| Mycoplasma gallisepticum is an important pathogen that causes chronic respiratory diseases in chickens.After infection,the immune system is weakened,When it is mixed with Escherichia coli the symptoms become worse and the death rate increases.Thus,the poultry industry has brought huge economic losses.Because of its intensive farming methods,the main way to control these diseases is to use antibiotics.Danofloxacin belongs to the third generation of synthetic animal quinolones antibacterial drugs,with a wide antibacterial spectrum,strong antibacterial activity,excellent pharmacokinetic characteristics.In the early stage,there have been reports on PK/PD in half body of E.coli with danofloxacin and PK/PD in body of Mycoplasma gallisepticum,but there have been no reports on mixed infection of E.coli and Mycoplasma gallisepticum with danofloxacin.The purpose of this study is to establish the E.coli and Mycoplasma gallisepticum infection in vivo PK/PD model,to determine pharmacokinetic and pharmacodynamic parameters of danofloxacin in chicks and the optimal PK/PD parameters were determined by fitting,determine the best PK/PD parameters,setting the recommended dose,which provided scientific basis for veterinary clinical medication.1.In vitro pharmacodynamics study of pathogenic E.coli and Mycoplasma gallisepticumThe minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC)and anti-mutation concentration(MPC)of danofloxacin against avian pathogenic E.coli and Mycoplasma gallisepticum in chicken serum and liquid medium were determined by microdilution method.The results showed that the MIC of danofloxacin on E.coli was 0.008 ?g/mL and MBC 0.008 ?g/mL,and the MPC was 0.113 ?g/mL.Using Mycoplasma gallisepticum culture medium as the matrix,MIC of danofloxacin on Mycoplasma gallisepticum was 0.004 ?g/mL,and MBC was 0.016?g/mL.Meanwhile,MPC was 0.05?g/mL.When chicken serum was used as the matrix,the MIC of danofloxacin against E.coli and Mycoplasma gallisepticum was 0.016 and 0.008 ?g/mL respectively,and MBC was 0.06 and 0.031?g/mL respectively.The in vitro bactericidal curve showed that for E.coli,when the drug concentration is less than MIC,Danofloxacin has a slight inhibitory effect.However,when the concentration of danofloxacin was 4 MIC,the bactericidal effect on E.coli was achieved in both chicken serum and in MH liquid medium.For Mycoplasma gallisepticum of chicken,the bactericidal effect can only be achieved when the concentration of danofloxacin is 8 MIC.2.Establishment of models of E.coli and Mycoplasma gallisepticum chicken infectionThrough different ways of tapping the poison and tapping toxic dose and clinical symptoms,mortality,pathogen isolation,fluorescence quantitative PCR identification way the poison of attack of the final solution for chicken poison Mycoplasma gallisepticum air bag injection of 0.4 mL 108 ccu/mL,twice a day for three consecutive days,chest muscle injection of 0.4 mL of E.coli 107 cfu/mL,The results show that obvious clinical symptoms and changes were observed after 6 h and the load of E.coli in the blood,liver,lung were 103 cfu/mL,106 cfu/g,106 cfu/g,trachea,airbags,pulmonary microbial load of Mycoplasma gallisepticum in 105 ccu/g.The model of mixed infection of Mycoplasma gallisepticum and E.coli with stable bacterial load was established,which indicated that the model could be used in subsequent experiments.3.Pharmacokinetic study of danofloxacin on the mixed infection of E.coli and Mycoplasma gallisepticum in chicks180 infected chicks of 8-day-old,randomly divided into 3 groups,oral 1,10 and 20 mg/kg b.w.of danofloxacin respectively.After dosing 0,0.5,1,2,4,6,8,12 and 24 h respectively from the jugular vein blood 0.5 mL and separation of serum.The concentration of danofloxacin in serum was determined by high performance liquid chromatography(HPLC).The pharmacokinetic parameters of danofloxacin in infected chicks were determined by non-atrioventricular model.The results show that the Cmax of low,medium and high doses were 0.077±0.005 ?g/mL,0.398±0.019 ?g/mL,and 0.887±0.049 ?g/mL,respectively.The Tmax was 3.458±0.958 h,4.833±0.458 h,and 3.458±0.562 h,respectively.The T1/2? was 15.987±2.400 h?9.070±1.022 h and 10.221±1.406 h,respectively.At the same time,the dosages were linearly fitted with AUC0-24 h and Cmax,respectively.The pharmacokinetic parameters AUC0-24h and Cmax were linearly correlated with the dosages of danofloxacin in the range of 1-20 mg/kg b.w.4.In vivo pharmacodynamics and PK/PD fitting of danofloxacin against E.coli and Mycoplasma gallisepticumFifty-four infected chickens of 8-day-old were randomly divided into 9 groups and given danofloxacin solution at concentrations of 20,15,12.5,10,7.5,5,2,1 and 0 mg/kg b.w.once a day for 3 days.The chicks were sacrificed 24 hours after the last administration of the drug,and the bacterial load of E.coli in the blood,lungs and liver and the bacterial load of Mycoplasma gallisepticum in the trachea,air sac and lungs were measured.Finally,Sigmoid Emax model was used to fit the correlation between PK/PD parameters and antibacterial effect.The results showed that in vivo pharmacodynamics studies,the in vivo bactericidal effect was enhanced with the increase of drug dosage.The parameters of danofloxacin AUC0-24 h/MIC fit well with the antibacterial effect of the drug in vivo.In the blood,liver and lungs,when AUC0-24h/MIC are 139.62?58.13?86.14,can inhibition of the number of E.coli,danofloxacin producing bactericidal while the AUC0-24 h/MIC ratio of is 61 8.56 and 623.72 in liver and lung.The AUC0-24h/MIC ratio of the danofloxacin producing bacteriostatic,and bactericidal effect were 462.45 and 2452.18 in trachea,air sacs and lungs for Mycoplasma gallisepticum.The above results show that:the AUC0-24h/MIC were 462.45 and 2452.18 when the bacteriostatic and bactericidal effects were achieved for the treatment of mixed infection of E.coli and Mycoplasma gallisepticum,the administration dose was 7 and 38 mg/kg b.w.once a day for three consecutive days to achieve bacteriostatic and bactericidal effects. |
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