Sera

The CBPP negative serum, positive serum of CBPP, Mycoplasma bovis, Mycoplasma bovirhinis, Mycoplasma agalactiae, Escherichia coli, infectious bovine rhinotracheitis virus, and Mmm immunized sheep sera were stored in our laboratory. The positive serum of bovine viral diarrhea virus, bovine tuberculosis, and bovine paratuberculosis were donated by the relevant teams of Harbin Veterinary Research Institute. Local sera were collected from Heilongjiang, Inner Mongolia, Guangxi, and Yunnan provinces of China.

Screening of diagnostic marker

Because the fact that Mmm is a class I pathogenic microorganism regulated by China, it is not possible to handle live Mmm in general laboratories in this country. To screen candidate diagnostic markers, this study obtained all protein sequences of Mmm strain PG1 from the NCBI database; the transmembrane region analysis software DeepTMHMM (DTU/DeepTMHMM – BioLib) (Alhaji et al. 2020) was employed to screen transmembrane proteins, and then the proteins that have an extracellular region with more than 100 amino acids and contain at least two transmembrane regions were selected. Then, the NCBI Protein BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) was used to select the protein regions that are conservative within Mmm and specific to other pathogens.

Expression and purification of candidate proteins

The coding gene of the candidate marker was synthesized according to the protein sequence and cloned into pGEX-4 T-1 vector (GE) and pMAL-c5X vector (NEB), respectively, and then expressed and purified according to the manufacturer’s instructions. The gene of the extracellular region which contains the candidate marker was synthesized and cloned into the pGEX-4 T-1 vector, expressed, and purified as mentioned above.

Antigenicity verification of candidate protein

The diagnostic marker expressed in pGEX-4 T-1 was tested by dot-ELISA with CBPP-immunized goat sera to confirm that the selected protein can react with CBPP positive sera and the negative serum as control.

Production and identification of Mab

Monoclonal antibodies were prepared according to the method established by Köhler and Milstein (Köhler et al. 1975). Briefly, five 6-week-old female BALB/c mice (supplied by the Laboratory Animal Center of the Harbin Veterinary Research Institute, CAAS) were immunized subcutaneously with 50 μg of recombinant maltose-binding protein (MBP) labeled protein mixed with Freund’s complete adjuvant (Sigma-Aldrich). The booster vaccines consisting of 50 μg purified MBP labeled protein in an equal volume of Freund’s incomplete adjuvant were administrated 2 and 4 weeks after the primary immunization. The enhanced immunization was performed after the purified MBP labeled protein was mixed with Freund’s incomplete adjuvant in equal volume; each mouse was immunized subcutaneously with 50 μg of recombinant protein.

After the second booster vaccination, blood was collected from the tail end and serum was separated; then, the serum titer was evaluated by ELISA as previously described (Xu et al. 2015). Two days after the final booster, the feeder layer cells were prepared from the abdominal cavity cells of non-immunized mice 1 day before the fusion. The next day, the immunized mice were euthanized, and the spleen cells fused with SP2/0 myeloma cells at a ratio of 5–10:1 using polyethylene glycol (PEG 4000; Sigma Aldrich). Hybridoma cells were resuspended in HAT medium (containing 20% FBS, 100 μg/mL streptomycin, 100 IU/L penicillin, 100 mM hypoxanthine, 16 mM thymidine, and 400 mM aminopterin) and seeded into a 96-well plate. On the 7th day after fusion, the medium was removed and replaced with fresh HT medium (containing 20% FBS, 100 μg/mL streptomycin, 100 IU/L penicillin, 100 mM hypoxanthine, and 16 mM thymidine). Following HAT/HT selection, the positive cells were subcloned for 3 rounds with limited dilution and screened for the production of immunogen-reactive antibodies by indirect ELISA.

Indirect ELISA

Indirect ELISA was used to determine the titer of mouse serum and ascites according to the protocol described previously with a few modifications (Xu et al. 2015). Briefly, microplates were sensitized at 4 °C overnight with the affinity-purified GST label protein at 5 μg/mL. The sensitized plates were incubated with test culture supernatants from hybridoma cells at 37 °C for 1 h, with goat anti-mouse IgG (H + L) conjugated with horseradish peroxidase (HRP) secondary antibodies (Sigma) at a 1:5,000 dilution at 37 °C for 1 h, followed by color development upon addition of tetramethylbenzidine substrate solution (Sigma). Color development was terminated with 2 M H2SO4, and the absorbance value of OD450nm was recorded.

Preparation and titration of ascites

The 10-week-old female BALB/c mice were intraperitoneally injected with 0.5 mL of Freund’s incomplete adjuvant. One week later, 1 × 105 hybridoma cells were injected into the abdominal cavity of mice. The ascites were collected when the abdomen of mice was swollen. The supernatant of ascites was 10 times continuous dilution from 1:1000 and then titrated with indirect ELISA. Unimmunized mice serum was set as a negative control. The highest dilution with OD450nm value ≥ 0.2 and P/N value ≥ 2.1 was considered the titer of ascites.

Monoclonal antibody subtype identification

The subtype of Mab was identified by the SBA Cloning System-HRP Kit (Southern Biotech) according to the manufacturer’s instructions. Briefly, add 0.1 mL of hybridoma supernatant to each well of GST label protein coating plate, and incubate for 1 h at room temperature. Dilute HRP-labeled detection antibodies (1:250), add 0.1 mL of these conjugates to appropriate wells of the plate, and incubate for 1 h at room temperature. Add 0.1 mL of ABTS substrate solution to each well of the plate. Read the optical density of each well at 405 nm after substrate addition.

The competitive ELISA

Costar ELISA plates (Corning) were coated with 0.1 ml of protein (1 μg/ml) diluted in 0.05 M carbonate buffer (pH 9.6) by incubation overnight at 4 °C. Plates were washed three times with PBST, and a 100 μl mixture of serum and Mab was added to each well. Place the plate in a 37 ℃ incubator for 1 h, discard the reaction liquid, and wash it three times with PBST. Then, add goat anti-mouse IgG antibody conjugated with HRP (Sigma) diluted at 1:5000, and react at 37 ℃ for another hour. After a final wash step, TMB (Sigma) was added to enable colorimetric analysis. The reaction was stopped by adding 50 μL of 2 M H2SO4, and optical density (OD) was measured at 450 nm. For each sample tested, the value of percentage inhibition (PI%) is calculated using the following formula: PI% = 100 x (ODMab—ODSample)/ (ODMab – ODBlank). The cut-off value between positive and negative sera was calculated with GraphPad Prism based on the analysis of a collection of 32 known CBPP-positive and 614 CBPP-negative serum samples.

Validation of the competitive ELISA

To determine the cross-reaction with the other relevant epidemic disease-positive serum, several positive sera were tested by the cELISA. These sera included the CBPP negative serum, positive serum of CBPP, Mycoplasma bovis, Mycoplasma bovirhinis, Mycoplasma agalactiae, Escherichia coli, bovine tuberculosis, bovine paratuberculosis infectious bovine rhinotracheitis virus, and bovine viral diarrhea virus, with one serum of each species. One hundred and four serum samples collected from four Mmm immunized goats were detected with the cELISA to analyze the seroconversion. To compare the cELISA established in this study with the CBPP commercial kit produced by IDEXX company, 52 bovine and 79 Mmm immunized sheep serum samples were tested using these two methods. The Kappa values were calculated based on the detection results for consistency analysis.

Seroprevalence of CBPP antibodies in bovine sera from Inner Mongolia, Heilongjiang, Guangxi, and Yunnan provinces, China

With the successful establishment of the cELISA, we collected bovine sera from Inner Mongolia, Heilongjiang, Guangxi, and Yunnan for CBPP antibody detection. These areas are located on the border of China. The monitoring of bovine serum samples in these areas is of great significance to prevent CBPP from being reintroduced and maintain the state of no epidemic.