Recombinant 20.8-kDa protein of Mycobacterium avium subsp. paratuberculosis-based sero-diagnosis of paratuberculosis

Johne's disease or paratuberculosis is a chronic infectious enteric disease of ruminants caused by the intracellular pathogen. The control of the Johne's disease is hampered by lack of specific diagnostic tests. In this study, we have cloned and expressed the N-terminal region of the locus tag Map 1637c encoding 20.8-kDa (r20.8) protein of Mycobacterium avium subsp. paratuberculosis. The recombinant protein r20.8 was expressed in high levels in Escherichia coli. The protein r20.8 was purified by single-step chromatography using Ni-NTA agarose. The protein r20.8 was reacted with anti-r20.8 antibodies as well as cattle sera infected with Map on Western blot. ELISA using well-characterized sera (both positive and negative; n = 60 each) Map-infected and non-infected cattle, respectively, yielded a sensitivity of 73.3% and a specificity of 98.3%. The 20.8 kDa protein expressed in the present study will prove useful as reagent in diagnostic test.


Introduction
Mycobacterium avium subspecies paratuberculosis (Map) is an intracellular pathogen causing paratuberculosis (Johne's disease), a chronic enteric disease of domestic and wild ruminants including primates (Chiodini et al. 1984). Johne's disease is widespread throughout the world and causes substantial economic losses to the farmers and the dairy industry (Hasonova and Pavlik 2006). Prevalence of bovine paratuberculosis in European countries was reported as 3.5-71.0% (Boelaert et al. 2000). Singh et al. (2005) reported sero-prevalence of 29.05 and 23.63% paratuberculosis in bovines and goats, respectively, in northern India.
To date, effective therapeutics or vaccine candidates are not available that can completely eliminate viable Map from the host. Early detection along with good management practices is the only way to control paratuberculosis (Harris and Barletta 2001).
Control of Map at the herd level requires identification of infected animals, at early subclinical level, and their removal from the herd (Van Roermund et al. 2007). A number of methodologies have been employed, including detection of Map by PCR in faeces and milk, bacterial culture of faecal samples, measurement of delayed-type hypersensitivity response, gamma interferon (IFN-c) assay and serodiagnosis by ELISA. However, variables such as antigen specificity and sensitivity, reproducibility, PCR inhibitors in faeces, and cross-reactivity among the antigens limit the efficacy of these methods (Wadhwa et al. 2012).
Previously, serological tests based on commercial ELISA kits for paratuberculosis infection and various studies have been compared for the accuracy (Fry et al. 2008;van Schaik et al. 2007). ELISA studies with different antigens have shown discrepancies in the ability of these tests to identify all infected animals (Costanzo et al. 2012).
It is universally accepted that major improvements are needed in currently available serodiagnostic tests for efficient control programs (Collins et al. 2005;Sweeney et al. 2006). Various laboratories have, therefore, chosen to improve the efficiency of serological assays by replacing the ''crude antigens'' by ''Map-specific antigens'' (Dupont et al. 2005;Willemsen et al. 2006). Bannantine et al. (2008) generated a partial protein array representing the Map coding sequences and showed that some Map-specific proteins were strongly detected in clinical sera early post infection in experimental model. To date, a number of Map-specific immune targets have been identified and the antigenicity of some of these proteins has been evaluated Facciuolo et al. 2013).
The protein tag Map 1637c encodes for immunodominant antigen of 53.2 kDa and contain epitopes specific for Map. The 40.8-kDa antigen from the tag Map 1637c was used for the detection of Map antibodies in sera from 18 Map-infected cattle with the sensitivity and specificity of 82 and 98%, respectively (Leroy et al. 2009). In the present study, we describe a cloning strategy for the N-terminus region of Map 1637c encoding 20.8-kDa protein, its purification and a preliminary evaluation using ELISA for its use as a diagnostic reagent in the detection of bovine paratuberculosis.

Bacterial strains and plasmid
Mycobacterium avium subsp. paratuberculosis (Map) strain 316F was obtained from Central Diengenees Kundig Tieh Institute, Lelystad, The Netherlands, and maintained at Gene Expression Laboratory, Veterinary Biotechnology Division, IVRI, Izatnagar, India. The organism was grown and maintained at 37°C on Middlebrook 7H10 agar (Difco laboratories, Detroit, USA) enriched with 0.1% glycerol (v/ v) and 10% oleic acid-dextrose-catalase (Difco laboratories and supplemented with of 20 mg/L mycobactin J (Allied Monitor, Fayette, USA) The. Escherichia coli strain (M15, pREP4) purchased from Qiagen (Valencia, USA) was maintained on Luria-Bertani agar containing kanamycin (25 lg/mL), as the strain carries kanamycinresistant plasmid. The plasmid pQE30 containing an ampicillin resistance marker was used as expression vector supplied by Qiagen.

DNA isolation, polymerase chain reaction and gene cloning
The genomic DNA from M. a. paratuberculosis was isolated from the grown culture by the method of Del Portillo et al. (1991). Plasmid DNA extraction from E. coli was carried out by the alkaline lysis method (Sambrook and Russel 2001).To amplify a gene fragment encoding 20.8-kDa protein of Map, a set of oligonucleotide primers 5 0 -TACGGATCC ATGCAACGGCAACGCGGC-3 0 and 5 0 -GTACTGCAGTCACCGATCATTTTGGGC-3 0 containing BamHI/PstI restriction endonuclease sites, respectively, were designed on the basis of sequence information of Map strain k10, complete genome GenBank Accession No. AE016958 tag Map 1637c. The primers were used to amplify about 546-bp PCR product encoding nearly 182 amino acids of the N? terminus of the whole gene encoding 53.2 kDa protein. For PCR amplification, 1 lL (100 ng) DNA of Map was added to 5 lL of Taq DNA polymerase buffer 10 mM/L Tris. HCl (pH 9.0), 50 mM/L KCl, 1.5 mM/L MgCl 2 and 0.01% (w/v) gelatin; 200 lM of each dNTP; 0.5 lM each primers; 2 units of Taq DNA polymerase (New England Biolabs Inc.). The final volume was made up to 50 lL with sterile distilled water. After the initial denaturation step (5 min at (94°C), 30 cycles of amplification were performed as follows: denaturation at 94°C for 1 min, annealing for 1 min at 58°C, and extension at 72°C for 1 min followed by final extension at 72°C for 3 min. The reaction was carried out in a PTC-100 reactor (MJ Research Inc., Waltham, USA). The PCR product was resolved in 1% agarose gel and stained with ethidium bromide. The purified PCR product was cut with BamHI/PstI and inserted in frame into the plasmid pQE30 digested with the same enzymes. The resulting plasmid pQ20.8 was transformed and recombinant clones were selected on LB agar containing ampicillin (75 lg/mL) and kanamycin (25 lg/mL) and subjected to direct colony PCR screening by insert-specific primers mentioned above.
Expression and purification of the recombinant protein and raising of antisera Escherichia coli M 15 cells harbouring the plasmid pQ20.8 were grown in LB medium containing ampicillin (75 lg/ mL) and kanamycin (25 lg/mL), and induced with 1.0 mM IPTG for 4-6 h. The purification of recombinant 20.8-kDa His fusion protein under denaturing conditions was carried out by one-step Ni-NTA (nickel-nitrilotriacetate) agarose (Qiagen) affinity chromatography and renatured as described previously (Basagoudanavar et al. 2004). The protein was analysed by SDS-PAGE. The protein solution was sterilized by filtration, and aliquots were stored at -70°C, until used. Concentration of the protein was determined spectrophotometrically (Lowry et al. 1951). The hyperimmune sera against the recombinant 20.8-kDa protein were raised in rabbits as described previously (Basagoudanavar et al. 2004). All the sera used in this study were preabsorbed with E. coli antigens.

Characterization of recombinant protein
The purified recombinant 20.8-kDa protein was separated on 12% (w/v) gel along with appropriate coloured protein marker range from 7 to 175 kDa (New England Biolab, USA).The resolved proteins from SDS-PAGE were transferred to nitrocellulose membranes (0.45 lM) using semidry electroblotting (Atto, Tokyo, Japan) at 0.8 mA/cm 2 . The blots were blocked with 5% skimmed milk powder in PBS-T buffer (PBS containing 0.1% Tween-20) for 2 h at room temperature. After washing thrice with PBS-T buffer, the membranes were incubated at 37°C for 2 h with antisera against 20.8-kDa protein (1:10,000 in PBS) raised in rabbit. Following further washing, the blots were incubated with a 1:10,000 dilution of HRP-labelled goat antirabbit IgG (Sigma) for 1 h. The blot was washed and dipped in substrate solution (0.02% diamino benzidine suspended in PBS containing 0.03% hydrogen peroxide in PBS pH 7.4), till brown colour appeared.

Serum samples
A total of 120 samples including 60 bovine paratuberculosis-positive serum and 60 negative samples were measured with the use of ELISA based on the r20.8 protein to determine the sensitivity of the assay. Healthy bovine serum samples (n = 60) were used to determine the specificity of the assay. Serum samples were collected from a total of 140 (cows/calves) from Gausadan, Ghumanhera village, New Delhi. The animals were tested by PPD as well as ELISA using commercial kit.

ELISA for the detection of anti-Map antibodies
Checkerboard titration was used to determine the optimum concentration of protein (0.1, 0.2, 0.3, 0.4 or 0.5 lg/well) and serum dilution for use in an indirect ELISA. The purified recombinant 20.8-kDa protein was diluted to 4 lg/ ml in 0.1 M Na 2 CO 3 /NaHCO 3 buffer, pH 9.6, and used for coating 96-well ELISA plates (Greiner, Germany) 50 ll/ well and incubated at 4°C overnight. The coated wells were washed thrice in PBS-T (0.05% Tween 20 in PBS, pH 7.2) and blocked with 5% skim milk powder in PBS for 2 h at 37°C to remove excess unabsorbed antigen. The wells were washed again with PBS-T and once with PBS and incubated for 1 h at 37°C with 50 ll of bovine sera samples at 1:100 dilution in PBS-T. Plates were washed with PBS-T thrice followed by incubation with 100 ll HRP-conjugated goat anti-bovine immunoglobulins (1:10,000 dilution in 5% skimmed milk) for 1 h at 37°C. After a final wash with PBS-T, colour was developed using chromogenic substrate 100 ll solution/well containing 1 mg OPD (o-phenylenediamine) and 10 ll of 30% H 2 O 2 in sodium phosphate/citric acid buffer, pH 5.0. The plate was incubated in dark at room temperature for 10-15 min in dark. The reaction was stopped using 2.5 M sulfuric acid and the plate was read at A492 using an ELISA reader (Tecan, Austria). The cut-off value for the assay was calculated as the mean specific absorbance plus 3 SD for 60 sera from healthy cattle assayed at a dilution of 1:100 and it was arbitrariy fixed at an A492 of 0.35.

Statistical analysis
The Kappa statistic was calculated using the standard formula (Cohen 1960). The kappa value was interpreted as described previously (Viera and Garrett 2005). Sensitivity (true positive rate) was calculated by the formula, sensitivity ¼ true positive=ðtrue positive þ false negativeÞ. The specificity (true negative rate) was calculated by the formula, specificity ¼ true negative=ðfalse positive þ true negativeÞ. Confidence intervals for sensitivity and specificity were calculated using free online tool at the website: https://www.medcalc.org/calc/diagnostic_test.php.

Results and discussion
Mycobacterium avium subsp. paratuberculosis (Map) is the etiologic agent of bovine paratuberculosis, also called Johne's disease. However, despite several years of development, currently available diagnostic tests still lack performance to be used in control programs. The early and specific diagnosis of Map is still a challenge. Experiments have revealed that antibodies appear much earlier, and therefore, the ELISA could be used as a diagnostic tool (Waters et al. 2003;Begg et al. 2011). Hence, it is necessary to characterize Map antigens to increase the sensitivity and specificity of the ELISA test for paratuberculosis diagnosis. To resolve this issue, we developed an antibodybased method using r20.8 antigen for diagnosis of paratuberculosis in cattle.
In the present study, we have used an efficient expression system, based on pQE30 vector to produce 20.8-kDa protein of M. a. paratuberculosis in E. coli so as to facilitate further characterization of the protein. Based on the sequence information of the locus tag 1637c N-terminus region of 546 bp encoding the 20.8-kDa protein of M. a. paratuberculosis and also the information on the multiple cloning site of the plasmid vector pQE30, restriction sites for BamHI/and PstI were incorporated into the specific primers, to facilitate the directional in-frame cloning into the pQE30 vector under the control of T5 promoter. The recombinant pQ20.8 clones were confirmed by colony PCR using specific primers. Earlier, a similar strategy of cloning in pQE expression vector system was used to produce 35-kDa protein as well as PPE protein of Map (Basagoudanavar et al. 2004;Deb and Goswami 2010). Production of r20.8 protein was induced by IPTG which binds to the lac repressor protein, inactivating it leading to transcription of sequences downstream of the promoter. High-level expression of the recombinant protein was observed in plasmid pQ20.8, 6 h post induction with 1 mM IPTG as resolved by predominant bands 20.8 kDa corresponding to that predicted for r20.8 protein on SDS-PAGE. No such protein band was observed with E. coli M15 cells or in uninduced E. coli M15 cells harbouring recombinant plasmid pQ20.8. Using pQE vector, Basagoudanavar et al. (2004) achieved maximum expression in 6 h post induction of IPTG. Therefore, it was decided to purify the proteins 6 h post induction from E. coli culture. The presence of 6xHis residues at N-terminal of the recombinant protein enabled us to purify the protein by single-step affinity purification using Ni-NTA resin. The 6xHis tag with 840 Da size is poorly immunogenic and may not interfere with structure and function of recombinant protein (Sisk et al. 1994). The yield of the pure recombinant protein was about 15-20 mg/l of culture at shake-flask level (Fig. 1a). High-level expression of the r20.8 protein indicates that the expression system is highly efficient.
Purified recombinant 20.8-kDa protein as well as lysate from IPTG-induced E. coli harbouring pQ20.8 plasmid, were probed with the rabbit polyclonal serum raised against the purified r20.8 kDa protein. It reacted strongly with the expressed protein on Western blot (Fig. 1b). No such band was found in E. coli cell M15 cells as well as in uninduced E. coli cell lysate harbouring plasmid pQ20.8 indicating the immunogenic nature of the r20.8 protein, which was not affected by expression in heterologous system. The immunoblot assays clearly identified the expression of the recombinant protein.
We have examined the ability of recombinant 20.8-kDa protein to detect Map infections in cattle using an ELISA. A total of 120 bovine sera samples (60 positive for Map and 60 negative for Map) were present in the laboratory earlier been tested by feacal examination, PCR, PPD and ELISA using commercial kit. The result demonstrated that the sensitivity of ELISA was 73.33% (95% CI 60.34-83.93%) and specificity was 98.33% (95% CI 91.06-99.96%), relative to infection status as indicated by the commercial kit test (Table 1). The ELISA did not have a reaction with sera from non-infected control.
Screening of serum 140 serum samples (cows/calves) from Gausadan with PPD and r20.8 protein revealed 11 and 18 positive reactors, respectively ( Table 2). The sensitivity was 100% (95% CI 71.51-100%), while the specificity was 94.57% (95% CI 89.14-97.79%). ELISA-based serological tests are easy to perform, cheap to produce and do not require dedicated facilities. In these current commercial ELISAs, antigens used for plate coating are different, and crude bacterial extracts require M. phlei pre-absorption of sera to remove cross-reactive antibodies. This pre-absorption step is not only time-consuming but also affects test sensitivity (McKenna et al. 2005). This is the main reason why we have chosen to focus our effort on the development of a sero-diagnostic test based on one or a handful of purified antigens rather than on a Map extract. In the present study, we have selected an N-terminus region of 546 bp from the locus tag MAP 1637 c because of having the most probable B-cell epitopes. A similar approach of using protein from locus tag MAP 1637 c has been used by Leroy et al. (2009) using 40.8-kDa protein and less number of serum samples. Obviously, the use of a 'golden' antigen should be able to discriminate all infected animals regardless of their clinical stage and with a very small false-positive rate. But it is now clear that such an antigen probably does not exist and that the most efficient diagnostic test would be based on a combination of purified antigens. Moreover, Bannantine et al. (2008) have demonstrated, using an experimental infection model, that diagnostic efficiency of an antigen could vary and decrease over the course of the infection. Thus, antigens effective in detection of clinical cases could be inefficient for early diagnosis. The ELISA test based on Map r20.8 protein shows substantial kappa value agreement with commercial ELISA kit (k = 0.72) as well as when compared with PPD assay (k = 0.78) (Viera and Garrett 2005). Therefore, the r20.8 protein is a good candidate to formulate an antigen combination useful to develop a highly-efficient ELISAbased serological test.

Conclusion
The use of gene expression techniques in the production of recombinant protein also completely eliminated the preparation and use of protein mixtures. The design of the r20.8 and its ease of expression and purification have the potential to make this a highly effective approach in the development of reagents for the diagnosis of bovine paratuberculosis. Comparing with the PPD skin test as the standard method, this ELISA method showed high specificity and sensitivity and can be used for routine diagnosis of paratuberculosis in cattle. Kappa agreement = 0.78