Synthesis of Escherichia coli OmpA Oral Nanoparticles and Evaluation of Immune Functions against the Major Etiologic Agent of Cow Mastitis

Abstract

Escherichia coli is a major etiologic agent of cow mastitis, a condition that results in huge economic losses. There is a lack of an oral vaccine for cow mastitis. Previous studies have confirmed that the outer membrane protein A (OmpA) of E. coli is immunogenic and can be used for vaccine design. In the present study, OmpA was encapsulated into nanoparticles (NP-OmpA) for an oral vaccine for cow mastitis. Methods: OmpA was purified with Ni-NTA flow resin and encapsulated with chitosan (CS) to prepare NP-OmpA nanoparticles. The gastrointestinal tract was simulated in vitro (PBS, pH 1.2) to measure the protein release rate. The optimal preparation conditions for NP-OmpA were determined by analyzing the concentrations of OmpA and CS, magnetic mixing speed, mixing time, and the ratio of tripolyphosphate (TPP)/CS (w/w). NP-OmpA safety was assessed by function factors and histopathological examination of livers and kidneys. The immune activity of NP-OmpA was determined using qRT-PCR to assess immune-related gene expression, leukocyte phagocytosis of Staphylococcus aureus, ELISA to evaluate antiserum titer and immune recognition of E. coli, and the organ index. The immune protection function of NP-OmpA was assessed by the protection rate of NP-OmpA to E. coli in mice, qRT-PCR for inflammation-related gene expression, assay kits for antioxidant factors, and visceral injury in the histopathological sections. Results: NP-OmpA nanoparticles had a diameter of about 700 nm, loading efficiency (LE) of 79.27%, and loading capacity (LC) of 20.31%. The release rate of NP-OmpA (0~96 h) was less than 50% in vitro. The optimal preparation conditions for NP-OmpAs were OmpA protein concentration of 2 mg/mL, CS concentration of 5 mg/mL, TPP/CS (w/w) of 1:1, magnetic mixing speed of 150 r/min, and mixing time of 15 min. Histopathological sections and clinical analytes of uric acid (UA), creatinine (Cr), alanine aminotransferase (ALT), aspartate transaminase (AST), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) showed NP-OmpA did not damage mice livers or kidneys. NP-OmpA could enhance the immune-related gene expression of IFN-γ and HSP70 in the spleen, liver, and kidney and the leukocyte phagocytosis of S. aureus. The antiserum titer (1:3200) was obtained from mice immunized with NP-OmpA, which had an immune recognition effect to E. coli. The immune protection rate of NP-OmpA was 71.43% (p < 0.05) to E. coli. NP-OmpA could down-regulate the inflammation-related gene expression of TNF-a, IL-6, and IL-10 in the spleen, liver, and kidney, and the antioxidant factors MDA and SOD in the liver, and reduce injury in the liver and kidney of mice induced by E. coli. Conclusions: A novel NP-OmpA nanoparticle was encapsulated, and the optimal preparation conditions were determined. The NP-OmpA was safe and had good immune functions. They are expected to induce a response that resists infection with the major etiologic agent (E. coli) of cow mastitis.

Escherichia coli Mastitis in Dairy Cattle: Etiology, Diagnosis, and Treatment Challenges

“…The outer membrane protein A (OmpA) has also been considered a potential antigen in the bovine mastitis vaccine associated with E. coli (Rainard et al, 2017;Chen et al, 2020;Liu et al, 2021). OmpA is a crucial virulence factor in the pathogenesis of E. coli, playing a key role in pore and biofilm formation, host cell invasion, and multidrug resistance (Confer and Ayalew, 2013).…”

Novel Proteoliposome-Based Vaccine against E. coli: A Potential New Tool for the Control of Bovine Mastitis

“…The proteoliposomes were able to retain several bacterial macromolecules with immunostimulatory potential, particularly membrane proteins, DNA and LPS. In SDS-PAGE gels, the presence of quantitatively important bands located between 35–45 kDa suggests the retention of highly antigenic outer membrane porins (Omp) [ 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ]. In addition, highly similar protein electrophoretic patterns in polyacrylamide gels were observed during the production process of E. coli proteoliposomes ( Supplementary Figure S1 ), suggesting that proteins retained in proteoliposomes are not denatured during production, contrary to formaldehyde treatment, which alters the chemical structure of proteins, induces protein crosslinking, limiting antigenic processing by APCs and, therefore, restricting antigenic presentation to T cells and the generation of memory cells [ 22 , 23 , 24 ].…”

Immune responses and protective efficacy of outer membrane protein ExbB of Pseudomonas fluorescens against Aeromonas hydrophila and Pseudomonas fluorescens affecting Carassius auratus

“…The organ index was performed, as previously described (Liu et al 2021). The mice were briefly weighed, and the liver, spleen, and kidney were weighed after anesthesia.…”

“…ExbB antiserum was obtained 7 days after the third immunization. The recognition between the antiserum and bacteria was evaluated with ELISA (Liu et al 2021). Briefly, after bacteria were harvested, oxymethylene (1%, W/V) was added to inactivate the bacteria at 80 °C for 90 min, and the bacterial solution was adjusted to 6 × 10 8 CFU/mL (OD 600 = 0.2).…”

“…According to the manufacturer's instructions, mRNA was obtained from the spleen, kidney, and gill tissues of C. auratus after 2 days of challenging bacteria by RNA extraction kits (TAKARA, Tokyo, Japan), and qRT-PCR was performed, as previously described in Liu et al (2021). A commercial kit (TAKARA, Tokyo, Japan) was utilized to prepare cDNA by the mRNA reverse-transcribed per the instructions.…”