected intramuscularly in both quadriceps muscles with 2 × 50 μg of control v1j . ns - tpa ( without histidine tag ) or v1j . ns - tpa - his - antigens . for map4308c , map0586c and map1693c - fl , a combined dna prime - protein boost protocol was compared to an exclusive dna vaccination protocol . for the protein boost immunization , mice were injected at the last time point with 20 μg of purified protein emulsified in incomplete freund adjuvant ( ifa ) in a volume of 100 μl subcutaneously ( s . c .) in the back . recombinant proteins used in protein boost were obtained as described under the subsection “ candidate antigen expression and purification ”. sera from c57bl / 6 and balb / c mice vaccinated with v1j . ns - tpa control vector , v1 . jns - tpa - his - map0586c , v1 . jns - tpa - his - map4308c were collected by tail bleeding three weeks after the last immunization . levels of total immunoglobulin g ( igg ) and igg1 , igg2a and igg2b specific antibodies were determined by an enzyme - linked immunosorbent assay ( elisa ) on individual sera , using purified recombinant antigens ( obtained as described under the subsection “ candidate antigens expression and purification ”) for coating ( 500 ng / well ), peroxidase - labeled rat anti - mouse igg , igg1 , igg2a , igg2b ( experimental immunology unit , universite catholique de louvain , brussels , belgium ) were used as secondary antibody and orthophenyldiamine ( sigma ) for revelation . data are presented as the optical density at 492 nm ( od492 ) for a serum dilution of 1 : 1600 . splenocytes was obtained as described before ( 4 ). indomethacin ( 1 μg / ml ; sigma ) was added to the medium only for infected mice . cells were incubated with purified recombinant antigens ( obtained as described under the subsection “ candidate antigens expression and purification ”) ( 5 μg / ml ) or synthetic peptides ( 10 μg / ml ). the lps eventually contaminating recombinant proteins were eliminated using “ endotrap ” column ( cambrex ) following manufacturer instructions . spleens from individual mice were tested for response against whole protein , and spleens were pooled for peptide testing . culture supernatants were harvested after 24 h for interleukin - 2 ( il - 2 ) assays and after 72 h for ifn - γ assays , when peak values of the respective cytokines can be measured . supernatants were stored frozen at − 20 ° c . until testing . peptides spanning of the entire map0586c sequence were synthesized as 20 - mer peptides overlapping by 10 residues . peptides spanning the entire map4308c sequence were synthesized as 15 - mer peptides overlapping by 9 residues . vaccinated b6 and balb / c mice were rested for weeks after the last immunization before challenge . luminescent map s - 23 or atcc 19698 ( 1 ) was grown in middlebrook 7h9 medium supplemented with oadc , mycobactin j ( allied laboratories inc , synbiotics europe , 2 μg / ml ) and hygromycin ( 100 μg / ml ), to an o . d . between 0 . 6 and 0 . 8 . bacteria were centrifuged for 30 minutes at 2000 rpm , suspended in pbs to a concentration of 8 . 5 × 10 6 rlu / ml ( measured in lb 9507 luminometer ) and mice were infected intravenously in a lateral tail vein with 0 . 2 ml of bacteria . the ratio cfu / rlu for exponentially growing axenic map cultures was determined to be 1 . 2 using lumat lb 9507 luminometer ( berthold technologies ) and the ratio cfu / mrlu 2 . 5 using a turner design 20 / 20 luminometer . the number of bioluminescent bacteria in spleen homogenates was determined using a bioluminescence assay with a lumat lb 9507 luminometer ( berthold technologies ) or a turner design 20 / 20 luminometer and 1 % n - decyl - aldehyde ( sigma ) in ethanol as substrate . in this assay , only live bacteria are enumerated , because emission of light is dependent on the presence of reduced flavin mononucleotide ( fmnh 2 ), co - factor which is only found in living cells . for statistical analysis ( one way anova , tukey &# 39 ; s multiple comparison test ), results obtained in relative light units ( rlu ) were converted to log 10 values . the number of cfu of map in spleen homogenates was determined by plating serial dilutions in pbs on middlebrook 7h11 - oadc agar supplemented with mycobactin j and with / without hygromycin . mice were inoculated intravenously with 0 . 05 mg of m . bovis an5 from a stock kept frozen at − 80 ° c . a first group of five 2 - 3 week old cattle were kept in isolation for 47 weeks . two calves originating from a paratuberculosis free herd were kept as controls . a third calf from the same origin was vaccinated subcutaneously at 2 months of age with 2 × 10 8 cfu of irradiated m . paratuberculosis ( map ) reference strain atcc 19698 in an oil adjuvant ( montanide isa 775 ; seppic ). finally , two calves , born from cows suffering from clinical paratuberculosis ( as confirmed by pourquier serology and by positive fecal and post - mortem organ map cultures ) were kept from the age of 1 week and used as presumably infected animals . a second group of 41 calves originating from a culture confirmed paratuberculosis herd , all aged 2 to 4 months , was blood sampled to assess antigen performance and sensitivity in the field conditions , and tested alongside pourquier serology and fecal culture . a third group of 39 calves originating from a culture confirmed natural bovine tuberculosis outbreak was used to assess antigen specificity in terms of m . bovis / map infection differentiation . blood was collected on heparin by venipuncture and proliferative responses were analyzed using a whole blood assay in 10 % autologous plasma . briefly , heparinized blood was diluted 1 : 10 in rmpi - 1640 medium supplemented with 5 × 10 5 m 2 - mercapto - ethanol . 200 μl of cells were mixed with 25 μl of antigen in 96 - well round - bottom microwell plates , and cultures were incubated in a humidified co 2 incubator for 7 days . avian and bovine purified protein derivative ( ppd ) were tested at a 20 μg / ml , and recombinant map proteins at a 5 μg / ml final concentration . recombinant ag85a ( map0216 ) and ag85b ( map1609c ) from map were included as controls , as described previously ( 4 ). after 6 days , cells were pulsed overnight with tritiated thymidine ( 0 . 4 μci / well ) and collected on a titertek cell harvester . radioactivity recovered on the filters was counted in a betaplate liquid scintillation counter and results expressed as mean cpm ± sd , or mean stimulation index ( si )± sd , of triplicate cultures . tests were performed as previously described ( 3 ), with the following modifications : heparinized blood was collected from the jugular vein and 200 μl aliquots were incubated in duplicate without antigen , with avian and bovine ppd at a 20 μg / ml final concentration , with esat - 6 and cfp - 10 synthetic oligopeptide pools at a 5 μg / ml final concentration each peptide ( 7 ), or with purified recombinant map proteins at a 5 μg / ml final concentration . cells were incubated for 20 hours at 37 ° c . in a humidified 5 % co 2 incubator . after centrifugation , plasma supernatants were collected and stored at − 20 ° c . until testing . bovine ifn - γ was determined using a bovine ifn - γ elisa ( biosource europe s . a ., nivelles , belgium ). heparinized blood was diluted 1 : 8 in rmpi - 1640 medium supplemented with 5 × 10 - 5 m 2 - mercapto - ethanol . 200 μl of cells were mixed with 25 μl of antigen ( concentrations as above ) in 96 - well round - bottom microwell plates , and cultures were incubated in a humidified co2 incubator for 6 days . supernatants were processed as above in the ifn - γ elisa . in the present invention , a postgenomic approach was implemented in order to identify new antigens that could be used for the serological diagnosis of johne &# 39 ; s disease . two different and complementary approaches were used for antigen identification , the first approach being based on sequence comparison with m . bovis and m . avium subsp . avium genome , and the second approach being based on an immunoproteomic approach , using sera from map infected cattle . in the first approach , a database of all proteins found in map cf ( sub - proteome ) has been established ( 125 proteins , not presented ), considering that map cf ( culture filtrate ) was representative of the map secretome . in the context of identifying non cross - reactive antigens , the most important characteristic of the proteins identified is their specificity . all proteins of the database have thus been compared to m . bovis complete and m . avium subsp . avium unfinished genome using blast at the tigr server . from this database , only 15 proteins out of the 125 cf proteins identified were found to be absent from the m . bovis genome . these proteins could be particularly useful to discriminate infections by map and m . bovis , in regions where they co - exist , and could thus represent valuable antigens for the diagnosis of paratuberculosis . table 1 presents the corresponding database of 15 proteins identified according to the first approach . a genomic comparison with publicly accessible , complete or unfinished , mycobacterial genomes indicated that two of these identified proteins were completely specific of map . these two proteins specific of map are : map2746 ( seq . id . no . 16 ) and map3680c ( seq . id . no . 21 ). in a further step , according to the second , immunoproteomic approach , proteins from ce and cf were resolved using both uni - and bidimensional electrophoresis so as to overcome the drawbacks related to the loss of information sometimes associated with 2de due , for example , to solubility problems or the pi range used . these proteins were assessed for antigenicity by western blot using sera of map infected cattle . three different positive reference sera were used in this approach and permitted detection of more than 40 proteins . for this reason , only proteins reacting with at least two of the three positive reference sera were considered antigenic . additionally , antigenic proteins reacting with sera from m . bovis infected cattle were eliminated . table 2 presents the database with these 14 proteins identified following the second approach . by combination of the data obtained in these complementary approaches , a database of 25 potential antigens candidates based on their specificity and / or antigenicity was finally established . tables 3 and 20 present the corresponding database of candidate antigens for paratuberculosis diagnosis and / or vaccination . from this database , three candidate proteins seq . id . no . 4 ( also referenced as map1693c ), seq . id . no . 3 ( map4308c ) and seq . id . no . 1 ( map0586c ), were then selected because they were specific and antigenic . two others candidate proteins , seq . id . no . 5 ( map3199 ) and seq . id . no . 2 ( map2677c ), were chosen because of their specificity . this means that these five candidate proteins could advantageously be used for paratuberculosis serological diagnostic , and thereby johne &# 39 ; s disease control , either alone and / or in partial combinations ( i . e . in combination with at least one of the other four proteins ) and / or in total combination (“ total ” meaning with the other four proteins ). other candidate proteins from the database could also advantageously be used for paratuberculosis diagnosis and / or vaccination , such as for example seq . id . no . 7 ( map3547c ), seq . id . no . 9 ( map0494 ), and / or seq . id . no . 6 ( map0907 ). this means that these three candidate proteins could advantageously be used for paratuberculosis diagnosis and / or vaccination , either alone and / or combined with each other , and / or in combination with at least one of the remaining 24 identified proteins , and particularly advantageously with at least one protein selected from the group consisting of seq . id . no . 4 ( map1693c ), seq . id . no . 3 ( map4308c ), seq . id . no . 1 ( map0586c ), seq . id . no . 5 ( map3199 ) and seq . id . no . 2 ( map2677c ). these three candidate proteins could also advantageously be used for paratuberculosis diagnosis and / or vaccination in combination with at least one , at least two , at least three , at least four , or with all the five candidate proteins mentioned hereabove ( i . e . selected from the group consisting of seq . id . no . 1 to seq . id . no . 5 ). candidate proteins from the database presented in tables 3 and 20 have been successfully cloned in e . coli , expressed , and efficiently purified by use of an imac strategy as illustrated on fig2 and 3 . genes encoding candidate antigenic proteins were amplified by pcr from map genomic dna using primers derived from the map genomic sequence as specified in tables 4 and 5 . high purity has been obtained by eluting the proteins with a linear gradient of competing agent rather than a stepwise elution . it was obviously critical in the present context to obtain high purity antigens . candidate proteins were tested for their applications in diagnosis and / or in vaccination . non limiting examples of such applications are presented hereafter . in fig4 , the antigenicity of candidate antigens is shown . antigenicity of these purified candidate antigens has been measured in elisa . it appears clearly that map1693c , map4308c and map2677c produce a high signal with some sera of map infected cattle . however , none of this three antigens could be used alone to detect all the tested sera . combinations of these three antigens increase the response and will so be used to investigate the sensitivity and specificity of the present assay with a larger panel of sera ( 21 map +, 48 control ). roc analysis of elisa tests ( see fig5 and 6 ) showed the following interesting results concerning part of the proteins from the database as summarized in table 6 . the panel of positive and negative sera used to challenge the elisa test using map1693c , map4308c and map2677c has also been tested with the commercially available pourquier test . results of this test are presented in table 7 . positive reference sera used in the elisa tests were from 21 naturally infected cows shedding map at the time of sampling , as shown by faecal culture . as shown in table 7 , among these positive reference sera , five sera were tested negative in the commercial kit of pourquier ( france ). control sera were from 48 cattle from two m . bovis - infected herds with no history of paratuberculosis . as shown in table 7 , six of these control sera were positive in the map pourquier test . the pourquier test obtained thus a sensitivity of 76 . 2 % for a specificity of 87 . 5 %. these results confirm that the elisa tests according to the present invention enhance the efficiency of the diagnosis comparatively to the commercial kit of pourquier ( france ). it is known that the initial stage of map infection is controlled by a th1 type immune response ( il - 2 , ifn - γ ) and that progression towards disease is accompanied by a loss of this th1 response and the apparition of a th2 type response as well as by the apparition of antibodies . it is also known that ifn - γ is the pivotal cytokine involved in protection against mycobacterial diseases in general . dna vaccines encoding either map4308c ( seq . id . no . 3 ), or map0586c ( seq . id . no . 1 ), or map2977c ( seq . id . no . 2 ), or map3199 ( seq . id . no . 5 ) or map1693c ( seq . id . no . 4 ) were tested for their capacity to induce this th - 1 type immune response . protection could be partially obtained in a mouse model using dna vaccines . the results are particularly interesting using map4308c and map0586c as illustrated hereafter . vaccination with dna encoding map4308c induced a strong antigen - specific cellular ( il - 2 and ifn - γ ) and humoral response in balb / c and c57bl / 6 mice ( fig1 ). t cell epitope mapping , using synthetic overlapping peptides , demonstrated that sequence 187 - 201 ( seq . id . no . 54 ) lvpiiepevtisiad encompassed an immunodominant h - 2 b restricted th1 epitope ( fig1 a ) and sequence 241 - 255 ( seq . id . no . 55 ) pliehpkvmrvvals and 247 - 261 ( seq . id . no . 56 ) kvmrvvalsggysre an immunodominant h - 2 d restricted epitope ( fig7 ). vaccination with seq . id . no . 3 ( map4308c ) dna induced strong total igg , igg1 , igg2a and igg2b antibodies in both mouse strains . levels were higher in serum from protein boosted mice , and levels were higher in balb / c ( fig1 ) than in b6 mice ( fig1 ). igg2a and igg2b antibodies were characteristics of th - 1 type immune response table 9 : bacterial replication in spleen from c57bl / 6 mice vaccinated with dna control , dna - map4308c and dna - map4308c boosted with recombinant map4308c protein in ifa adjuvant and challenged of bioluminescent m . avium subsp . paratuberculosis s23 and analysed 8 weeks postinfection . vaccination with dna encoding map0586c induced a strong , antigen - specific il - 2 and ifn - γ response in balb / c and c57bl / 6 mice as illustrated in fig1 . epitope mapping showed that peptides 291 - 310 llpigydasspipaadyvaa ( seq . id . no . 57 ) and 301 - 320 igydasspipaadyvaahpq ( seq . id . no . 58 ) were very strong h - 2 b restricted il - 2 and ifn - γ epitopes ( fig8 a ) and peptides 121 - 140 elidvydrnvdarrqltalt ( seq . id . no . 59 ) and 171 - 190 sgvgwnylaainfietrfgs ( seq . id . no . 60 ) very strong h - 2 d restricted epitopes ( fig8 b ). vaccination with plasmid dna encoding map 0586c induced only weak total igg , igg1 , igg2a and igg2b antibody levels in both mouse strains ( fig1 ). such vaccinal response could be particularly interesting since it will not impair a subsequent serological detection of jonhe &# 39 ; s disease . protein boosting induced significant antibody levels . dna encoding map0586c protected balb / c mice partially against a challenge with luminescent map atcc16968 * at 8 weeks post infection ( table 11 ). table 11 : bacterial replication in spleen from balb / c mice vaccinated with dna control , dna - map0586c and dna - map0586c boosted with recombinant map0586c protein in ifa adjuvant and challenged with bioluminescent m . avium subsp . paratuberculosis atcc 19698 and analysed 8 weeks postinfection a vaccination with dna encoding map3199 and map2677c induced a significant antigen - specific production of il - 2 and ifn - γ in c57bl / 6 ( table 12 ). significant antigen - specific production of ifn - γ immune responses were obtained after vaccination of balb / c and c57bl / 6 mice with either full - length or the mature construct , both using the classical dna vaccination regimen or the dna prime - protein boost regimen ( table 13 ). with the aim to confirm the specificity of the antigens , immune response against the different purified recombinant proteins was studied in spleen cell cultures from c57bl / 6 mice infected with the intravenous route with either map - s23 or m . bovis ( fig9 and 10 ). as show in fig3 and 4 , none of the proteins induce production of ifn - γ in b6 infected m . bovis , confirming the specificity of these antigens . responses against map0586c ( seq . id . no . 1 ), map3199 ( seq . id . no . 2 ) and map1693fl ( seq . id . no . 4 ) proteins induced strong ifn - gamma responses in map . map2977 ( seq . id . no . 2 ) induced a intermediary and map4308c ( seq . id . no . 3 ) a low ifn - γ responses . these responses confirmed the immunogenicity of this five antigens in infection context in b6 mice . at least three antigens have been shown to be efficient for cellular diagnosis of map . these three antigens are map2677c , map0586c and map4308c . this means that they could be exploited in an ex vivo ifn gamma - based diagnostic assay . at least four antigens have been shown to have an interesting immunizing potential . these four antigens are map1693c , map2677c , map0586c and map4308c . four antigens , seq . id . no . 1 ( map0586c ), seq . id . no . 3 ( map4308c ), seq . id . no . 2 ( map2677c ), and seq . id . no . 5 ( map3199 ) have been shown to be specific as follows . thirty - nine cattle from a culture - confirmed m . bovis outbreak were tested in ex vivo 20h - ifn . they were tested in the bovine tb ifn - gamma assay based on avian and bovine ppds and results analysed using previously validated interpretation criteria ( 3 ). two cattle were classified paratuberculosis reactors based on these criteria and the remaining yielded tb - specific , aspecific , non - interpretable ( high background ), or negative results . all animals were simultaneously tested in the ifn - gamma assay using the mycobacterium tuberculosis complex - specific esat - 6 and cfp - 10 synthetic oligopeptide pools . the m . bovis infected status was confirmed by culture in 26 of the 37 non - paratuberculosis reactor cattle , and esat - 6 / cfp - 10 specific responses were measured in 8 of the remaining animals ( table 14 ). results shown are m . bovis isolation (+: successful ; −: unsuccessful ), od readings in the ifnγ elisa for the tested antigens and a pbs control , the standard ppd - based assay output ( b = tb - positive ; p = paratuberculosis positive ; asp = non - specific mycobacterial sensitisation ; ni = non interpretable ; blank = negative ), and all readings above the elisa cutoff calculated following the supplier &# 39 ; s specifications ( neg = below ; pos = above the cutoff ). the two paratuberculosis reactor cattle remained negative for m . bovis in culture and in the esat - 6 / cfp - 10 based ifnγ assay . seq . id . no . 2 ( map2677c ) remained undetected by any of the 37 non - paratuberculosis reactors . the remaining three antigens were however also detected by one animal out of the 37 non - paratuberculosis reactors , or two animals in the case of map3199 , as shown in table 14 and fig1 . assuming the two paratuberculosis reactors are truely map infected based on their recognition of the four antigens , and that none of the 27 remaining animals would be map infected , yields the single antigen specificities listed in the following table ( column “ calculated specificity ”). assuming all reactors would be false positives would yield the minimal specificities listed in the following table 15 ( column “ minimal specificity ”): a first group of five cattle kept in isolation from the age of 2 weeks , and either immunized with irradiated map ( n = 1 ), naturally infected at birth ( n = 2 ) or kept as controls ( n = 2 ), were tested twice at the age of 18 months , in the ifn - gamma assay . all except the controls , showed consistent reactivity to avian ppd as shown in table 16a . duplicate ex vivo 20 h - ifn readouts at 1 week interval , in 5 calves kept in isolation , including two presumed infected at birth (# 3154 and 3702 ), one immunised with map (# 7 ), and 2 paratuberculosis free calves (# 22 and 24 ). antigens tested are a pbs negative control , avian and bovine ppds , a staphylococcal enteroxin β ( seb ) positive results shown in the left panel are od readings , in the right panel are the readouts applying a stringent cutoff as per the supplier &# 39 ; s specifications ( neg = below ; pos = above the cutoff ), and in the central panel are colour - coded od indices ( odi = test antigen od / pbs control od ) ( grey = odi & lt ; 2 ; bold = odi & gt ; 2 and & lt ; 4 ; shaded bold = odi & gt ; 4 ). antigen - specific responses were weak and only detectable for seq . id . no . 1 ( map0586c ) in the immunized animal applying standard cutoffs ( table16a , right panel ). adjusting the cutoff to less stringent conditions however resulted in three antigens being detected at one sampling point , by one calf born from an infected cow ( table16a , central panel ). this shows the importance of optimising diagnostic cutoffs for optimal diagnostic performance . the same animals were simultaneously tested in the 6 - day proliferation assay to assess memory t cell responses , as shown in table 16b . in vitro proliferative responses in 5 infected , immunised and control cattle , as described in table 16a . tritiated thymidine incorporation in whole blood cultures antigen - stimulated for results expressed as cpm ( left panel ), and stimulation indexes ( si ; right panel ) using a cutoff of si = 10 ( neg = below ; pos = above the cutoff ). ( grey = si & lt ; 10 ; shaded bold = si & gt ; 10 ). the detection of map4308c observed in the ex vivo ifn assay was confirmed in the immunized animal . thirty - six out of a second group of 41 calves originating from a known paratuberculosis herd were screened in a proliferation assay against 3 of the 5 map antigens : map4308c , map0586c and map2677c ( fig1 ). none of these 2 - 4 month old calves appeared positive in serology or by fecal culture . however , eight animals showed significant memory t cell responses against at least one antigen ( si ≧ 18 ). seven calves from this group were selected , based on their avian ppd vs bovine ppd bias and their antigen - specific reactivity , and resampled 3 weeks later for testing against the 5 map antigens in the ex vivo ifn and proliferation assays . an effector t cell response was detected in four calves out of seven against one or more of the map antigens ( table 17 ). results shown are od readings in the ifnγ elisa with a pbs negative control , avian ( ppda ) and bovine ppds , a staphylococcal enteroxin β ( seb ) positive control and the tested antigens , alongside ifnγ detection readout as per the supplier &# 39 ; s specifications ( neg = below ; pos = above the cutoff ). as in the tb group , map1693c remained undetected by all animals , as well as map3199 . map4308c , map0586c and map2677c were detected by 1 , 3 and 4 calves respectively ( fig2 , diamond symbols ). using a si cutoff of 10 , memory t cell responses to map1693c were detected , unlike effector responses , in 2 animals out of 7 ( fig1 & amp ; table 18 ). two animals similarily responded to map4308c , and only one to map0586c and map2677c each . when comparing the proportions of animals responding to single antigens in the effector t cell - based ex vivo ifn assay and in the memory t cell - based proliferation assay , the 5 antigens tested thus rank differently in terms of diagnostic potential ( map2677c & gt ; map0586c & gt ; map4308c ) versus immune memory potential ( map4308c & gt ; map1693c & gt ; map0586c & gt ; map2677c ) as illustrated in table 19 . as a result , it appears from the cellular immune assays carried out in cattle that , whereas map2677c and map0586c show the best cellular diagnostic potential , map1693c and map4308c , with a lower diagnostic potential , present a better immunizing potential , for cattle . of course , the experimental results presented here are not limiting results and other candidate antigens listed in tables 3 and 20 either alone or in combination with at least one , at least two , at least three , at least four , at least five , at least six , at least seven , at least eight , at least nine , or at least ten other candidate antigens listed in these table 3 and 20 could also have an interesting potential both in diagnosis ( serological and / or cellular ) and / or in vaccination ( and namely in dna vaccination ). 1 . rosseels , v ., roupie , v ., zinniel , d ., barletta , r . g ., and huygen , k . ( 2006 ) development of luminescent mycobacterium avium subsp . paratuberculosis for rapid screening of vaccine candidates in mice . infect immun 74 , 3684 - 3686 2 . noel - georis , i ., bernard , a ., falmagne , p ., and wattiez , r . ( 2002 ) database of bronchoalveolar lavage fluid proteins . j chromatogr b analyt technol biomed life sci 771 , 221 - 236 3 . walravens , k ., marche , s ., rosseels , v ., wellemans , v ., boelaert , f ., huygen , k ., and godfroid , j . ( 2002 ) ifn - gamma diagnostic tests in the context of bovine mycobacterial infections in belgium . vet immunol immunopathol 87 , 401 - 406 4 . rosseels , v ., marche , s ., roupie , v ., govaerts , m ., godfroid , j ., walravens , k ., and huygen , k . ( 2006 ) members of the 30 - to 32 - kilodalton mycolyl transferase family ( ag85 ) from culture filtrate of mycobacterium avium subsp . paratuberculosis are immunodominant th1 - type antigens recognized early upon infection in mice and cattle . infect immun 74 , 202 - 212 5 . tanghe , a ., content , j ., van vooren , j . p ., portaels , f ., and huygen , k . ( 2001 ) protective efficacy of a dna vaccine encoding antigen 85a from mycobacterium bovis bcg against buruli ulcer . infect immun 69 , 5403 - 5411 6 . moss , m . t ., e . p . green , m . l . tizard , z . p . malik , and j . hermon - taylor ( 1991 ) specific detection of mycobacterium paratuberculosis by dna hybridisation with a fragment of the insertion element is900 . gut 32 : 395 - 8 7 . vordermeier , h . m ., whelan , a ., cockle , p . j ., farrant , l ., palmer , n ., and hewinson , r . g . ( 2001 ) use of synthetic peptides derived from the antigens esat - 6 and cfp - 10 for differential diagnosis of bovine tuberculosis in cattle . clin diagn lab immunol 8 , 571 - 578 8 . magnusson , m ., and m . w . bentzon ( 1958 ) preparation of purified tuberculin rt 23 . bull world health organ 19 : 829 - 43 . 9 . sugden , e . a ., stilwell , k ., and michaelides , a . ( 1997 ) a comparison of lipoarabinomannan with other antigens used in absorbed enzyme immunoassays for the serological detection of cattle infected with mycobacterium paratuberculosis . j vet diagn invest 9 , 413 - 417 10 . walravens , k ., wellemans , v ., weynants , v ., boelaert , f ., debergeyck , v ., letesson , j . j ., huygen , k ., and godfroid , j . ( 2002 ) analysis of the antigen - specific ifn - gamma producing t - cell subsets in cattle experimentally infected with mycobacterium bovis . vet immunol immunopathol 84 , 29 - 41
the present invention discloses new antigens of mycobacterium avium subsp . paratuberculosis , antigenic compositions comprising at least two of said antigens , as well as epitopes , antibodies or hypervariable fragments thereof and nucleotide sequences coding for them . the present invention also concerns their use in diagnosis and / or vaccination against mycobacterium avium subsp . paratuberculosis , in mammals , and in particular in cattle , but also in sheep and caprines . the invention also concerns their potential application in diagnosis and / or vaccination against crohn &# 39 ; s disease in human .