Synthesis of Escherichia coli OmpA oral nanoparticles and evaluation of immune function against the main pathogenic bacteria of cow mastitis
Authors: Liu Xiang , Sun Wei , Wu Nana , Rong Na , Kang Chao , Jian Sijie , Chen Chunlin , Chen Chen , Zhang Xiaoying 
Published: February 2021

Abstract
Background: Escherichia coli is a main pathogenic bacteria that causes cow mastitis, a condition that results in huge economic losses. There is lack of orally delivered prevention for cow mastitis. The outer membrane protein A (OmpA) of E. coli is immunogenic and can be used in a vaccine. In the present study, OmpA was synthesized into nanoparticles (NP-OmpA) for oral delivery and prevention of 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 ratio of tripolyphosphate (TPP)/CS (W/W). NP-OmpA safety was detected by function factors and histopathological examination of livers and kidneys. Immune activity of NP-OmpA was determined using qRT-PCR to detect immune-related gene expression, leukocyte phagocytosis of Staphylococcus aureus, ELISA to detect 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 nanodiameter of about 700 nm, loading efficiency (LE) of 79.27%, and loading capacity (LC) of 20.31%. The release rate was less than 50% in vitro. The optimal preparation conditions for NP-OmpA 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 factors of uric acid (UA), creatinine (Cr), alanine aminotransferase (ALT), aspartate transaminase (AST), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) showed NP-OmpA was safe for mice liver and kidney. 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 the injury in mice liver and kidney induced by E. coli. Conclusion: A novel NP-OmpA nanoparticle was synthesized, and the optimal preparation conditions were determined. The nanoparticles were found to be safer and have better immune function. They are expected to induce a response that resists infection with the main pathogenic bacteria (E. coli) of cow mastitis.