Isolation And Culture Of Lawsonia Intracellularis In Swine And Dose Regimen Formulation Of Tilmicosin Based On PK-PD

Porcine proliferative enteritis?PPE?caused by Lawsonia Intracellularis?L.intracellularis?has brought great losses to pig industry at home and abroad.Tilmicosin has been used as one of the important drugs in the clinical treatment of PPE because of its strong antibacterial activity,excellent cell membrane permeability and good intracellular accumulation capacity.In order to scientifically standardize the application of tilmicosin in the treatment of PPE and avoid resistance,it is particularly important to establish a reasonable dose regimen of tilmicosin against L.intracellularis through pharmacokinetic-pharmacodynamic?PK-PD?model,and provide reference for the study of PK-PD model of intracellular bacteria.Based on the establishment of isolation,culture and identification system of L.intracellularis,PK-PD model was used to develop the clinical dose regimen of tilmicosin suspension with good palatability and long action time,so as to achieve the best therapeutic effect and avoid resistance,which provided a new method for the effective control of L.intracellularis.And it provided a new dose regimen for the effective control of L.intracellularis.It is of great significance to give full play to the clinical application value of tilmicosin suspension and promote the healthy development of animal husbandry.1 Establishment of isolation,culture and identification system of L.intracellularisThree methods for the identification and detection of L.intracellularis were established by polymerase chain reaction?PCR?,real-time PCR?real-time PCR?and peroxidase monolayer assay?IPMA?;The isolation system was optimized by the number of bacteria in the sample,digestion and incubation time and membrane pore size;By optimizing the culture system of infection dose and infection time,L.intracellularis was isolated from the terminal ileum of pigs.The results showed that the three methods were sensitive,specific and reproducible.When the infection dose was 5×10⁵L.intracellularis/m L and the infection time was 3 h,the optimal infection efficiency was obtained.The growth curve of L.intracellularis in intestinal porcine epithelial cell?IPEC-J2?cells showed that L.intracellularis was in the slow phase,logarithmic phase,stable phase and decay phase at 0-1,2-5,6 and 7-8 d,respectively.When the number of L.intracellularis in ileum was more,the digestion and incubation time was 30 min,and the pore size of filter membrane was 5,1.2 and 0.65?m,respectively,the success rate of bacteria isolation was higher.A 319 bp target fragment was amplified by PCR.According to the kinetic curve amplified by real-time PCR,the concentration of the isolated strain was 5×10⁵L.intracellularis/m L.IPMA stained L.intracellularis in IPEC-J2 cells with red fluorescence.2 Pharmacodynamics of tilmicosin against L.intracellularisThe pharmacodynamics of clinical isolates of L.intracellularis was studied.The minimal inhibitory concentration?MIC?,in vitro and in ex vivo intracellular killing curves,mutant prevention concentration?MPC?and post-antibiotic effect?PAE?of tilmicosin against L.intracellularis were determined.The effect of tilmicosin on the viability of IPEC-J2 cells was detected by methyl thiazolyl tetrazolium?MTT?assay.The results showed that the cell viability decreased to 79.5%after cultured with 64?g/m L tilmicosin and IPEC-J2 for 3 days.And the cell viability decreased to 79.1%after cultured with 32?g/m L tilmicosin and IPEC-J2 cells for 4 days.The intracellular MIC,extracellular MIC and MPC of tilmicosin against L.intracellularis were 2,4 and 6.4?g/m L,respectively.Therefore,the range of mutant selection window?MSW?was 2-6.4?g/m L.The PAE of tilmicosin incubated with L.intracellularis for 1 h and 2 h were0.82-5.03 h and 2.53-6.92 h,respectively.In vitro and in ex vivo intracellular killing curves showed strong concentration-dependenttendencies.Therefore,the area under the concentration time by MIC?AUC/MIC?was selected as the PK-PD fitting parameter.3 Pharmacokinetics of tilmicosin premix and suspension in pig plasma and ileum contentTwenty four healthy weaned piglets weighing about 22 kg were randomly divided into 4 groups with 6 pigs in each group.The model of ileal fistula was established by installing T-shaped fistula in ileum.PPE model was established by using isolated L.intracellularis.The piglets in the infected group and the healthy group were administered with tilmicosin premix and suspension at the dose of 10 mg/kg.Blood and ileum content were collected at 0,0.5,1,2,3,4,5,6,8,10,12,24 and 48 h after administration.The concentrations of free tilmicosin premix and suspension in plasma and ileum content were determined by high performance liquid chromatography?HPLC?.The non compartment model in winnonlin software was used to simulate the data.The results showed that the peak time(T_max)of tilmicosin premix and suspension in plasma and ileum contents of healthy and infected piglets were 2 h.The peak concentration(C_max)and area under the concentration time curve from 0 to 24 h(AUC_0-24h)of tilmicosin premix in healthy and infected piglets were 1.08±0.04 and 0.99±0.03?g/m L,9.61±1.47and 9.30±1.43 h·?g/m L,respectively.And the C_maxand AUC_0-24hwere 3.78±0.03 and3.41±0.06?g/m L,20.41±1.64 and 22.65±1.32 h·?g/m L,respectively.The C_maxand AUC_0-24hof tilmicosin suspension in healthy and infected piglets were 1.23±0.02 and1.16±0.13?g/m L,14.39±0.35 and 14.84±0.42 h·?g/m L,respectively.And the C_maxand AUC_0-24hin ileum contents of healthy and infected piglets were 4.57±0.37 and 3.68±0.43?g/m L,25.81±0.24 and 24.54±0.34 h·?g/m L,respectively.4 In ex vivo PK-PD model fitting and formulation of dose regimenThe sigmoid Emax model was used to fit the relationship between AUC_0-24h/MIC value and logarithm value of the concentration change of L.intracellularis in ex vivo intracellular killing curves,and the PK-PD parameters(AUC_0-24h/MIC)were calculated when E=0,-3 and-4.Combined with MIC value,clearance rate by bioavailability?CL/F?and AUC_0-24h/MIC value,Monte Carlo simulation was used to predict the daily dose of prevention,treatment and eradication effect.PK-PD model based on bacterial growth kinetics was established to predict the dose regimen.When E=0,-3 and-4,the AUC_0-24h/MIC values of the corresponding antibacterial,bactericidal and eradication effects were 6.87,26.80 and 36.02 h?tilmicosin premix?,5.77 h,23.32 h and 36.18 h?tilmicosin suspension?,respectively.According to the dose calculation formula and Monte Carlo simulation,the daily dose of tilmicosin premix for clinical prevention,treatment and eradication of porcine proliferative enteritis was 2.95,14.39 and 15.48mg/kg,respectively,and that of tilmicosin suspension was 2.48,12.53 and 15.54 mg/kg,respectively.The results of PK-PD model based on the mechanism of bacterial growth kinetics showed that the treatment and eradication dose could achieve the expected treatment and eradication effect at the interval of 24 h,while the recommended dose regimen for prevention effect was once every 24 h.In order to facilitate the large-scale pig farm population administration,the daily dose of tilmicosin premix was converted to60,280 and 300 mg/kg respectively,and the daily dose of tilmicosin suspension was 25,125 and 150 mg/L,respectively.In this study,the isolation,culture and identification system of L.intracellularis were established,and the in ex vivo PK-PD model of tilmicosin premix and suspension against L.intracellularis was established by using ileum content,and the scientific and reasonable dose regimen of tilmicosin and suspension in the prevention,treatment and eradication of PPE were formulated.It will provide corresponding clinical trial data for the registration of new veterinary drugs.At the same time,this study provided a reference for PK-PD model of other intracellular bacteria.