Cloning of mhp366-N gene fragment
Plasmid pGEX-6P-2-mhp366 was extracted from recombinant bacteria GST-Mhp366 [26] using HiPure Plasmid Micro Kit (Magen, China). Nucleotide fragment mhp366-N which contains the corresponding peptide segment recognized by the convalescent serum but not by hyperimmue serum was amplified with two primers 5'-CGCGGATCCATGAAAAAAATGGTAAAATATTTTCTAG-3' (BamH I) and 5'-CCGCTCGAGCCAAAATGGGCCACCGTT-3' (XhoI) by using PrimeSTAR® Max DNA Polymerase (Takara, China). After that, the PCR product was ligated into vector pET-28a(+) to construct the recombinant plasmid. Finally, the ligation product was transformed into E. coli DH5α competent cells, and was identified by double restriction enzyme digestion and sequencing.
Expression and purification of recombinant protein Mhp366-N
Recombinant plasmids were transformed into E. coli BL21(DE3) competent cells. Transformed clone was grown at 16℃ for 20 h with shaking supplemented with 50 μg/mL kanamycin and 1 mM IPTG. Recombinant Mhp366-N protein was purified by Ni affinity chromatography (GE Healthcare, USA) using a gradient of 0.1-1 M imidazole, and identified by SDS-PAGE and Western blot. The concentration of Mhp366-N protein was determined by BCA protein assay kit (Beyotime, China).
Animal source
The experiment was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. All experimental protocols were approved by the Institutional Animal Ethics Committee of Southwest University (Approval no. IAECSWU20170921) and performed accordingly. The objectives, protocols and potential risks were clearly explained to all participating farm owners. Written informed consents were obtained from all participating farm owners.
Serum samples used in this study were collected from 4 farms. Pigs from farm A were M. hyopneumoniae-free and no EP-like clinical syndromes occurred or lung lesions were observed. Pathogen and serology detection were carried out in recent 2 years. M. hyopneumoniae organism and nucleotide are free by culture and nested PCR. Also, the sera are negative by immunological diagnosis with commercial ELISA kit (IDEXX laboratories, Westbrook, Maine, USA). While, pigs from farm B, C and D had a history of EP according to the clinical observation and serological surveillance in last 2 years. For the farm B, about one quarter of pigs showed EP-like clinical syndromes. However, EP sporadically occurred at farm C and D. All pigs were weaned on 21st day.
Sample collection and preparation
Twenty pigs from farm A were immunized with a commercial M. hyopneumoniae inactivated vaccine (MYPRAVAC SUIS, Hipra Lab) on the 7th day and 21st day after their bearing. MYPRAVAC SUIS is a whole-cell, inactivated bacterin based on J strain, with mineral oil and aluminum hydroxide as adjuvants. Fifty six days after the last immunization, serum samples were collected from the front cavity veins of immunized pigs from farm A. Meanwhile, laryngeal swabs were obtained from the laryngeal cartilages with the help of snares and mouth gags for pig restraint as described previously [16]. Pigs from farm C and D were also vaccinated with MYPRAVAC SUIS on the 7th and 21st day. Twenty pigs of 21 weeks old and 100 pigs of 10-11 weeks old were chosen from farm B and C, respectively. Five piglets of 7 days old before immunization and other 5 piglets of 14 days old shot on 7th day from farm D were picked up randomly. Laryngeal swabs were collected from corresponding pigs at farm B [16], while, nasal and laryngeal swabs were collected from corresponding pigs at farm C and D, as previously described [9, 16].
Glycerol was added to the collected sera and the final concentration was 50%. Then, the sera were kept in aliquots at -20°C until further use. The contents of laryngeal swabs were concentrated by centrifugation at 12 000 g for 10 min after releasing into 1 mL sterile PBS at 4℃ overnight. M. hyopneumoniae was determined by nested PCR from laryngeal swabs as described previously [9]. Each nasal swab was put into 1.5 mL microcentrifuge tube containing 1 mL sterile PBS and stored at 4℃ overnight. After centrifugation at 10 000 r/min for 10 min, sIgA was detected from the supernatant according the procedure of sIgA ELISA kit [9].
All pigs used in this study were released after sample collection.
Optimization of ELISA procedure and working condition
The 96-well microtiter plates (Corning incorporated, USA) were coated with 100 μL Mhp366-N protein (from 0.25 μg/mL to 8 μg/mL) in 0.5 M carbonate buffer (pH 9.6) overnight at 4℃ after 37℃ for 1 h. Unbound antigen was discarded, and the wells were washed five times with PBS containing 0.05% Tween-20 (PBST). Non-specific bindings were blocked with 200 μL PBST, 1% BSA, 2.5% skim milk, 10% FBS, 1% gelatin or 1% ovalbumin at 37℃ for 0.5 h, 1 h or 2 h. After five washes with PBST, 100 μL serum samples diluted from 1:50 to 1:8000 were added and incubated at 37℃ for 0.5 h, 1 h or 2 h. Following five washes with PBST, the plates were conjugated with 100 μL of HRP-conjugate rabbit anti-pig IgG (H+L) secondary antibody (Invitrogen, USA) diluted in blocking buffer (from 1:10000 to 1:80000) at 37℃ for different times (0.5 h, 1 h and 2 h). The plates were washed as described above, 50 μL of substrate A (100 mL H2O containing anhydrous sodium acetate 2.72 g, citric acid monohydrate 0.2078 g, 30% hydrogen peroxide 0.06 mL) and substrate B (100 mL H2O containing EDTA·Na2 0.04 g, citric acid monohydrate 0.2078 g, glycerol 10 mL, TMB·2HCl 0.0391 g) were added, respectively. After incubation for different time periods (5 min, 10 min and 20 min) at RT, the reaction was terminated by adding 50 μL 2 M H2SO4. The optical density at 450 nm (OD450) was recorded using an automatic ELISA plate reader (ThermoFisher Scientific, Ratastie 2, FI-01620 Vantaa, Finland). All samples were run in triplicate, and each experiment was performed at least twice. Each working condition was optimized and determined with the highest P/N ratio between convalescent serum samples (P) and hyperimmune serum samples (N).
Calculation of cut-off value
The cut-off value was obtained by determining the OD450 calculated from the mean of hyperimmune serum control plus 3 standard deviations (SD), as described previously [27, 28].
Evaluation of reproducibility
Reproducibility of intra- and inter- assay variation between runs was performed as described by Feng et al. [9] with minor modification. In brief, 2 hyperimmune and 2 convalescent sera were selected randomly for the reproducibility experiments. Five replicates of each sample in the same batch were chosen for intra-assay (within plate) reproducibility and 3 plates from different batch were chosen for inter-assay (between runs) reproducibility. Mean values, SD and coefficient of variations (CV) were calculated.
Estimation of specificity and sensitivity
The specificity of this assay was investigated by using positive sera of M. hyorhinis (Mhr), A. pleuropneumoniae (App), S. suis serotype 2 (SS2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and pseudorabies virus gB protein (gB-PRV). Two hyperimmune and 2 convalescent sera were used as negative and positive controls, respectively.
Five convalescent sera were diluted with blocking buffer as follows: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000. Then, ELISA was carried out with the optimal working conditions except the optimal dilution of convalescent sera. The sensitivity of the ELISA assay was accessed according to the cut-off value.
Application and comparison of ELISA discriminating hyperimmune sera and convalescent sera with commercial kits
Samples from farm C and D were processed for the detection of M. hyopneumoniae IgG and sIgA. Serum samples were used for the detection of IgG with both commercial IDEXX kit and our established ELISA method. sIgA-ELISA kit was applied to decide sIgA from nasal swabs. Each sample was conduct in duplicate. M. hyopneumoniae DNA was tested by nested PCR from laryngeal swabs as described previously (Feng et al., 2010).
SDS-PAGE and Western blot
Pretreated bacteria or purified protein were mixed with loading buffer and loaded onto SDS polyacrylamide gels. After electrophoresis, gel was used for staining with coomassie brilliant blue, or transferred to polyvinylidene difluoride membrane (Roche Diagnostics, German) for 2 h at 100 V using a trans-blotting apparatus (Bio-Rad, USA). The membrane was blocked overnight at 4℃ in 5% skimmed milk-TBST and was detected by His-tag (4C2) monoclonal antibody (Bioworld Technology, China) with a 1:8000 dilution at RT for 1 h. The primary antibody binding was incubated with a 1:20000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Proteintech, China) at RT for 1 h and visualized with an enhanced chemiluminescence kit (CWBio, China).