The bovine P. multocida serotype A PmCQ2 and PmCQ6 strains were isolated from lungs of feedlot calves suffering from fatal pneumonia at several cattle farms in Chongqing, China between May 2012 and December 2013. The bovine P. multocida serotype F PmF strain was isolated from lungs of feedlot calves suffering from disease of respiratory system at Gaojiazhen farms in Fengdu (Chongqing, China, longitude/latitude 107.70/29.89) in October 2013. The serotype B strain PmB is a well-characterized pathogenic strain and was purchased from the Chinese Veterinary Drug Supervision (cat. no. CVCC450; Beijing, China). The animal studies were performed with approval from the ethics committee of Southwest University (Beibei, China; permit no. 11-1025) and in compliance with the Laboratory Animal Care principles of the National Institutes of Health (Bethesda, MD, USA). The P. multocida strains were aerobically cultured in Martin's broth medium (Qingdao Hope Biol-Technology Co., Ltd., Qingdao, China) at 37°C for 12 h. E. coli DH5α was aerobically cultured in Luria Bertani broth (LB; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 20 h. The E. coli strain genomic DNA was used as a negative control.

A total of 120 Female Kunming (KM) mice (8 weeks-old, body weight 18–22 g) were obtained from the Chongqing Academy of Chinese Materia Medica (Chongqing, China). The animals were housed in pathogen-free conditions (temperature, 20–30°C; relative humidity, 45–60%; 12-h light/dark cycle) and were given free access to food (standard rodent diet) and drinking water.

OMPs were prepared in accordance with previously described protocols (19–21). The bacteria cell cultures were harvested by centrifugation at 10,000 × g for 15 min at 4°C. The pellets were washed 3 times with cold, sterile PBS (pH 7.4) and resuspended in PBS (pH 7.4) containing 8% Triton X-114, 0.5% 3-(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 1 mmol/l phenylmethylsulfonyl fluoride, and maintained at 4°C for 3 h. The cell debris was removed via centrifugation at 10,000 × g and 4°C for 10 min, and the aqueous phase was collected and 10 µg OMPs were detected by using 12% SDS-PAGE gel. The gelatinous decontamination phase was added into 1:9 volume absolute ethanol, and the pellets were collected by centrifugation at 10,000 × g and 4°C for 30 min, washed twice with PBS, and dissolved into PBS. Concentration of OMPs was determined using the Bradford method (Bradford Protein Assay kit; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The 10 µg OMPs were separated by using 12% SDS-PAGE gel and blotted electrophoretically onto a polyvinylidene difluoride membrane. The membrane was incubated with infected PmCQ2 bovine serum (PmCQ2-IS; 1:16) and horseradish peroxidase-conjugated rabbit anti-bovine IgG antibodies (cat. no. SAB3700018; 1:20,000; Sigma-Aldrich; Merck KGaA). An Enhanced Chemiluminescent reagent (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was applied for a chromogenic reaction. Proteins were visualized using a LTQ Mass Spectrometer (Shanghai Applied Protein Technology Co. Ltd., Shanghai, China). Proteins in P. multocida species were detected using BioWorks Browser software version 3.3 (bioworks.software.informer.com).

The target genes were amplified from the genomic DNA of strain PmCQ2. DNApol was from Takara Biotechnology Co., Ltd. (Chongqing, China) and the primers for the selected genes are presented in Table I. The products of polymerase chain reaction (94°C for 3 min, 94°C for 1 min, 50°C for 1 min, 72°C for 1 min, 35 cycles at 72°C for 10 min, stored at 4°C) were cloned into pET-28a plasmid (Takara Biotechnology Co., Ltd.) in E.coli DH5α (Takara Biotechnology Co., Ltd.), expressed in E. coli BL21 (DE3; Takara Biotechnology Co., Ltd.), cultured at 37°C in LB broth or on agar supplemented with 100 µg/ml kanamycin (Sigma-Aldrich; Merck KGaA) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich; Merck KGaA) at 30°C or 37°C for 4 h. The recombinant proteins were purified using affinity chromatography and Ni-NTA Superflow cartridges (Qiagen GmbH, Hilden, Germany).

A total of 4 P. multocida-antibody-free calves were purchased from a farm in Chongqing. One calf was immunized intramuscularly in the neck with 4.8×10¹⁰ colony forming unit (CFU)-inactivated PmCQ2, which was fully emulsified with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich; Merck KGaA) following 3 administrations with 10-day intervals between each. The blood was collected for anti-serum (PmCQ2-VS) separation. A second calf was intramuscularly injected in the neck with 4.8×10¹⁰ CFU live PmCQ2. Following 3 injections with the same dose at 10-day intervals for each dose, the antiserums (PmCQ2-IS) obtained from the calf were separated post-infection at 30 days. The remaining two calves were intranasally infected with 4.8×10¹⁰ CFU live PmB and PmF 3 times. The calves were injected 3 times with the same dose, at 10 day intervals for each dose. Subsequently, the antiserums (PmB-IS and PmF-IS) were separated post-infection at 30 days.

Recombinant protein was dissolved in coating buffer (0.5 M carbonate buffer; pH 9.6) and added to the well (10 µg protein/well) of 96-well microplates (Nunc-Immuno™ MicroWell; Merck KGaA) and then incubated at 4°C overnight. Plates were washed three times with PBS containing 0.05% Tween 20 (PBST), blocked for prevention of non-specific binding with 200 µl 2% skimmed milk (Sigma-Aldrich; Merck KGaA) at 37°C for 1 h. A 1:100 dilution of bovine serum in blocking buffer was added to each well and incubated at 37°C for 1 h. Following washing three times each for 20 min with PBST, a 1:20,000 dilution of horseradish peroxidase-conjugated rabbit anti-bovine IgG antibodies (SAB3700018; Sigma-Aldrich; Merck KGaA) in blocking buffer was added to each well and incubated continuously at 37°C for 1 h, then washed three times each for 20 min with PBST. Then, 100 µl substrate solution 30 mg o-phenylenediaminedihydrochloride (Takara Biotechnology Co., Ltd.) in 100 ml substrate buffer (0.2 M Na₂HPO₄-12H₂O; 0.1 M C₆H₈O₇; pH 4.8)] was added to each well and incubated without light for 15–30 min at room temperature. The reaction was stopped by adding 100 µl 2N sulfuric acid (H₂SO₄; Sigma-Aldrich; Merck KGaA) and absorbance values were recorded at a wave length 450 nm by ELISA reader (Spectrophotometer 1510, Thermo Fisher Scientific, Inc.).

A total of 120 female KM mice were equally divided into 12 groups (n=10/group). Each mouse was subcutaneously inoculated with the 100 µg/dose recombinant proteins. The control group mice were inoculated with 100 µl PBS. The initial two inoculations occurred once in two consecutive weeks and the third and final inoculation occurred in week 4. The mice were intramuscularly injected with 10 LD₅₀ PmCQ2 (LD₅₀=6.48×10⁶ CFU), PmB (LD₅₀=3.87×10⁶ CFU) and PmF (LD₅₀=1×10⁷ CFU) strains 2 weeks after the third and final inoculation.

Data were analyzed by Microsoft Office Excel 2010 and SPSS version 20 (IBM SPSS, Armonk, NY, USA). Sequence homology analysis was employed using ClustalW2 (http://www.ebi.ac.uk/tools/clustalw2). Survival rates of animals were evaluated using Kaplan-Meier analysis and Life test of SAS 9.2 (SAS Institute Inc.) P<0.05 was considered to indicate a statistically significant difference.

The OMPs were extracted using Triton X-114 from P. multocida serotype A, B and F strains. The OMPs were identified by SDS-PAGE (Fig. 1A) western blot analysis (Fig. 1B) of PmCQ2-IS serum. A ~57 kDa common OMP band was observed in PmCQ2, PmCQ6, PmB and PmF (indicated by black arrows in Fig. 1), and a clear 34 kDa OMP band in PmCQ2 and PmCQ6 (indicated by red arrows in Fig. 1). To identify the candidate cross-protective antigens for P. multocida serotype A, the two gel bands were cut out and used for LTQ/MS analyses.

Based on the LTQ/MS analysis, the protein sequences were compared with the Uniprot database (www.uniprot.org) and our bovine P. multocida genome database. A total of 6 protein sequences were matched and are presented in Table II. These protein sequences were predicted as secretory proteins using TMHMM Server.2.0 (22), SignalPv3.0 (23), PSORTb (24) and GPI-SOM (25). A total of 6 protein sequences contained a signal peptide, and 3 secretory proteins were predicted from the bioinformatics analysis (Table I). Using the aforementioned combined analyses, 6 protein sequences were cloned and purified.

The sera for PmCQ2-VS, PmCQ2-IS, PmB-IS and PmF-IS were used to test the reactivities of 6 recombinant proteins using an indirect ELISA (Table III). rPmCQ2_3g0306, rPmCQ2_2g0128, rPmCQ2_3g0121 and rPmCQ2_2g0046 had the high reactogenicity to PmCQ2-IS; however, only rPmCQ2_1g0327 and rPmCQ2_1g0020 were able to react with the 4 sera. Therefore, the present study used r-PmCQ2_2g0128, r-PmCQ2_1g0327 and r-PmCQ2_1g0020 to verify potential cross-protective antigens and diagnostic antigens for P. multocida serotype A.

The present study extracted genomic DNA from the PmCQ2, PmB, PmF and E. coli strains to amplify recombinant proteins (Fig. 2). PmCQ2_1g0020 and PmCQ2_2g0128 DNA were amplified in PmCQ2, PmB and PmF (Fig. 2A and C, respectively). However, PmCQ2_1g0327 DNA was not amplified in PmB and PmF (Fig. 2B). The purity of recombinant proteins was determined using SDS-PAGE, which revealed single bands with the molecular masses 34, 35 and 57 kDa (Fig. 2D). The identity and integrity of the recombinant proteins (rPmCQ2_1g0020, rPmCQ2_1g0327 and rPmCQ2_2g0128) were determined using western blotting (Fig. 2E).

The homology analysis was performed with Clustal X software version 2.0 (http://www.ebi.ac.uk/tools/clustalw2) on PmCQ2_1g0327 and different P. multocida strains that contained PmCQ2, PmCQ6, PmB, PmF, 5 additional bovine P. multocida (isolated from the lungs of diseased cattle in our laboratory), 1 poultry P. multocida (isolated from the lungs of diseased anseriformes in our laboratory) and 27 additional P. multocida genome sequences from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Sequence analysis revealed that PmCQ2_1g0327 exhibited a 100% sequence match to Pmu_17770 and PmCQ6_c4301g0004 in P. multocida serotype A Pm36950 and PmCQ6 strains (Fig. 3).

In order to evaluate the protective efficacy of these proteins, mice were immunized with 100 µg/dose purified r-PmCQ2_2g0128, r-PmCQ2_1g0327 and r-PmCQ2_1g0020. Therefore, it was determined that up to 7 days after a challenge with PmCQ2, r-PmCQ2_1g0327 and r-PmCQ2_2g0128 proteins provided improved survival rates (50 and 40%, respectively) compared with PBS alone (control group) and r-PmCQ2_1g0020 (0%). No statistically significant difference was identified between the protection percentages (30–40%) of these three proteins against PmB. However, the protection percentages against PmF were low or zero (Fig. 4).