Pharmacokinetics And Pharmacodynamics Of Florfenicol In Ridgetail White Prawn Exopalaemon Carinicauda

Drug residues of aquaculture is a worldwide problem.In recent years,people realized that using antimicrobial made the drug residue seriously and had already led to the environmental pollution and threatened people’s health.And what is more,antibiotic resistance can transfer to human pathogens through the food chain with potential hazards to food safety and human health.Florfenicol was a kind of new type antibiotic,that there was a strong antibacterial activity to most gram-positive and gram-negative bacteria,and it was widely used to aquaculture,especially in breeding and seedling of shrimp.However,the florfenicol residues occur more and more frequently and florfenicol-resistant bacteria increase significantly in aquaculture waters.Therefore,to promote the development of bacterial resistance risk assessment,to protect aquaculture development and food safety and to safe guard human health are urgent demands to monitor the florfenicol residues and florfenicol-resistant bacteria in aquaculture.In this thesis,E.carinicauda was chosen as the experimental material,which is one of the emerging aquaculture species in China.In this study,the first aim was to investigate the pharmacokinetics profiles of florfenicol in the E.carinicauda after intramuscular and oral administration(10 mg/（kg·BW）and 30 mg/（kg·BW）at different water temperatures（22°C and 28°C）.The second aim was to evaluate the distribution of florfenicol in the E.carinicauda treated with 10,20 and 40 mg/（kg·BW）of florfenicol for 5days and measured the uptake and elimination dynamics of florfenicol in different organs and tissues.The third aim was to develop a physiological based pharmacokinetic model（PBPK）for estimating florfenicol concentrations in E.carinicauda and to calibrate and test the accuracy of the developed PBPK by the florfenicol concentrations in L.vannamei.The fourth aim was to determine the in vivo pharmacodynamics and the immune-antioxidant related enzymes of florfenicol.At last,we investigate the florfenicol resistance of Vibrios isolate from shrimp ponds with different mariculture patterns.The main results are as follows:1:Pharmacokinetics of florfenicol in the E.carinicauda at two water temperaturesIn this study,the pharmacokinetics profiles of florfenicol in the E.carinicauda were investigated following intramuscular and oral administration（10 mg/（kg·BW）and 30 mg/（kg·BW））at 22°C and 28°C water temperatures.Plasma and tissue samples（hepatopancreas,muscle,gill,intestine and carapace）were analyzed using an HPLC method.The results showed that the plasma concentration-time data for florfenicol were described commendably by two-compartment open model at the two water temperatures.In the same administration,temperatures did greatly effects on the pharmacokinetics florfenicol in the E.carinicauda.When oral administration was conducted,the distribution and elimination half-lives t_1/2α and t_1/2β were found to be 3.623 h and 19.038 h at 22°C.While 1.569 h and 11.358 h at 28°C.When intramuscular administration was conducted,the elimination half-1ives(t_1/2β)were found to be 10.26 h at 22℃and10.648 h at 28℃.In the same temperature and different adminsrations,differences were also showed.At 22°C,the elimination half-lives(t_1/2β)were found to be 19.038 h in oral administration and 18.26 h in intramuscular administration,meanwhile,there were the same situation at28°C.These datas indicated that both temperature and drug administration had significant effect on the elimination of florfenicol,and lower temperature or oral administration resulted in lower elimination.2:Pharmacokinetics of florfenicol in the E.carinicauda after oral administrationThe shrimps were fed with medicated feed at 10,20 and 40mg/（kg·BW）daily,and sampled in 1,2,3,4,5,7,10,15 and 20 day after the end of the drug exposure period.Results of the HPLC analysis showed highest florfenicol concentrations in hepatopancreas.The rank order of the mean concentrations of florfenicol in shrimp tissue and organs was hepatopancreas>intestine>plasma>muscle≈gill>carapace.The results also demonstrated a significant dose-dependent accumulation of florfenicol in shrimp.Florfenicol decreased gradually with the time.A withdrawal period of 7 d or more is proposed for E.carinicauda orally treated with florfenicol in order to meet the current food safety standards.3:Establishment of physiological based pharmacokinetic（PBPK）model of florfenicol in E.carinicaudaA physiological based pharmacokinetic model for estimating florfenicol concentrations from E.carinicauda was developed,which included hepatopancreas compartment,muscle compartment,gill compartment,carapace compartment and others compartment.E.carinicauda was assumed to be no metabolism.The residue elimination was described by the PKPB model.The PKPB model parameters were corrected through maximum likelihood method.And then the sensitivity analysis of model parameters ware compled.It shows that some parameters such as clearance,organ volume ratio are important to the whole PKPB model.The PKPB model predicted value has agreement with the actually measured value.The model can successfully predict the residue elimination of E.carinicauda.The extrapolation of the developed PBPK to L.vannamei was studied.The predicted concentrations of the model could describe the residue and elimination.The results show that the developed PBPK has a resliable ability to predicte the residue and elimination.4:In vivo pharmacodynamics of florfenicol in E.Carinicauda to V.parahaemolyticus infectionV.parahaemolyticus is conditioned pathogen which has high pathogenicity,and can infect shrimp,crab even human beings.The aim of this study was to investigate the in vivo pharmacodynamics of 10mg/（kg·BW）,20 mg/（kg·BW）and 40 mg/（kg·BW）concentration of florfenicol through blank control group（no bacterial infection or drug given）and hepatopancreas damaged control group（infected by bacterial but no drug given）and by establishment of a pathological model in E.Carinicauda infected by V.parahaemolyticus.The cumulative mortality rates were 0 in blank control group and 65.01%in hepatopancreas damaged control group 5 d after V.parahaemolyticus infection,indicating that the pathological model was successful,the cumulative mortality rates were 58%in 10 mg/（kg·BW）,51%in 20 mg/（kg·BW）and 45.35%in 40mg/（kg·BW）（P<0.05）.Compared with hepatopancreas damaged control group,the survival rates of E.carinicauda were significantly increased7.01%,14.01%and 19.66%in 10,20 and 40 mg/（kg·BW）,respectively.The levels of bacterial amount in hemolymph and hepatopancreas had a decreasing trend in hepatopancreas damaged control group,10,20 and 40mg/（kg·BW）.The bacterial amount in hemolymph and hepatopancreas were 1.73×10² CFU/m L,0,0 and 2.81×10² CFU/g,1.13×10² CFU/g,0 in10,20 and 40 mg/（kg·BW）at 12 d,and they were not significant difference compared with blank control group（P>0.05）.Histopathology observation revealed that florfenicol could improve some abnormal histological changes in hepatopancreas caused by V.parahaemolyticus,such as cellular swelling and vacuolated.The results suggested that the florfenicol had better effects in treatment of E.Carinicauda infected by V.parahaemolyticus and it can be used for the prevention and cure of shrimp vibrio disease.5:Effects of florfenicol on the immune-antioxidant capacity of E.CarinicaudaThe aim of this study was to investigate the effects of florfenicol on immune-antioxidant activities of the E.carinicauda.In this study,effect of different concentrations of florfenicol on HEM concentration,ACP,AKP,T-SOD,CAT and T-AOC activities in plasma were measured.In 10 and 20mg/（kg·BW）,HEM concentrations were significant higher than the control group at 3 h（P<0.05）,and it was significant higher than the control group from 3 to 48 h at 40 mg/（kg·BW）（P<0.05）.ACP activities were significant higher than the control group at 3 and 6 h（P<0.05）at 10 and 20mg/（kg·BW）,while it was significant lower than the control group from 3to 168 h at 40 mg/（kg·BW）（P<0.05）.AKP activities were significant higher than the control group at 3 h（P<0.05）,followed by a decreasing trend,which were lower than the control group from 6 to 48 h（P<0.05）at 10 and20 mg/（kg·BW）,while it was significant lower than the control group from3 to 48 h at 40 mg/（kg·BW）（P<0.05）,then all of the three experimental groups recovered to the control group at the end of the experiment.In 10and 20 mg/（kg·BW）,T-SOD activities were higher than the control group during the experiment and peaked at 6 and 24 h respectively.While it was significant lower than the control group at 24 and 48 h at 40mg/（kg·BW）（P<0.05）.CAT activities were higher than the control group at3 and 6 h（P<0.05）,then were significantly lower than the control group till96 h（P<0.05）,which recovered to the control group at the end of the experiment at 10,20 and 40 mg/kg.And T-AOC activities were significantly lower than the control group till 96 h in all of the three experimental groups（P<0.05）,which showed a dose-dependent manner.The results suggested that these parameters were potential biomarkers for evaluating the physiological health of florfenicol in E.carinicauda.6:Investigation on florfenicol-resistance in Vibrios from shrimp ponds with different mariculture patternsIn order to explore the structure composition and florfenicol resistance status of Vibrios from shrimp ponds with different mariculture patterns.Vibrio strains were isolated from aquaculture water and identified by TCBS specific medium combined with biochemical identification and molecular methods.Subsequently,we tested the flo R,cfr genes and integron-related elements in Vibrios through the PCR detection method,and then the drug-resistant gene cassette was amplified.Results showed that:142 vibrio strains were isolated,Rizhao region isolated 69 strains of 18 kinds,Weifang region isolated 56 strains of 9 kinds and Qingdao region isolated17 strains of 4 kinds.The numbers of V.harveyi 26.06%（37 strains）and V.alginolyticus 21.83%（31strains）were the most.Susceptibility results showed that 21 strains were isolated from the 142 vibrio strains,and the resistance rates of florfenicol in the isolated strains was 14.79%,Rizhao was 10.14%（7 strains）,Weifang was 23.21%（13 strains）and Qingdao was5.88%（1 strains）,respectively.Resistance genes detection result shown that,positive strains detection rate of flo R was 19.01%（27 strains）,while the cfr was not detected.Class 1 integrase positive rate was 100%（21/21）,class 2integrase and class 3 integrase positive rate were 0（0/21）.Then,when for the amplification of six strains which shown positive results in 5’-CS and3’-CS,the study found that stree strains of vibrio carried different gene cassettes:R-3（V.splendidus）and R-5（V.cyclitrophicus）carrying arr-3-dffr A27 gene cassettes,and R-17（V.alginolyticus）carrying dhfr gene cassette.The results of this study provide theoretical basis for the safety assessment of florfenicol in mariculture environment.