Animals and bacterial strains
Kunming mice (4 weeks old) were purchased from Chongqing Tengxin Biotechnology Co. Ltd., China. All animal procedures were performed in accordance with the guidelines prescribed in the Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Ethics Committee of the Shaanxi University of Technology, China (No. 2019-015).
E. coli and S. aureus isolated from cow mastitis and the E. coli OmpA expression strain were all preserved in the biochemistry and molecular laboratory of Shaanxi University of Technology.
Expression, purification, and preparation of nanoparticles of OmpA
Expression and purification of OmpA were performed as described previously . Briefly, the OmpA expression strain was cultured overnight and transferred to 600 mL LB medium until OD600 nm = 0.5. Isopropyl-β-D-thiogalactoside (IPTG) was then added and induced at 20℃ for 24 h. Bacterial cells were harvested by centrifugation and disrupted by sonication with an ice bath. Finally, OmpA was purified with the Ni-NTA flow resin (Sigma, USA).
The OmpA nanoparticles (NP-OmpA) were prepared by CS encapsulation. Briefly, TPP (3 mL, 1 mg/mL) was added dropwise to a CS solution (10 mL, 1 mg/mL), and stirred for 10 min at 700 r/min. After centrifugation (15 min at 9,500 r/min), the precipitate was added to 25 mL of water and subject to ultra sound (2 min at 50% power). Then 3 mL of OmpA was added dropwise. After centrifugation, 10 mL of water was added to the precipitate to obtain the NP-OmpA. Nanoparticle diameter and zeta potential were analyzed using a Laser Particle Size Analyzer (Beckman, USA), and the morphology was observed using a scanning electron microscope (Phenom Pro, Netherlands) .
In vitro release of NP-OmpA
NP-OmpA was tested for in vitro protein release to simulate the digestive function of the gastrointestinal tract. Briefly, the NP-OmpA solution was transferred to a dialysis bag (MW 14–20 kDa) that was placed into the pH 1.2 solution. At each assigned time point (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 84, and 96 h), 200 μL of supernatant was taken from the solution and analyzed for protein content using Bradford diagnostic kits .
The optimal preparation conditions for NP-OmpA
Nanoparticles were prepared as described by Li et al. , with minor modifications. The factors that were optimized for the preparation of the NP-OmpA were the concentrations of OmpA and CS, magnetic mixing speed, mixing time, and the ratio of TPP/CS (W/W). Each is briefly described here. (1) The concentration of OmpA: TPP (3 mL, 0.5 mg/mL) was added dropwise into a CS solution (10 mL, 0.5 mg/mL) with stirring for 10 min at 700 r/min. After centrifugation (15 min at 9,500 g), 25 mL of water was added to the precipitate under continuous ultrasonication for 2 min, and 3 mL OmpA solution (0.5, 1.0, 1.5, 2.0, 2.5 mg/mL) was added dropwise with stirring (150 r/min for 15 min). After centrifugation (15 min at 9,500 r/min), nanoparticles were obtained. (2) The concentration of CS: TPP was added into the CS solution (1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL), and mixing (10 min at 700 r/min). After centrifugation, 25 mL of water was added to the precipitate under continuous ultrasonication, and 3 mL of OmpA solution was added. Finally, nanoparticles were obtained by centrifugation. (3) The ratio of TPP/CS: TPP was added into the CS solution at the ratios of TPP/CS (W/W) of 1:1, 1:2, 1:3, 1:4, and 1:5. After centrifugation, the precipitate was re-suspended and OmpA solution was added. Finally, nanoparticles were obtained by centrifugation. (4) Magnetic mixing speed: After centrifugation, the precipitate was re-suspended with dH2O, and OmpA solution was added dropwise with magnetic mixing speed at 100, 150, 300, 500, or 700 r/min. Finally, nanoparticles were obtained by centrifugation. (5) Mixing time: After centrifugation, the precipitate was re-suspended and OmpA solution was added with mixing times of 10, 15, 20, 30, 60, and 120 min. Finally, nanoparticles were obtained by centrifugation. Also, loading efficiency (LE), loading capacity (LC), and particle diameter were measured to determine the optimal factors for preparation of NP-OmpA.
Immunoprotective effect of NP-OmpA
Kunming mice were divided into four groups with 20 mice in each group. Groups 1 and 2 were vaccinated with NP-OmpA and OmpA, respectively. Group 3 was vaccinated with CS nanoparticles without OmpA (NP-Empty), while Group 4 received normal saline (NC). All vaccines were orally administered at 6 µg/g of mice body weight four times. The first immune interval was 14 days, and the subsequent immune interval was 7 days. Mice were challenged 7 days post-vaccination with E. coli, and mouse mortality was counted after 15 days. At that point, the immune protection rate (RPS) of the mice was calculated, RPS (%) =1- (% vaccinated mortality/% control mortality) × 100. SPSS software was used to test statistical significance .
Organ index, white blood cell (WBC) count, and leukocyte phagocytosis
The organ index was implemented as follows: the mice were weighed after cervical dislocation. The spleen and thymus were removed and weighed. The organ index = organ weight / mice weight.
WBC counts were conducted as follows: mice anticoagulant was collected to prepare blood smears. Wright's and Giemsa's dye solutions were used for staining, and samples were washed slowly for 3 min with water. After drying, microscopic counting was performed.
Leukocyte phagocytosis was performed as described previously . Briefly, 0.2 mL of mice anticoagulant was added to 2 mL of S. aureus (6×108 CFU/mL) and shaken for 60 min at 25℃ in a water bath. The mixed liquid smears were drawn with a pipette. Each sample was fixed with methanol for 3–5 min, stained (Giemsa) for 30 min, washed and air dried, and then observed by oil microscope. Phagocytic percentage (PP %) = no. of WBCs involved in phagocytosis per 100 leukocytes / 100 ×100%. Phagocytic index (PI %) = no. of bacteria phagocytized / no. of WBCs phagocytizing bacteria. The results were analyzed by variance analysis (ANOVA) and the Tukey test (P < 0.05) with SPSS 19.0 software.
Detection of the interaction between the antiserum and E. coli, and the antiserum titer
Interaction between the antiserum and bacteria was assessed by ELISA as described previously . Briefly, after E. coli were harvested, 1% oxymethylene (W/V) was added for 90 min at 80°C to inactivate the bacteria, and the solution was adjusted until OD600 nm = 0.2. The bacterial solution was transferred to 1.5 mL tubes, and antisera at various dilutions were added before incubation for 1 h at 37°C. After washing with PBS, rabbit anti-mouse antibody (Sigma, USA) was added, and the solution was washed with PBS again. The bacteria were suspended with 20 μL of PBS and transferred to an enzyme-linked plate. Coloration liquid (50 µL H2O2 and 50 µL TMB) and stop solution (50 μL 2M H2SO4) were added to the wells, and absorbance was read at OD450 nm with a microplate reader (Bio-Rad, USA).
Serum antibody titer was detected by ELSA as described previously . Briefly, the purified OmpA was added to an enzyme-linked plate and incubated with blocking solution (5% skim milk), and various dilutions of antiserum were added before incubation for 1 hour at 37°C. After washing, rabbit anti-mouse antibody (Sigma, USA) was added to the plate. Coloration liquid (50 µL H2O2 and 50 µL TMB) was added to each well and the absorbance read at OD450 nm with a microplate reader (Bio-Rad, USA).
Biochemical indexes for physiological function of visceral organs
Four-week-old mice were divided into six groups. Groups 1–5 received orally administered vaccine candidates. Group 1 received NC (300 μL). Group 2 received NP-Empty (without OmpA). Group 3 received OmpA (4 μg/g). Groups 4 and 5 received NP-OmpA (4 μg/g and 8 μg/g, respectively). Mice in Group 6 received intraperitoneal injections of NP-OmpA (4 μg/g). After 7 days of continuous oral administration, the serum and liver of the mice were taken. After homogenizing in ice-cold PBS, the liver tissues were centrifuged (900 g, 4°C, 10 min), and the supernatants were assayed for alanine aminotransferase (ALT), aspartate transaminase (AST), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) using assay kits. Serum uric acid (UA) and creatinine (Cr) were measured according to the kit instructions (Jiancheng Institute of Biotechnology, China).
Determination of immune-related gene and inflammation-related gene expression by qRT-PCR
First, mRNA was isolated from the spleen, liver, and kidney tissues using an RNA isolation kit (TAKARA, Japan) and according to the manufacturer’s instructions, as described previously . Briefly, the mRNA was reverse-transcribed to cDNA using a PrimeScript RT Master Mix kit (TAKARA, Japan), and cDNAs were amplified using the primers shown in Table 1. The qRT-PCR was performed using an Applied Biosystems StepOnePlusTM Real-Time PCR System (ABI Applied Biosystems, USA) with a SYBR® Green Permix Pro Taq HS qPCR kit (TAKARA, Japan). The mRNA expression was analyzed by the 2-(ΔΔCt) formula and GAPDH was included as an internal control gene.
Histopathological morphology of injury to visceral organs
The preparation of pathological sections of mice liver and kidney involved dehydration, transparency, sectioning, and H & E staining . Briefly, the liver and kidney were dehydrated using an alcohol gradient for 1 h and then placed in an alcohol: xylene mixture (1:1, V/V) for 30 min, xylene for 8 min, xylene: paraffin solution (1:1, V/V) for 30 min, and paraffin for 1 h. Slices with a thickness of about 5 μm were cut, dried, H & E stained, observed under a microscope, and photographed (Leica, Germany).