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1Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, Private Bag 11 222, New Zealand 2Unite de Genetique Mycobacterienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
Correspondence Chris Dupont cdupont{at}sciborg.uwaterloo.ca
Received May 19, 2005
Accepted July 12, 2005
An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75 % identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.
Present address: Department of Biology, University of Waterloo, 200 University Ave. W., Waterloo, Ontario, Canada N2L 3G1.
Abbreviations: IFN-
, interferon-gamma; MAP, Mycobacterium avium subsp. paratuberculosis; PPD, purified protein derivative.
The GenBank/EMBL/DDBJ accession number for the sequence of p22 in Mycobacterium avium subsp. paratuberculosis is AY956313.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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A logical step towards the design of improved Johne's disease vaccines and diagnostic reagents is the identification and characterization of the bacterial components involved in the host immune response. Cell-surface or secreted mycobacterial proteins are attractive candidates for inclusion in diagnostic tests and subunit vaccines because they are major immune targets (reviewed by Andersen, 1997). Experiments using subunit vaccines based on exported proteins such as Ag85A have shown some promising results in animal models for Mycobacterium tuberculosis infection (Huygen et al., 1996; Lozes et al., 1997; Tanghe et al., 2000), and it is conceivable that similar proteins could be identified in MAP for use in a new vaccine against Johne's disease.
The aim of this work was to identify secreted or surface proteins of MAP that elicit immune responses in vaccinated and naturally infected cattle and sheep. We have previously constructed a MAP library of secreted proteins fused to alkaline phosphatase (Dupont & Murray, 2001) using DNA from a New Zealand clinical isolate cloned into the shuttle vector pJEM11 (Lim et al., 1995). The library was expressed in the surrogate hosts Escherichia coli and M. smegmatis in order to identify potential exported proteins of MAP. Several gene regions were identified as possessing potential signal peptides, of which a selection was chosen for further analysis (Dupont & Murray, 2001). One of the resulting M. smegmatis PhoA+ clones (pTB-16) was partially sequenced and found to contain the N-terminal region of a putative lipoprotein. The complete ORF corresponding to this protein (P22) was identified, cloned and expressed as a histidine-tagged protein in M. smegmatis. Recombinant P22 was found to elicit interferon (IFN)- secretion from whole blood collected from sheep vaccinated with a live attenuated strain of MAP. Western blot analysis demonstrated that antibody to P22 was also present in serum from vaccinated sheep and naturally infected cattle.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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DNA manipulation.
Construction of the alkaline phosphatase gene fusion library in the vector pJEM11, transformation of E. coli and M. smegmatis and expression in M. smegmatis mc2155 have been described previously (Dupont & Murray, 2001). Plasmid DNA was isolated from E. coli using commercial kits. Automated DNA sequencing was carried out by Massey University DNA Analysis Service. The primer JEM2 (5'-TCGCCCTGAGCAGCCCGGTT), which binds to the coding sequence of the phoA gene downstream from the BamHI site of pJEM11, was used for partial sequencing of the plasmid pTB-16. PCR amplification of the mycobacterial 16S rRNA gene was done using the primers 246 and 264 (Boddinghaus et al., 1990).
PCR reactions were routinely carried out using Taq DNA polymerase (Invitrogen). For cloning or production of PCR products for sequencing, Platinum Pfx DNA polymerase (Invitrogen) was used including 10 % (v/v) DMSO. Cycling parameters typical for a standard PCR reaction were denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and chain elongation at 72 °C for 30 s, followed by a final elongation step at 72 °C for 10 min. For Platinum Pfx, chain elongation was carried out at 68 °C.
Purified genomic DNA from the mycobacterial isolates was prepared as previously described (Dupont & Murray, 2001).
Cloning of the MAP lprG ORF.
Genomic DNA extracted from MAP ATCC 53950 was used as a template for PCR amplification of the lprG ORF. The forward primer p22-fBam (5'-GATGGGATCCATGCA GACCCGCCGCCGCCT) was designed to the 5' end of the predicted ORF using the sequence obtained from the pJEM11 construct pTB-16. A BamHI site, shown underlined, was incorporated for directional cloning in the mycobacterial expression vector pMIP12 (Le Dantec et al., 2001). The reverse primer p22-rKpn (5'-TGAGGGTACCCGAGCTCACCGG GGGCTTGG) was designed to the 3' end of the ORF using a sequence obtained from the Institute for Genomic Research (TIGR) MAP database (http://www.tigr.org). A KpnI site, shown underlined, was incorporated for directional cloning and the TGA stop codon was omitted to allow read-through to produce the C-terminal hexahistidine tag encoded by pMIP12. The 725 bp product was inserted into pMIP12 to obtain the plasmid pMIP-p22 for constitutive expression of the recombinant protein in M. smegmatis. Insert identity and confirmation of correct insertion for expression were done by sequencing, using the primers BlaF3 (5'-TCGCGGGACTACGGTGCC) and R2 (5'-TCGA ACTCGCCCGATCCC). M. smegmatis cells were transformed using electroporation with pMIP-p22 and confirmed by PCR and sequence analysis of the 900 bp product using the primers R2 and BlaF3.
Databases accessed through the National Centre for Biotechnology Information (NCBI) BLAST server (http://www.ncbi.nlm.nih.gov/blast/) and European Bioinformatics Institute (EBI) Fasta3 server (http://www2.ebi.ac.uk/fasta3/) were used for completed genomes. Preliminary sequence data for Mycobacterium avium subsp. avium and MAP were obtained from TIGR. Analyses of MAP sequences were done with the ExPASy Molecular Biology Server (http://www.expasy.ch/).
Protein preparation.
The MAP vaccine strain 316F was grown in Middlebrook 7H9 broth (Difco), supplemented with 1 mg mycobactin J l–1 (Allied Monitor), 0.2 % (v/v) glycerol, 1 : 1000 (v/v) Middlebrook ADC enrichment (Difco) and 2 g glucose l–1, with vigorous shaking at 37 °C to late exponential phase (OD600 1.7). All mycobacteria cultures were harvested by centrifugation at 3500 g for 20 min. MAP culture supernatants were sterilized by filtration using a 0.22 µm filter and concentrated by ultrafiltration in a stirred cell apparatus (Millipore) using a 3000 molecular mass cut-off Diaflo YM membrane (Amicon). This was followed by further concentration using a 3000 molecular mass cut-off Centriplus 3 filtration device (Amicon). The resulting 200-fold-concentrated preparations were stored at –20 °C. The mean yield from 1 l culture filtrate was approximately 500 mg total protein, the majority of which was bovine serum albumin from the ADC enrichment, with an estimated 5–10 % being actual MAP protein. For preparation of recombinant protein from M. smegmatis, cell pellets were washed three times in PBS and were frozen at –70 °C. To each pellet, 40 ml 20 mM Na2HPO4, 500 mM NaCl and 15 mM imidazole, pH 7.4, was added, and the suspension was sonicated on ice. The sonicated material was centrifuged at 14 000 g for 30 min and the lysate was collected. The pellet was washed three times in PBS and frozen at –70 °C. Protein concentrations were estimated using the Bio-Rad Protein Assay reagent according to the manufacturer's instructions against standard dilutions of bovine serum albumin.
For purification of recombinant protein from M. smegmatis, lysate was used directly for affinity chromatography. Ni2+-affinity chromatography was carried out using 5 ml HiTrap chelating columns (Amersham Pharmacia Biotech) connected in series. Pooled fractions containing recombinant P22 were concentrated and buffer exchanged into PBS by centrifugation using a 10 000 molecular mass cut-off Centricon 10 filtration device (Amicon). Protein concentration was estimated by absorbance at 280 nm. For further purification of recombinant P22, size exclusion chromatography using a Sephacryl S-100 (16/60) gel filtration column (Amersham Pharmacia Biotech) was used. The purified recombinant protein was shown to be free of any significant amount of M. smegmatis protein by SDS-PAGE followed by staining with Sypro Ruby protein gel stain (Molecular Probes).
N-terminal sequencing.
Recombinant P22 protein was electrophoresed in 15 % SDS-PAGE gels and transferred to PVDF membrane (Gelman). Following transfer, the protein was stained with Ponceau S and the 23 kDa band was excised. Approximately 1 pmol of recombinant P22 was used for automated Edman degradative N-terminal sequencing, carried out by Massey University Protein Sequencing Services.
Immunodetection of Western blots.
Protein samples (typically 0.5 µg) were prepared in sample buffer containing ß-mercaptoethanol (Sambrook et al., 1989) prior to electrophoresis in 15 % SDS-PAGE gels. Protein was transferred to PVDF or nitrocellulose membranes (Bio-Rad) and stained with Ponceau S. For immunodetection, membranes were preincubated in blocking solution, [20 mM Tris/Cl (pH 7.4), 100 mM NaCl, 0.1 % (v/v) Tween 20, and 5 % skimmed milk powder] for 1 h at ambient temperature, then incubated with appropriately diluted primary antibody in the same blocking solution for 2 h with shaking or, in the case of serum, overnight at 4 °C. Blots were washed six times for 5 min in wash buffer [20 mM Tris/Cl (pH 7.4), 100 mM NaCl, 0.1 % (v/v) Tween 20] and appropriate secondary peroxidase-conjugated antibody [goat anti-rabbit (A 6154), donkey anti-sheep (A 3415) or rabbit anti-bovine (A 7414) (Sigma)] was added in blocking solution and incubated for a minimum of 1 h. Following washing, blots were developed by chemiluminescence with the substrate SuperSignal West Femto (Pierce) as per the manufacturer's recommendations and exposed to radiographic film (BioMax MR, Kodak). For detection of histidine-tagged P22, anti-hexahistidine peroxidase-conjugated antibody (Roche Molecular Biochemicals) was used at 1 : 500 dilution.
IFN- assay.
IFN- assays were performed using the whole blood Bovigam EIA bovine interferon test, which is suitable for the detection of ovine IFN- (Commonwealth Serum Laboratories). The test was performed as per the manufacturer's instructions, with some modifications. Briefly, blood samples were collected in lithium heparin tubes and processed within 4 h of collection. One millilitre aliquots of blood were dispensed into 24-well tissue culture trays. Routinely, antigens were tested in duplicate. Each antigen was added in a standard volume of 67 µl to the blood aliquots and mixed for 5 min on a rotating platform shaker. The trays were incubated for 22 h at 37 °C in a humidified atmosphere with 5 % CO2. From each well, 50 µl plasma was harvested and assayed singly for IFN- using Bovigam EIA plates. Avian purified protein derivative (PPD; Commonwealth Serum Laboratories) was used at 12.5 µg ml–1 as a control for specific stimulation. PBS was included as a negative control. For all assays, the non-specific T-cell mitogen concavalin A (Sigma) was included for all animals at 20 µg ml–1 to check cell viability. Results were expressed as ‘corrected’ absorbance at 450 nm. For duplicate samples, this was defined as the mean A450 of the stimulated wells (Avian PPD or P22) minus the mean A450 of the PBS control wells. Differences between groups were calculated by the Mann-Whitney test.
Preparation of rabbit antibody raised to P22.
For immunization, P22 antigen was prepared by transferring approximately 0.05 mg Ni2+-affinity-enriched recombinant protein onto a nitrocellulose membrane following SDS-PAGE gel electrophoresis. The membrane was stained with Ponceau S and the P22 band was excised and fragmented in 300 µl PBS. Freund's incomplete adjuvant (500 µl) was added and the mixture was injected subcutaneously in the mid-scapular skin fold of an adult New Zealand White rabbit. This was repeated 3 weeks later. Serum was harvested at 3 weeks following the last immunization and was used for immunodetection at 1 : 1000 dilution followed by anti-rabbit IgG peroxidase-conjugated antibody at 1 : 20 000 dilution.
Sheep and cattle.
Twenty 4-month-old Romney-cross wethers were housed on pasture with water ad libitum for the duration of the study. At 5 months of age, 11 sheep were chosen at random and vaccinated subcutaneously in the right side of the upper neck with Neoparasec (Merial) as per the manufacturer's instructions. The remaining nine sheep were kept as unvaccinated controls. A blood sample from each animal, taken prior to vaccination, was tested using a Bovigam kit to establish immune status to MAP. Blood samples were routinely taken at 4-weekly intervals and subjected to IFN- assays using PPD antigen to monitor immune response after vaccination. Recombinant P22 was included in two assays. Two vaccinated and four unvaccinated animals died of unrelated causes.
In a separate experiment, five sheep of similar age and breed were similarly injected with 40 mg MAP culture filtrate in 1 ml Neoparasec adjuvant.
Serum and faecal samples of 19 dairy cows from four naturally infected dairy herds in the proximity of Palmerston North were used in this study. Blood and faeces were collected twice at 6-monthly intervals and another blood sample was taken 6 months later. A serum ELISA was carried out and interpreted as positive or negative by Gribbles Veterinary Pathology (formerly Alpha Scientific). Faecal samples were cultured by AgResearch and positive cultures were tested by PCR for MAP. In addition, sera from two clinically affected cows from two other local herds were used in Western blot analyses. Animal 27 had acid-fast organisms in the faeces and animal 25 was confirmed at slaughter with gross lesions typical of Johne's disease.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Current information on the genome sequence for MAP can now be obtained from http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=41400296. However, when this study was undertaken the complete genome sequence of MAP was not available; therefore the closely related M. avium subsp. avium (TIGR) was searched using the 69 bp 5' end of the ORF. A 100 % identity was obtained, and the M. avium subsp. avium sequence was examined for the first in-frame stop codon, thus defining the 3' end of the ORF. This M. avium subsp. avium ORF sequence was then used to search the incomplete MAP database (TIGR). The match was 99 %, with a single base change that did not alter the translated sequence (GenBank accession no. AY956313).
To define the start codon of the ORF, the translated MAP sequence was used in protein database searches. Comparison with the N-termini of the resulting aligned sequences (Fig. 1) allowed prediction of the N-terminal methionine, encoded at 60 bp in the pTB-16 sequence. A possible ribosome-binding site represented by a G-rich motif was found just before this ATG codon at 60 bp. This is in agreement with the predicted ribosome-binding site for the similar M. bovis lprG (Bigi et al., 1997). The ORF encoded a precursor protein of 235 amino acids and a calculated molecular mass of 24.4 kDa. The G+C content of the ORF was 66.4 %, which is consistent with the published G+C content for MAP (Imaeda et al., 1988).
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The translated DNA sequence was used in database searches to find the extent of similarity to other proteins. The identity to M. avium subsp. avium (TIGR) was 100 %. The next two highest scoring alignments are presented in Fig. 1. These proteins belong to the mycobacterial LppX/LprAFG family of lipoproteins. All have putative signal sequences and potential lipid attachment sites, based on the motif around the cleavage sites in their mature N-termini (von Heijne, 1989). The predicted protein sequence was examined for similar features and was found to possess a potential signal sequence. At positions 21–25 of the precursor protein was the sequence IAGCS (see Fig. 1), which is similar to the consensus sequence for lipidation and cleavage by signal peptidase II based on predicted and known lipoproteins of Gram-negative and Gram-positive bacteria (Braun & Wu, 1994; Sutcliffe & Russell, 1995). The N-terminal sequencing result of the recombinant protein, LIAGCS at positions 20–25, showed that this cleavage site was not used in M. smegmatis. The cleavage site used, ATA-LIA at positions 17–22, appeared to be typical of cleavage sites used by signal peptidase I enzymes, which cleave non-lipoprotein precursors. It contained the conserved sequence AXA at positions 17–19, immediately preceding the cleavage site (von Heijne, 1985).
Expression and purification of recombinant protein
The ORF was cloned into the shuttle vector pMIP12 and expressed in M. smegmatis. The mean yield of eluted recombinant P22 was approximately 1 µg (ml cell lysate)–1. Western blot analysis using a monoclonal anti-hexahistidine peroxidase-conjugated antibody showed that the recombinant protein was present in both the soluble and insoluble fractions of cell sonicates (Fig. 2). The protein was further isolated from the soluble fraction by Ni2+-affinity chromatography followed by elution with imidazole. The theoretical size of the cleaved mature recombinant product as expressed from pMIP12 was 23.6 kDa and a band of approximately this size was seen following SDS-PAGE analysis (Fig. 2). The native mature protein of 216 amino acids was predicted to be 22.3 kDa and was therefore designated P22.
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Cellular localization of recombinant and native P22
Western blot analysis using anti-hexahistidine antibody (Fig. 2) showed that recombinant P22 was detected in culture filtrates of M. smegmatis containing pMIP-p22, and not in M. smegmatis containing vector only. The apparent molecular mass of 23 kDa was consistent with the size determined from SDS-PAGE using M. smegmatis sonicates.
Polyclonal rabbit antisera raised against recombinant mature P22 was used to probe Western blots of equivalent amounts of MAP strain 316F culture filtrate and soluble and insoluble cell fractions produced by sonication. A single band of apparent molecular mass 24.2 kDa was detected in all fractions (data not shown). This distribution was consistent with other studies of the subcellular location of weakly associated envelope lipoproteins of mycobacteria (Andersen et al., 1991; Cameron et al., 1994; De Kesel et al., 1993; Florio et al., 1997; Orme et al., 1993; Wiker et al., 1991), including the similar 22 kDa antigen of M. bovis BCG (Lefevre et al., 2000). The proportionately larger amount of P22 present in the sonicated cell fractions compared to culture filtrates from both recombinant M. smegmatis and MAP suggested that the protein was not actively secreted to the environment in vitro. Previous assays in this laboratory for the activity of isocitrate dehydrogenase, an indicator of cell lysis, have shown lysis of cells in MAP cultures grown through stationary phase to be minimal. As the cultures used in this study were mid-exponential phase, release of proteins due to lysis was unlikely and the presence of P22 in the culture filtrate was probably due to release from the envelope during growth.
Mature, native P22 from MAP consistently migrated on SDS-PAGE at a larger apparent molecular mass than was calculated from its amino acid composition using either of the potential signal sequence cleavage sites. Although further composition analysis was not performed with the native protein, differential migration on SDS-PAGE gels is commonly employed to assay for the presence or absence of lipid modification of proteins (Sankaran et al., 1995). The apparent size difference may therefore reflect post-translational modification of P22 in MAP. Reduced mobility in SDS-PAGE gels has been reported for MK35, a predicted lipoprotein from Mycobacterium kansasii (Armoa et al., 1995), and the closely related 27 kDa antigen of M. bovis (Bigi et al., 1997). The latter was found to be post-translationally modified by acylation in recombinant E. coli (Hovav et al., 2003). Although the presence of the lipobox consensus remains the hallmark for prediction of bacterial lipoproteins (Sankaran & Wu, 1994), more evidence would be required to confirm lipid modification of P22 in MAP.
Species distribution of lprG
In addition to the alignments shown in Fig. 1, PCR using primers p22-fBam and p22-rKpn was undertaken for a limited number of mycobacterial species (Table 1), including several MAP isolates representing five IS900 RFLP types (Collins et al., 1990) and 11 strains from the M. tuberculosis complex, including Mycobacterium pinnipedii. Amplification of the genus-specific 16S rRNA gene was carried out in parallel for all DNA species and served as a positive control for the reactions. PCR products of the expected size for lprG (725 bp) were amplified from all 13 isolates of MAP. Given the varied geographical and host sources of these isolates, this suggested that lprG is highly conserved in MAP strains. PCR products of a similar size were also detected from Mycobacterium scrofulaceum and Mycobacterium intracellulare, which are members of the so-called ‘MAIS’ complex (Wayne, 1984), a group of closely related mycobacteria including MAP, M. avium subsp. avium and Mycobacterium avium subsp. silvaticum. PCR products were not detected from any of the other mycobacterial species tested (data not shown).
Genes similar to the related lprG of the M. tuberculosis complex have been previously reported to possibly exist in Mycobacterium vaccae, MAP and M. avium subsp. avium, based on PCR analysis using primers designed to the lprG gene of M. bovis (Bigi et al., 1997). The present study has confirmed that these genes share significant identity with lprG of MAP and M. avium subsp. avium. The role of P22 is unknown at this stage; however, recent work by Bigi et al. (2004) has demonstrated that knockout of the lprG-Rv1410 operon produces attenuation of M. tuberculosis. In addition, Gehring et al. (2004) have shown that LprG (Rv1411c) is a TLR-2 ligand that inhibits human macrophage class II MHC antigen processing. It will be interesting to investigate the biological activity of P22 and its relatives in the MAIS complex.
Immune responses to P22
Evaluation of the capacity of P22 to induce humoral and cellular responses in relevant host species was undertaken. Serum samples from Neoparasec-vaccinated sheep were used in individual immunoblot assays to determine their humoral response to P22. Strong bands corresponding to P22 were consistently produced by 10 of the 11 Neoparasec-vaccinated sheep in Western blot analyses (Fig. 3). Very faint bands were produced by sera from three of the sheep (507, 578 and 560) prior to vaccination and also in two unvaccinated animals (599 and 569). As animals were kept on open pasture, it is possible that this is from exposure to environmental mycobacterial species such as M. avium subsp. avium and M. intracellulare. Control sera from the remaining animals prior to vaccination and from unvaccinated control animals did not react with the protein.
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In a separate experiment, serum samples from sheep immunized with MAP strain 316F culture filtrate were used to probe individual Western blots of recombinant P22. The culture filtrate-immunized animals did not have antibody to P22 prior to vaccination but at 1 month post-immunization all five animals had antibodies to P22 (Fig. 4), further suggesting that native P22 is present in culture filtrate and is immunogenic in sheep.
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To investigate whether antibody to P22 was present in naturally infected cattle, Western blots were performed with sera from animals belonging to herds confirmed to have Johne's disease. A summary of the results for serum ELISA, faecal culture and P22 Western blot analysis is shown in Table 2. Antibody to P22 was present in sera from 11 of 13 (sensitivity approximately 85 %, 95 % confidence interval 54–97 %) subclinically infected cattle that were positive on at least one faecal culture. The four cows that were positive on all ELISA and faecal culture tests carried out (24, 144, 49 and 2) were also positive in the Western blot. Antibody to P22 was also present in both clinically affected cows (27 and 25). Two subclinically infected cows (181 and 68) did not have detectable antibody to P22 on Western blot analyses and were also negative on the last ELISA and the last faecal culture. Of interest was the observation that four of the subclinical cows (327, 517, 58 and 115) that were negative on all three ELISA tests had detectable antibody to P22.
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Of the six cows that were negative on all ELISA tests and faecal cultures, one (53) produced a positive signal to P22 on the Western blot (specificity approximately 80 %, 95 % confidence interval 36–99 %). Based on the reported ELISA sensitivity and specificity of 20 % and 99 %, respectively (Sockett et al., 1992a), and the faecal culture sensitivity and specificity of 45 % and 100 %, respectively (Sockett et al., 1992b), the probability that these six cows were truly negative was estimated as 85 % (1–[{1–sensELISA}3 x {1- sensfaecal culture}2]) using a parallel test interpretation for the MAP infection status and assuming conditional independence of the two tests (Dohoo et al., 2003).
This preliminary study suggests that P22 might be a candidate for future investigation as a serodiagnostic reagent for the detection of MAP infection. Serological tests have the advantage of simplicity and produce rapid results. Although few defined proteins from MAP have been investigated for this purpose, some studies indicate that specific proteins are useful to diagnose MAP infection and may allow increased sensitivities compared to commercial ELISAs (De Kesel et al., 1993; El-Zaatari et al., 1997). The presence of genes similar to lprG in other mycobacterial species may limit the specificity of the P22 protein as a diagnostic tool. Screening of a larger number of MAP-infected and uninfected animals under different environmental conditions will be needed to assess the specificity and sensitivity of P22 under field conditions.
Development of accurate tests for the detection of MAP at all stages of infection would greatly assist in eradication programmes. Given the present knowledge that the early immune response to MAP infection appears to be cell-mediated, it may be advantageous to use a reagent capable of eliciting specific IFN- production. Results of IFN- assays are shown in Fig. 5. There was a significant difference (P < 0.01) between Neoparasec-vaccinated (n = 9) and unvaccinated (n = 4) groups in the IFN- responses to P22 at the three concentrations of P22 used. Blood from one of the control animals did not respond to the concavalin A positive control (not shown) and was removed from calculations. Eight of the nine vaccinated animals showed IFN- production to all three concentrations of P22, often in a concentration-dependent manner. Similar results were obtained using P22 purified by gel filtration (data not shown). Animal 136 had very low IFN- production to P22 and to Avian PPD compared to the other vaccinated animals. None of the unvaccinated animals had high IFN- responses to P22; however, three of these animals (128, 133 and 569) had comparatively larger responses to Avian PPD, especially animal 569. This may suggest an overall higher specificity for P22 antigen compared to Avian PPD.
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Notable responses of the control animals to Avian and Johnin PPD had been previously observed on several occasions during this study. Since infection of domestic hoofstock with environmental mycobacteria is considered to be rare (Thorel et al., 2001), responses may have been due to transient sensitization as the animals were kept on open pasture. Others have reported such responses to PPD antigens in uninfected cattle (de Lisle & Duncan, 1981; Hintz, 1981), and these are often attributed to cross-reactions with other environmental organisms (Buergelt et al., 1977). This is an illustration of the poor diagnostic ability reported for these crude antigens in cellular tests under field conditions for individual animals (Harris & Barletta, 2001; Hope et al., 2000).
Only a small number of individual proteins of MAP have been isolated and characterized, and few of these have been subjected to immunological studies. In this study, a novel MAP gene encoding an exported protein of approximately 22 kDa was partially characterized and investigated for its immunological activity. The results described here show that P22 is a highly immunogenic protein of MAP, producing both antibody and cell-mediated immune responses in cattle and sheep. Thus, P22 may prove to be useful as a defined antigen for improved immunodiagnostic tests for MAP infection. In addition, its ability to induce cell-mediated immune responses suggests that it may also be a candidate for inclusion in a subunit vaccine against Johne's disease.
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