ABSTRACT
Mycoplasma hyopneumoniae (M. hyopneumoniae) infection affects the swine industry. Lithium chloride (LiCl), is a drug used to treat bipolar disorder and has also shown activity against bacterial and viral infections. Herein, we evaluated the antibacterial activity of LiCl on PK-15 cells infected with M. hyopneumoniae. Incubation of LiCl (40mM) with cells for 24h, did not significantly affect the cell viability. The qRT-PCR showed ~80% reduction in M. hyopneumoniae genome when LiCl added post-infection. A direct effect of LiCl on bacteria was also observed. However, treatment of cells with LiCl prior infection, does not protect against the infection. Anti-bacterial activity of LiCl was further confirmed by IFA, which demonstrated a reduction in the bacterial protein. With 40mM LiCI, the apoptotic cell death, production of nitric oxide and superoxide anion induced by M. hyopneumoniae, were prevented by ~80%, 60% and 58% respectively. Moreover, caspase-3 activity was also reduced (82%) in cells treated with 40mM LiCl. LiCl showed activity against various strains of M. hyopneumoniae examined in our study. Collectively, our data showed that LiCl inhibited the infection of M. hyopneumoniae through anti-apoptotic mechanism.
Subject(s)
Adjuvants, Immunologic/pharmacology , Lithium Chloride/pharmacology , Mycoplasma hyopneumoniae/drug effects , Animals , Apoptosis/drug effects , Bacterial Proteins/metabolism , Caspase 3/metabolism , Cell Line , Cell Survival/drug effects , Gene Expression Regulation, Bacterial/drug effects , Mycoplasma , Nitric Oxide/metabolism , Superoxides/metabolism , SwineABSTRACT
Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR), and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curve was established. This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml, with the half maximal inhibitory concentration (IC(50)) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml, respectively. The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested, and the IC(50) values to enoxacin, ciprofloxacin, and pefloxacin were 3.1, 3.4, and 4.1 ng/ml, respectively. It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%. When spiked in milk at 5, 20, and 50 ng/ml, the recoveries for NOR, enoxacin, ciprofloxacin, and pefloxacin ranged 90.5%-98.0%, 84.0%-95.2%, 94.0%-106.0%, and 89.5%-100.0%, respectively. The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.
