Animal and housing
Experimental procedures and animal use were approved by the Northwest A&F University Animal Care and Use Committee. Arbor Acres broiler chickens were purchased from Dacheng Poultry Industry Company (Xianyang, China) and raised in clean and sterilized rooms under standard conditions until they were 7 days old. Each room was provided with filtered, non-circulated air, and air pressure differences and strict sanitary conditions were maintained.
Bacterial strains and preparation of MOMVs
Three most common APEC strains that cause chicken colibacillosis, including O1, O2 and O78 serogroups, were obtained from China Veterinary Culture Collection Center. Bacterial OMVs were prepared from these three APEC strains using the protocol as described previously [52, 53]. Briefly, the bacterial strain was grown in LB to the logarithmic phase at 37°C shaking at 180 rpm. Bacteria-free supernatant was collected by centrifugation (15 min, 12,000 g, 4°C) and then filtered through a 0.45-μm bottle top vacuum ﬁlter (Corning, NY, USA). The filtered supernatant was concentrated using an Amicon Ultrafiltration system (Merck Millipore, Billerica, Massachusetts, USA) with a 100 kDa-exclusion filter, and subsequently subjected to ultracentrifugation (2 h, 150,000 g, 4°C) in a Beckman type 70 Ti rotor (Beckman, CA, USA). The pellet containing OMVs was resuspended in sterile PBS (pH 7.4) and further purified by OptiPrep density gradient centrifugation (16 h, 180,000 g, 4°C) with Optiprep (Sigma-Aldrich) concentrations ranging from 10% to 55% (w/v) . After centrifugation, each fraction from the top of the gradient to the bottom was collected to determine the particle number by nanoparticle tracking analysis (NTA). These fractions enriched with OMVs were pooled, diluted in sterile PBS and then centrifuged (2 h, 150,000 g, 4°C) to remove OptiPrep. Purified OMVs pellet was resuspended in sterile PBS, sterilized by ﬁltration (0.45 μm; Millipore, Bedford, MA), and finally stored at -80°C until future use.
The protein concentration of OMVs was measured by a bicinchoninic acid assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The purified OMVs from these three APEC strains were uniformly mixed in equal proportions to formulate the final MOMVs. Outer membrane proteins (OMPs) were prepared from each APEC strain using the Sarkosyl method as described previously . Muti-serogroup OMPs (MOMPs) were formulated in the same way as MOMVs and used to measure the anti-MOMPs IgG titer in sera. To remove vesicular lipopolysaccharide (LPS) and proteins, native MOMVs were treated with an equal amount of polymyxin B (PMB_MOMVs) and 50 μg/mL (3 U/mL) proteinase K (PK_MOMVs) according to previously reported methods, respectively [55, 56]. Inactivation of proteinase K was performed by raising the temperature (75°C for 30 min) and adding proteinase K inhibitor (Cocktail Set I, Sigma-Aldrich). Limulus Amebocyte Lysate (LAL) and SDS-PAGE electrophoresis assays were performed to confirm the effectiveness of the treatments. These MOMVs were used for subsequent vaccination of chickens.
Characterization and proteomic analysis of MOMVs
Purified MOMVs were visualized to detect their morphology and integrity by scanning electron microscopy and transmission electron microscopy using a Field Emission Scanning Electron Microscope (S-4800, Hitachi, Tokyo, Japan) and FEI Tecnai™ G2 Spirit BioTWIN (FEI Company, OR, USA), as described previously . The diameter size distribution of MOMVs was assessed by NTA using a Nanoparticle Analyser (NanoSight, Malvern, Worchestershire, UK) with the operating parameters as follows: 15 for camera level, five 60-second videos for each sample and 6 for detection threshold. To determine the proteome of MOMVs, proteins (10 μg) of MOMVs were separated by 10% SDS-PAGE gel followed by staining with Coomassie Brilliant blue G250 (Sigma-Aldrich). Protein lanes were extracted from the gel, and then digested with trypsin. The obtained peptides were analyzed by the UPLC coupled to tandem mass spectrometry (MS/MS) (LC-MS/MS; Thermo Scientific) . The resulting MS/MS data from three independent experiments were processed separately using Maxquant search engine (v.22.214.171.124). For protein identification, mass spectra were matched with typical E. coli K-12 strains in the UniProt database. All searches were ﬁltered using the parameter settings described in a previous study . The identiﬁed proteins were analyzed by subcellular localization as well as Gene Ontology (GO) biological processes and molecular functions using CELLO (http://cello.life.nctu.edu.tw/) and InterProScan (http://www.ebi.ac.uk/interpro/), respectively.
In vitro studies of chicken macrophage
The HD11 cells, a transformed chicken macrophage cell line, were used to investigate whether MOMVs could induce innate immune responses in vitro. We first explored the uptake of MOMVs by HD11 macrophages using a co-culture experiment as described previously . Briefly, dialkylcarbocyanine iodide (DiI, Sigma-Aldrich)-labeled MOMVs were co-cultured with HDl1 cells in complete PRMI-1640 medium (Gibco) containing 10% heat-inactivated FBS (HyClone) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Sigma-Aldrich) at 37°C in a 5% CO2 atmosphere. After incubation, the cell nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then visualized with High-speed spinning-disk confocal microscope (Andor Revolution XD, Andor Technology, UK). The cells that were not treated with MOMVs were used as the control. We next performed a stimulation assay to evaluate the immune responses of chicken macrophage to MOMVs. HD11 monolayers (1 × 106 cells/mL) were cultured with various doses of MOMVs (0-100 ng/mL) in cell culture medium described above. After 16-h stimulation, the cell culture supernatants were collected for determining the production of cytokines.
Determination of the lethal doses for APEC strains
Three doses (1 × 108, 5 × 108 and 1 × 109 CFU) of each APEC strain in 100 μL PBS were administrated into chickens by the intratracheal route to determine the lethal dose (LD). The survival rate was recorded every day for 10 days.
Immunization and challenge
Prior to conducting animal experiments, specific PCR tests were used to ensure that the chickens were not infected with these three APEC strains . To investigate the cross-protective efficacy of MOMVs immunization against APEC infections, 7-day-old chickens were vaccinated three times with 10, 50 and 100 μg of MOMVs in 100 μL PBS at a 1-week interval via the intramuscular route, respectively (Fig. 6a, upper panel). Seven days after the third vaccination (day 28), the birds were infected with the LD of each APEC strain by the intratracheal route. The survival rate was monitored every day for 10 days. To examine the long-term protective effect of MOMVs immunization, chickens were immunized with an optimal dose of MOMVs, and then infected with 2 × LD of each APEC strain 5 weeks after the last immunization (day 56). To evaluate the role of vesicular proteins and LPS in MOMVs-mediated protection, we used PMB_MOMVs and PK_ MOMVs to immunize chickens, respectively, and observed the survival rate after infection with the LD of each APEC strain.
Determination of specific antibody titer
One week after each vaccination, sera from chickens were sampled for determining the levels of specific IgG against these three OMVs of the mixed MOMVs using an indirect ELISA method as described previously . Brieﬂy, the 96-well plates were coated with 200 ng of each OMVs overnight at 4°C and then blocked with 1% bull serum albumin. The sera were diluted by 200-fold in PBS, and used as the primary antibody, which was then added in the blocked wells and incubated at 37°C for 1 h. The specific IgG was detected after the plates were incubated with secondary HRP-conjugated rabbit anti-chicken IgG (Sigma-Aldrich) followed by the addition of tetramethylbenzidine substrate. The absorbance at 450 nm was detected using a Microplate Reader (Epoch 2, Biotek, Winooski, USA). Each sample was detected in triplicate. The anti-LPS and anti-MOMPs IgG titer in sera were determined by the same method using puriﬁed LPS and MOMPs.
Expression of immune genes
Total RNA was extracted from spleen tissues of MOMVs- and PBS-immunized birds at 1 week after the third immunization using a total RNA kit I (Omega BioTek, Norcross, GA, USA), and then reverse-transcribed into cDNA using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology, Dalian, China). Quantitative real-time PCR (qRT-PCR) for immune-related genes (Table.1) was performed in a Real-Time PCR Detection System (CFX96 Touch, Bio-Rad, Hercules, CA, USA). The primers for the target genes and reference gene (β-actin) are listed in Table S1. Each PCR reaction was conducted in triplicate, as follows: 95°C for 1 min, 40 cycles of 95°C for 15 s and 60°C for 30 s. Relative gene expression was presented as fold-change compared with the control using the 2−ΔΔCt method .
Growth performance and blood parameters
One week after the final immunization, daily feed intake (DFI), average daily weight gain (ADG), feed conversion ratio (FCR) and mortality for the entire period of immunization (day 7-28) were measured as described previously . Blood samples were collected from MOMVs- and PBS-immunized chickens for determination of the number of white blood cells and platelets using an automatic blood cell analyzer (XFA6100; Perlong new technology Co., Ltd., Nanjing, China).
Measurement of bacterial burden
After APEC infection, bacterial burdens of liver and lung tissues were estimated at indicated times by qRT-PCR as described previously . Briefly, DNA was isolated and purified from liver or lung tissues using a QIAamp DNA Kit (Qiagen, Shanghai, China), and bacterial burden was detected by using specific primers and a probe derived from 16S rDNA sequences of E. coli, including the forward primer (5’-CATGCCGCGTGTATGAAGAA-3’), the reverse primer (5’CGGGTAACGTCAATGAG CAAA-3’), and the detecting probe (5’-TATTAACTTTACTCCCTTCCTCCCCGCTGA A-3’). Bacterial burden was presented as the number of 16S rDNA gene copies per unit of total DNA after normalization of total DNA content per unit of tissue for the same sample.
Measurement of chicken cytokines
The cytokine levels in serum and cell-culture supernatant were determined, including interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) in serum collected at day 22 (24 h after the last immunization) and day 29 (24 h after challenge), IL-6, TNF-α and IL-12 in the supernatant of HD11 cells. Since chicken IL-12 has functional homologue and bioactive similarity with human IL-12, we used a human IL-12 ELISA kit (R&D System) to determine chicken IL-12 levels . The production of chicken IL-6 and TNF-α were estimated using IL-6 and TNF-α activity bioassays, respectively .
Graph Pad Prism software 5.0 was used for data analysis. Data are shown as the mean ± standard error of the mean (SEM). Student’s t-test was used for pairwise comparisons. Significant differences (P < 0.05) of means among three or more groups were analyzed using one-way ANOVA with the Newman-Keuls test as the post-hoc test. The survival rates after bacteria challenge were compared by the log-rank test.